Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

Sara Thornby Bak; Eva Bang Harvald; Ditte Gry Ellman; Sabrina Bech Mathiesen; Ting Chen; Shu Fang; Kristian Skriver Andersen; Christina Dühring Fenger; Mark Burton; Mads Thomassen; Ditte Caroline Andersen

doi:10.1007/s00395-023-00979-2

Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

Bak, Sara Thornby; Harvald, Eva Bang; Ellman, Ditte Gry; Mathiesen, Sabrina Bech; Chen, Ting; Fang, Shu; Andersen, Kristian Skriver; Fenger, Christina Dühring; Burton, Mark; Thomassen, Mads; Andersen, Ditte Caroline 2023-03-02 00:00:00 Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid prolifera- tive CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes. Keywords Heart development · Cardiomyocytes · Proliferation · Endoreplication · Zinc Finger E-Box Binding Homeobox 1 (Zeb1) Introduction Cardiovascular diseases are among the leading causes of death worldwide [68], and one major explanation is the inability to regenerate the heart after myocardial infarction (MI), leaving the affected subjects with impaired cardiac Sara Thornby Bak, Eva Bang Harvald and Ditte Gry Ellman have function [52]. Whereas, the mammalian heart forms through contributed equally to this work. cardiomyocyte (CM) proliferation during fetal development * Ditte Caroline Andersen [58], CMs enter cell cycle arrest around birth [4]. As in gen- dandersen@health.sdu.dk eral, CM proliferation requires the CM to go through the four cell cycle phases: G1, S, G2 and M [57]. Yet, around birth Andersen Group, Department of Clinical Biochemistry, Odense University Hospital, Odense, Denmark (P3 in mice) a portion of CMs exit the cell cycle and become quiescent in the G0 phase [28, 61, 71], whereas another CM Clinical Institute, University of Southern Denmark, Odense, Denmark fraction undergoes a final round of mitosis peaking at post- natal stage P5 (in mice), but indeed fails to complete karyo- Amplexa Genetics, Odense, Denmark 4 and/or cytokinesis. This is often referred to as the G2/M Department of Clinical Genetics, Odense University challenge which results in polyploid and binucleated CMs Hospital, Odense, Denmark Vol.:(0123456789) 1 3 8 Page 2 of 24 Basic Research in Cardiology (2023) 118:8 [72, 79, 80], and is considered a hindrance for heart regener- 130–098-373) according to the manufacturer’s recommen- ation through CM proliferation [20, 32]. The mechanism for dations. Following viable cell counting (NC-200, ChemoM- this switch is unclear, but has been associated with reduced etec), dissociated cells were stained with a fixable viability expression of cyclins and cyclin dependent kinases (CDKs) stain (Fixable viability stain 570; BD Biosciences, 564,995) in parallel with induced expression of several CDK inhibi- prior to fixing in methanol for 15 min followed by rehydra- tors [29, 44], changes in miRNA expression, extracellular tion to reverse the RNA to its original state. During rehydra- signaling pathways, centrosome integrity, telomere dysfunc- tion, the RNase inhibitor, RNasin Plus (Promega; N2615), tion, epigenetics as well as extracellular matrix compositions was added to prevent RNA degradation and included in all [51]. In the adult mouse and rat heart, ~ 80–95% [29] of CMs subsequent steps. After rehydration, samples were stored at are binucleated while ~ 5% CMs are mononucleated [6]. In -80 °C until analysis. All reagents were high grade, RNase adult humans, controversies remain regarding the ratio of free and the environment was kept strictly RNase free to binucleated CMs compared to mononucleated, and it has avoid degradation of the RNA. For comparison of fresh and been suggested that ~ 60% human CMs remains mononu- fixed scRNA-seq profiles, mouse myoblasts (C2C12; ATCC, cleated [47], whereas the percentages of binucleated range CRL-1772) were used and maintained as recommended. between 12 and 75% [35]. Even so, a large portion of the mononucleated human CMs is polyploidy [47]. The termi- Fluorescence‑activated cell sorting (FACS) nally differentiated and polyploid CMs are often referred to as dormant with respect to cell division [32, 38], although Fixed cardiac cells were stained for the CM marker MYH1 recent evidence indeed contradicts this [36]. As with CM (Mouse IgG2b,k; 1:300; MF20-c; DSHB) and visualized by proliferation, current mechanistic insight to understanding donkey anti-mouse IgG Alexa Fluor 488 (1:200; Invitro- polyploidy also remains deficient [32, 35]. More systematic gen, A21202), whereas Hoechst 33342 (Sigma) was added approaches are therefore required to identify factors mediat- 5 min before sorting (FACSAriaIII, BD Biosciences). Prior ing CM cell cycle activity, and which on a longer term may to FACS, cells were filtered (Falcon, 352235) to avoid cell be used to tackle the challenge of a non-regenerating heart. clumps. Strict RNase free conditions as described above Herein, we developed a new protocol combining fluores- including new tubing were prioritized throughout the pro- cence-activated cell sorting (FACS) of diploid and tetraploid cedure. Analysis and sorting gating strategy (Supplemen- murine CMs around birth with high-throughput single cell tary Fig. 1c) included hierarchical gating using the FACS- RNA sequencing (scRNA-seq) to uncover transcription fac- Diva software v8.0.1 (BD Biosciences) based on FSC/SSC, tors (TFs) that regulate the G2/M CM cell cycle process. viability Alexa 570, and MYH1-Alexa 488, and Hoechst 33342. For each developmental stage (E16.5, P1, P5), three independent sortings (n = 3, each consisting of cells from Methods one litter) were performed after carefully checking and vali- dating the FACS setup using FMO controls (Supplemen- Mice tary Fig. 1c). Prior to scRNA-seq analysis CM purity, nuclei number, and cell clumping of sorted cells were assessed C57BL/6 J mice were obtained from Taconic Europe, using immunofluorescence microscopy whereas the RNA housed with a 12/12 h light/dark cycle, and fed ad libitum. integrity number (RIN) was determined using an Agilent For scRNA-seq, mice were plug bred, and litters for each 2100 Bioanalyzer (Agilent Technologies) combined with the of the three timepoints were obtained from different breed- Agilent RNA 6000 Nano Kit (Agilent Technologies) [56]. ing pairs. Plug was checked in the morning and evening. Sorted cells were stored at -80 °C until scRNA-seq. For E16.5 primary cultures, mice were plug bred as well, whereas for primary P0 cultures continuous breeding was ScRNA‑seq used. All animal experiments were approved by the Dan- ish Council for Supervision with Experimental Animals For scRNA-seq, cells originating from three independent (#2016–15-0201–00,941 and #2022–15-0201–01119). FACS were pooled (14–20 pups/sample) to account for bio- logical diversity in the scRNA-seq analysis. Single Cell 3’ Preparation of CMs for scRNA‑seq RNA-Seq libraries were prepared using Chromium Single Cell 3′ Reagent Kits v2 (10 × Genomics) according to the For scRNA-seq, the left heart ventricle was dissected under user guide. In brief, cellular suspensions of approx. 1200 a stereomicroscope at E16.5, P1, and P5 (n = 3 litters, each cells/µl were mixed with master mix reagents and loaded counting 4–8 pups), and enzymatically dissociated using on a Single Cell A Chip (10 × Genomics) together with the semiautomatic GentleMACS tissue dissociator system Single Cell 3’ Gel Beads (10 × Genomics) and partitioning (MACS Miltenyi Biotec; Neonatal Heart Dissociation Kit oil to generate single cell gel beads-in-emulsion (GEMs). 1 3 Basic Research in Cardiology (2023) 118:8 Page 3 of 24 8 The GEM generation took place in a Chromium Control- visualization by Uniform Manifold Approximation and ler (10 × Genomics). Single cell reverse transcription was Projection (UMAP) clustering [69, 70]. Cell clustering by performed in a standard thermal cycler, and the GEMs were expression pattern was performed by first calculating the subsequently broken using Recovery Agent (10 × Genom- k-nearest neighbors and constructing the shared nearest ics). The resulting cDNA was cleaned up with DynaBeads neighbor (SNN) and next optimizing the modularity func- MyOne Silane Beads (Thermo Fisher Scientific) and tion to determine clusters. SPRIselect Reagent Beads (Beckman Coulter), and then Clustering and Heatmaps The Seurat v 2.3.0 and 3.1.5 [7, amplified by PCR using Single Cell 3′ Reagent Kit v2 (No. 62] were used for cluster visualization by UMAP and for dif- of cycles: 8). After another cDNA clean-up with SPRIselect ferential gene expression of marker genes between clusters. Beads, the fragment sizes and concentrations were measured For each of the samples, a Seurat object was created, and using QIAxcel DNA High Resolution Kit (1200) (Qiagen) the cells filtered based on whether they expressed a com- and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), bination of the CM-specific markers Tnni3, Tnnt2, Actc1, respectively. Enzymatic fragmentation, end-repair, and and Tnnc1. Each sample was then log-normalized, variable A-tailing were performed in one-step using the Single Cell features were identified using “vst” as selection method and 3′ Reagent Kit v2, and fragments of approx. 200 bp were 2000 nfeatures, and the data was scaled using nCount_RNA selected by double sided-size selection using SPRIselect for vars.to.regress. PCA was run and based on jackstraw- Beads. NGS libraries were then constructed by adapter liga- and PC elbow plots the optimal number of dimensions tion and PCR mediated sample indexing (No. of cycles: 13). was determined (range 9–14). Moreover, when applied, all After a final double-sided size selection, the NGS library samples were merged, integrated using FindIntegrationAn- concentrations were measured using Qubit dsDNA Assay chors and IntegrateData, filtered, normalized, and scaled as Kit. Libraries were sequenced on the Illumina NextSeq 500 described above with generated UMAP plots depicting cell platform using NextSeq 500/550, high output Reagent Car- cycle phases, clusters, and original data affiliation as for each tridge V2, Illumina Kit (Read 1 = 26 cycles, i7 Index = 8 individual sample. cycles, Read 2 = 130 cycles), and the second analysis was Visualization, clustering, and cell cycle analysis UMAP performed on a Illumina NovaSeq 6000. plots and clusters were generated as described above using PCA as reduction type and resolution = 0.6; based on the ScRNA‑seq data analysis top 30 marker genes for each cluster. Subsequent GO term enrichment was evaluated using clusterProfiler::enrichGO Read alignment and construction of gene expression matrix [75, 78] and the “org.Mm.eg.db” library with ont = “BP”, Base calls were converted to FASTQ format and demulti- pAdjustMethod = “BH”, and cutoff values = 0.01. Fea- plexed using the cellranger mkfastq function embedded in tures witg avg_log2FC > 0.5 were used, where each cluster the 10 × Genomics cellranger software package using default was named according to biological identity. Finally, each settings (https://suppo r t.10xg enomics. com/ sing le-cell- g ene- dataset was split into three groups (G1-, S-, or G2/M- expre ssion/ softw are/ overv iew/ welco me). Single cell gene phase) based on the expression of cell cycle markers [67] counts matrices were generated using the cellranger count and each cell was assigned with a cell cycle score using command. During this step, FASTQ files generated by the Seurat::CellCycleScoring. cellranger mkfastq step, were aligned to the Mus musculus Analysis across developmental stages After merging and genome (mm10/GRCm38) using the splice-aware aligner integration as described above, two clusters of cell cycle STAR [12]. Subsequently, STAR used the Mus musculus active E16.5 and P5 cells, respectively, were subtracted from transcriptome reference (GRCm38.84) to segregate the the data and compared using FindMarkers. The resulting list mapped reads into exonic, intronic and intergenic regions of features was used for generating cnetplot and TF analysis. and for assessment of how confidently the reads have been Mouse single site analysis was used for TFs (oPOSSUM mapped to these regions. Only non-duplicated reads which version 3.0) [25, 26, 33], all genes in current dataset as back- were confidently mapped to the transcriptome, and which ground, all vertebrate profiles with a minimum specificity of had barcodes and unique molecular identifiers (UMIs) were 8 bits, conservation cutoff 0.40, matrix score threshold 85%, used for UMI counting. The expression matrices were gen- up/downstream sequence 5000/5000). In addition, oPOS- erated by counting the number of strand-specific UMI for SUM results were supported by GSEA using the Molecu- each cell mapping to either the exonic or intronic regions lar Signature Database [41, 42, 63] and transcription factor of each gene. targets (TFT). Clustering and UMAP visualization Using the R pack- Trajectory analysis Data were prepared in Seurat (filtered age Seurat[62] v 2.3.0 and 3.1.5 dimensionality reduction and cell cycle assigned; since UMI data were used, nor- by principal component analysis (PCA) was performed; malization was avoided in agreement with recommendations subsequently the PCA data analysis was used as input for by the Monocle platform) in merged pools of either 2n-, 1 3 8 Page 4 of 24 Basic Research in Cardiology (2023) 118:8 4n-, or all samples, respectively. Subsequently, phenotype promoter; synthesized by GeneArt (Thermo Fisher); the data and feature data were extracted from the Seurat object DNA sequence was kindly provided by Professor Brent A. and converted to a Monocle CellDataSet (CDS) object. French, University of Virginia, USA) was inserted for CM Next, dispersion estimates for count dataset were obtained specificity by PCR amplification in two steps: first, plasmid using monocle::estimateDispersions and cells were sorted pAAV-EF1a-mCherry-IRES was PCR amplified by primers according to num_genes_expressed (500 < num_genes_ (F or w ard: GG A A TT CC A T A T GGG T A C CGG A TC CG T G A G expressed < 3000). A set of ordering genes was isolated C and R e v erse: GCT CT A G AA A TT CCC A CT CCT TT CAA G using differentialGeneTest and used to order the CDS by ACCT AG) containing the XbaI and NdeI restriction sites to the monocle::setOderingFilter. Next, the dimensions were excise the EF1a promoter. Secondly, the cTnT promoter was reduced and cells were ordered along the trajectory using inserted between the two restriction sites (Forward: GCT monocle::reduceDimension and monocle::orderCells, CT A G A G C A G T CTG and R e v erse: GG A A TT C C A T A T G A G respectively. The trajectory was plotted depicting origi- GTC ). The resulting pAcTnT-mCherry-IRES plasmid was nal identity, cell cycle phase, and pseudotime state. The then sequenced (Eurofins Genomics, Ebersberg, Germany) monocle::BEAM function was utilized in each branch point for validation (Data not shown). Genes (Origene plasmids of the trajectory plots to evaluate branch point dependent and Nfya) were inserted into the pAcTnT-mCherry-IRES gene expression. plasmid between the SgfI and MluI restriction sites. Since the SgfI restriction site was already included in the Egr1 Plasmids and AAV9 packaging sequence, Egr1 was amplified by the following primers For - war d: AA T GG T GG T T CT GG T GCG A T C GC A T GG C AG To determine the most efficient AAV serotype for CM trans- CGG CCA AG and Reverse: TT G A T A T CG AA T CTA ACG duction, pilot studies with both AAV6 and AAV9 transduc- CG T GC A AA T TT C AA T T G T C. N e xt t he Egr1sequence tion were performed, as these serotypes have previously was added to pAcTnT-mCherry-IRES by NEBuilder® HiFi shown efficient in CM transduction [53]. In our study design DNA Assembly Master Mix (NEB). Proper gene insertions we found the AAV9 serotype to be much more efficient in were validated by enzymatic digestion at the respective transducing CMs, as compared to the AAV6 serotype (data restriction sites and size determined by gel electrophoresis not shown). (Data not shown). Generation of plasmids Plasmids harboring the genes For plasmid packaging in an AAV9 serotype capsid we of interest were purchased from Origene (Mouse Tagged used the Rep/Cap plasmid, pAAV2/9n, a gift from James M. ORF Clones; Supplementary Table 1), except for Nfya, Wilson (Addgene plasmid # 112,865; http:// n2t. ne t/ addg ene: which were synthesized by GeneArt (Thermo Fisher; Sup- 112865; RRID:Addgene_112865) and the helper plasmid plementary Table 1). The AAV backbone transfer vector pHelper (a kind gift to our collaborator Per Svenningsen, was derived through modifications of the plasmid pAAV- University of Southern Denmark, from Ben Deverman, EF1a-mCherry-IRES-Cre (a gift from Karl Deisseroth; Caltech, Pasadena, USA). Addgene plasmid # 55,632; http:// n2t. net/ addge ne: 55632; Virus generation Large-scale AAV generation for in vitro RRID:Addgene_55632) [15], allowing simultaneous tran- use was performed in HEK293T cells (ATCC; CRL-3216) scription of mCherry and the gene of interest through the by co-transfection with pAcTnT-mCherry-IRES (empty vec- internal ribosome entry site (IRES). Thus, due to the IRES tor) or pAcTnT-mCherry-IRES harboring the gene of inter- site, transcription of the gene of interest correlates to the est, pAAV2/9n and pHelper. Transfection efficiency was level of mCherry. To unify the process of gene insertions, addressed by mCherry visualization using immunofluores- the restriction sites SgfI and MluI were inserted into the plas- cence microscopy. Five days after transfection, recombinant mid. Briefly, the already existing MluI restriction site was AAV was isolated by PEG 8000 precipitation and purified by removed by introducing a point mutation in the plasmid by iodixanol gradient ultracentrifugation followed by centrifu- PCR amplification using the following primers: Forward: gation through an Amicon Ultra Centrifugal filter (50 K). C GC A CG G G T A A G C TT T GC A AA G AT G G A TAA A G T Recombinant AAV yields were determined by quantitative TTTAAA C AGA GAGG A and Reverse: AAGCTT A CCCG T real-time PCR (qRT-PCR) through a titration of pAcTnT- GCG GCC GCA GGA ACC CCT AGT GAT . The Cre site was mCherry-IRES plasmid using the primers Forward: AGT then removed and the SgfI and MluI restriction sites were G TT GCA TT C CT C T CT GG and Reverse: AGC GCA T GA hereafter inserted by PCR amplification using the prim-ACT CCT TGA T. ers Forward: TCT GGT GCG ATC GCC TAG ACG CGT TAG Adenoviral constructs were generated by Vector Bio- ATT CGA TAT CAA GCT TAT CGA TAA TCA ACC TCT and labs (PA, USA) using Adenoviral Human Type 5 (dE1/ Reverse: CTAGGC G ATCGC A CCA GAA CCA CCA TTA TC E3) as backbone. For ZEB1 knockdown experiments, a U6 A TC G TG TTT TT C AAA GG A AAA CC A CG T CCC . Finall y , a promoter was driving ZEB1 short-hairpin RNA (shRNA) truncated chicken cardiac Troponin T promoter [53] (cTnT e xpr ession of t he seq uence 5´CCG G AT AG A GGC TAC 1 3 Basic Research in Cardiology (2023) 118:8 Page 5 of 24 8 AAG CGC TTT A-CTC GAG -TAA AGC GCT TGT AGC CTC were counted (NC-200; ChemoMetec). Cells were seeded in TA-TTT TTT G-3´ and a targeting sequence of ATA GAG 12-well plates pre-coated with ECM at a density of approx. GCT ACA AGC GCT TTA. An eGFP reporter was expressed 236,500 cells/cm and placed in an incubator (37 °C, 5% under a separate CMV promoter. Ad-GFP-U6-scrmb-shRNA CO ) or approx. 88,235 cells/cm on 4-well chamber slides (cat. no. 1122N) containing a scrambled shRNA and an (cat.no. 154917, Lab-Tek™ II) for confocal microscopy. eGFP reporter was used as control. For ZEB1 overexpres- Before deciding which concentration of virus to use, sion experiments, the backbone vector contained a CMV both for AAV9 and adenovirus transduction, titration tests promoter to drive expression of the gene of interest. Ad- were performed and the concentrations resulting in the most GFP-Zeb1 was generated using mouse cDNA (GenBank: efficient transduction without causing cytotoxicity were BC139768.1) and eGFP, and ZEB1 were expressed under used. After 24 h the number of cells in each experiment separate CMV promoters. Ad-GFP (cat.no. 1060) was used were estimated (NC-200; ChemoMetec), and cell cultures as empty control. were transduced with either 750,000 viral genomes (vg)/ cell of the desired AAV9 or 50 MOI of adenovirus. For E16.5 CM cultures and Zeb1 knockdown AAV9 experiments, six, 24, and 48 h after viral transduc- tion, medium was refreshed with medium containing 10 µM On embryonic day 16.5 (E16.5), the pregnant mice were EdU. For adenovirus, EdU was added together with the virus sacrificed by cervical dislocation and the hearts from the and replenished every 24 h. All cells for qRT-PCR were pups were quickly removed and placed in a cardioplegic replenished with medium without EdU. Cells were either buffer (MIB; 1.2 mM KH PO (pH 7.4); 0.25 g/l Na CO fixed in 2.5% Neutral Buffered Formalin (NBF) diluted in 2 4 2 3; 6.44 g/l NaCl; 2.6 mM KCl; 1.2 mM M g SO ; 11 mM HBSS/5%FBS/1%PS for flow cytometry analysis 72 h post 2 4 glucose) supplemented with 1% Bovine Serum Albumin transduction (see below), fixed in the wells in 10% NFB (BSA; MIB/1%BSA). The heart ventricles were dissected or 4% Paraformaldehyde (PFA), or the RNA was isolated under a stereomicroscope before enzymatically dissocia- for qRT-PCR 48 h post transduction for adenovirus or 72 h tion into a single cell suspension using the semiautomatic post transduction for AAV9 (see below). Transduction effi- GentleMACS tissue dissociator system as described by the ciency was addressed by immunofluorescence microscopy manufacturer. Dissociated cells were counted (NC-200; for mCherry during culture, and by qRT-PCR (mCherry and/ ChemoMetec), plated on extracellular matrix (ECM) at a or gene of interest) and flow cytometry for mCherry or GFP. density of approx. 118,500 cells/cm and cultured in growth Furthermore, we consistently observed lower levels of GFP medium (79.5% DMEM (supplemented with 1% PenStrep with the Ad-GFP-Zeb1 compared to Ad-GFP suggesting (PS)), 19.5% Medium 199 (supplemented with 1% PS), and correlation between GFP and ZEB1 expression. 1% newborn calf serum). After 24 h, the number of cells were counted in some wells to calculate the amount of virus Flow cytometry required. Optimal MOI was determined from titrating the virus and quantifying transduction efficiency as well as Fixed cells were permeabilized with phosphate buffered observing for immediate cytotoxicity. saline (PBS) containing 1% BSA and 0.1% Triton X-100 After 24 h of culturing, cells were transduced with 10 (TX100) and stained with primary antibodies in different MOI of either Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1. combinations (mouse anti-MYH1, 1:300, MF20-c, DSHB; In addition, 10 µM of 5-ethynyl-2′-deoxyuridine (EdU) was rat anti-mCherry, 1:500, M11217, Thermo Fisher; and rab- added to assess for cell cycle activity. The medium, with or bit anti-GFP, 1:500, ab290, Abcam) for 1 h in the dark on without EdU, was replenished every 24 h, and experiments ice while shaking. After washing, cells were incubated with were terminated as indicated at 96 h after transduction for EdU Click-it reaction cocktail according to the manufac- analysis. turer’s protocol (Invitrogen, C10419), and washed before incubation with secondary antibodies in different combina- Neonatal CM cultures and viral transduction tions (488-donkey anti-mouse, 1:200, A21202, Invitrogen; 555-donkey anti-rat, 1:200, Ab150154, Abcam; 555-don- Neonatal (P0) mouse pups from each litter were sacrificed key anti-mouse, 1:200, A31570, Invitrogen; and 488-donkey by decapitation, whereafter the hearts were quickly removed, anti-rabbit, 1:200, A21206, Invitrogen) for 30 min in the and the ventricles dissected under a stereomicroscope. Dis- dark on ice while shaking. Three final washes were per - sected ventricles were pooled in a tube with MIB/1%BSA formed in PBS/1% BSA/0.1% TX100 and Hoechst 33342 before enzymatic dissociation into a single cell suspension was added 5 min before flow cytometry using the LSRII using the semiautomatic GentleMACS tissue dissociator flow cytometer (BD Biosciences). Data was analyzed using system as described by the manufacturer. Dissociated cells the FACSDiva software v8.0.1, and initially gated according were resuspended in growth medium and the number of cells to the CM marker MYH1 and then sub-fractionated based 1 3 8 Page 6 of 24 Basic Research in Cardiology (2023) 118:8 on the antibody amplified mCherry or GFP signal. Cells and each heart was processed individually, or prepared for positive or negative for a reporter (mCherry or GFP) were paraffin embedding (see below). gated according to EdU incorporation to determine cell cycle activity. Ploidy was addressed in subpopulations by Hoechst Immunohisto‑ and immunocyto‑chemistry 33342 using gates (2n, 4, and > 4n) defined by the entire CM and microscopy population. Each analysis as indicated consisted of at least three to nine independent experiments designated n, each P8 hearts from virus injected pups were fixed in 10% NBF comprising cells from one litter. Within an experiment, 1–3 overnight, rinsed in PBS, dehydrated, and finally embed- replicates (n*) were performed and used as an average of the ded in paraffin. Embedded specimens were then cut into n for further statistical analysis as indicated. 10 µm sections before mounting on glass slides and stored at 4 °C. Sections were deparaffinized and rehydrated before staining. Immunostainings were performed on NBF or PFA RNA isolation, RNA integrity, and qRT‑PCR fixed cell cultures or paraffin embedded sections as previ- ously described in Andersen et al. [2] with the following RNA was isolated from each sample and qRT-PCR was per- primary antibodies: rabbit anti-GFP (1:500, ab290, Abcam), formed as described previously [3]. Briefly, the cells were mouse anti-MYH1 (1:300, MF20-c, DSHB), rabbit anti- lysed with TriReagent and the RNA was isolated using Poly- ZEB1 (1:500, PA5-28,221, Thermo Fisher), rabbit anti- acryl carrier, 1-Bromo-3-Chloro-Propane and 2-propanol. Mef-2c (1:500, 5030S, Cell Signaling Technology), mouse The RNA was rinsed using 75% ice cold ethanol. Finally, anti-actinin (1:200 A7811, Sigma), mouse anti-α-tubulin the RNA was dissolved in nuclease-free water and the RNA (1:250, 3873, Cell Signaling Technology), and 647-phalloi- concentration was determined using a nano-drop. For qRT- din (1:800, A30107, Thermo Fisher). Secondary antibodies PCR, cDNA was generated using the High-Capacity cDNA used were: 488-donkey anti-rabbit (1:200, A21206, Invitro- Reverse Transcriptase kit (Applied Biosystems; 4368814) gen), 555-donkey anti-rabbit (1:200, A31570, Invitrogen), according to the manufacturer’s recommendations. Each 647-donkey anti-mouse (1:200, A31571, Invitrogen). All sample for qRT-PCR contained 2–4 ng cDNA (Supplemen- sections were mounted with DAPI (Vectashield, Vector Lab., tary Table 2) in a total volume of 10 µl and were analyzed for Phalloidin and α-tubulin staining, Fluoroshield, Abcam in technical triplicates of qRT-PCR using a mixture of was used). Microscopy was performed on a Leica DMI 4000 Power SYBRGreen PCR Master Mix (Applied Biosystems, B microscope with a Leica CTR4000 illuminator and Leica 4367659) and appropriate forward and reverse primers (Sup- DFC300FX/DFC 340 FX cameras, and confocal micros- plementary Table 2). The qRT-PCR was run on a 7900HT copy was performed on an Olympus FV1000MPE confocal Fast Real-time PCR system (Applied Biosystems) under the laser scanning microscope equipped with an UPlanSApo following conditions: holding for 10 min at 95 °C, hereafter 60x/1.20 water objective. During analysis, all camera set- 40 cycles consisting of 15 s of denaturation at 94 °C, 30 s tings and picture processing were applied equally to samples of annealing at 57–60 °C (Supplementary Table 2) and 30 s and controls. of elongation at 72 °C. The obtained data was analyzed as previously described [3] by normalization to multiple stably Statistics and reproducibility expressed endogenous gene according to the qBase Plus 3.2 platform (Biogazelle). All statistics were performed using the GraphPad Prism (v 9.0.0) software and the appropriate tests, number of inde- Injection of adenovirus in P0 pups pendent experiments (n) and replicates (n*) are defined in the corresponding figure legends. We used the significance The litter was gently taken from their home cage. Pups level α = 0.05 for identifying significant results marked by were then anesthetized by induction of hypothermia before asterisks, yet have indicated throughout the exact p-value 7.60 × 10 PFU (in a total volume of 20 µl, diluted in sterile if between 0.05 and 0.1 to enable objective evaluation of PBS) of the desired adenovirus was injected into the super- trends. For scRNA-seq, each timepoint includes three bio- ficial temporal vein. Pups were reheated and placed together logically independent experiments each comprising mouse with their littermates before the litter was gently put back pups derived from three distinct litters. Cells were pooled into their home cage. Following the pups received a 50 µl just before library generation and scRNA-seq. ScRNA-seq subcutaneous injections of EdU (2.5 mg/ml) at P4 and P6 and subsequent gene expression analysis and transcription before they were sacrificed by decapitation at P8. Hearts factor binding site analysis was performed using two dif- were dissected and either dissociated for flow cytometry ferent sequencing platforms (NextSeq and NovaSeq) with as described above for P0 pups, except cells were strained similar results. All transduction experiments were success- (100 µm nylon cell strainer, 352360) prior to NBF fixation, fully reproduced with at least three biologically independent 1 3 Basic Research in Cardiology (2023) 118:8 Page 7 of 24 8 experiments obtaining similar results, thereby confirming of this method (Supplementary Fig. 1a) includes avoid- the design and robustness. ance of the long-term susceptibility of CMs to tissue dis- sociation, improvement of logistics with the ability to store scRNA-seq samples obtained at different timepoints, and Results application of FACS to enrich CMs using intracellular CM markers such as myosin heavy chain 1 (MYH1) and DNA FACS of fixed CMs combined with high‑throughput (Hoechst; Supplementary Fig. 1a–c). Based on a viability scRNA‑seq efficiently distinguishes diploid stain prior to fixation we excluded and reduced the num- and tetraploid CMs during heart development ber of dead cells (Supplementary Fig. 1c) and in general checked for clumps of cells (Supplementary Fig. 1e, f) To address the mechanisms underlying CM proliferation in prior to generating cDNA libraries for scRNA-seq. Over- the G2/M phases, our focus was fairly strict on tetraploid all, scRNA-seq data from fresh and fixed cells show a –16 CMs in the G2/M phases of the cell cycle (Fig. 1a). To that high degree of correlation (R = 0.98; p < 2.2 × 10 , Sup- end, we developed an approach that combines FACS of plementary Fig. 1d) confirming data consistency between fixed heart cells with scRNA-seq of the sorted CMs (Sup- the methods. plementary Fig. 1a), while preserving the RNA integrity We next aimed to use this approach for identifying TFs (RIN; Supplementary Fig. 1b) to increase resolution spe- specific for regulating the G2/M phases of CMs around cifically of CMs in the scRNA-seq analysis. Advantages birth. Accordingly, we isolated diploid (2n) and tetraploid Fig. 1 High scRNA-seq CM resolution of embryonic and neona- test; ****P ≤ 0.0001). d Heatmap visualizing differential expression tal CMs a Top: Schematic of the CM binucleation process during of known marker genes for different cell types (CMs, fibroblasts, heart development (E16.5, P1, and P5). Middle: Images of mouse endothelial cells, macrophages, and smooth muscle cells) in scRNA- hearts used for scRNA-seq with the left ventricle being dissected and seq analyzed heart cells (2n- and 4n, E16.5, P1 and P5) prior to ana- used (encircled by the dotted line). Bottom: FACS dot plots show- lytical CM-filtering. e Table displaying the number of cells prior ing the CM marker MYH1-488/Hoechst-stained cells (N = 3 litters/ to and after analytical CM filtering according to expression of the age; 14–20 pups/age). b–c Quantification of percentage of b non- CM marker genes Tnni3, Actc1, Tnnc1 and Tnnt2 at E16.5, P1 and myocytes (NMs) and CMs, c and 2n- and 4n-CMs, at E16.5, P1, and P5, and stratified by ploidy (2n and 4n). f UMAP-plots showing the P5 based on flow cytometric data and cell counts (mean, SD, N = 3, expression of the CM marker Tnni3 prior to filtering (blue: Tnni3 , Statistical analysis included two-way ANOVA with Tukey’s post grey: Tnni3 ) 1 3 8 Page 8 of 24 Basic Research in Cardiology (2023) 118:8 (4n) CMs from the left ventricle of mice before (E16.5 Gradual CM maturation and terminal differentiation and P1 samples) and after (P5 samples) (Fig. 1a) the is a dynamic process occurring already at E16.5, known proliferative stop at P3 [61]. At E16.5, the left leaving P1 hearts almost devoid of proliferating ventricle comprised of 80.8 ± 2.4% CMs and 17 ± 2.8% CMs non-myocytes (NMs) (mean, SD; n = 14–20). This CMs/ NMs ratio remained constant from E16.5 to P1 with To evaluate the biological activity of the sorted CMs, all 77.5 ± 5.8% CMs and 19.6 ± 5.3% NMs at P1, whereas scRNA-seq samples were integrated and CMs were clus- the percentage of NMs as expected increased rapidly from tered and visualized based on their gene expression pro- P1 to P5 (55.3 ± 6.9% NMs) (Fig. 1b). No difference was file, using Uniform Manifold Approximation and Projec- observed in the percentage of diploid CMs (2n-CMs) tion (UMAP) plots combined with Gene Ontology (GO) between E16.5 (59.7 ± 4.9%) and P1 (67.4 ± 7.5%), but term designation (Fig. 2a, b). In agreement with prior this percentage declined (36.2 ± 5.7%) at P5 (Fig. 1c). knowledge [13, 45], we observed that embryonic CMs Likewise, the percentage of tetraploid CMs (4n-CMs) switch from pyruvate metabolism to fatty acid metabo- remained constant from E16.5 (20.2 ± 0.8%) to P1 lism soon after birth (Fig. 2a, b), validating the scRNA- (15.5 ± 2.7%), but increased to 41 ± 7.3% at P5 (Fig. 1c). seq design. Moreover, we identified two major “Cell divi- Thus, as expected karyo-/cytokinesis failure in the G2/M sion” clusters (Fig. 2a, b), one mainly composed of E16.5 phases of P5 CMs were apparent. By examining the CMs and one dominated by P5 CMs, both embracing expression of MYH1 and the number of visible nuclei mainly tetraploid CMs. Using trajectory analysis (Fig. 2c, in FACS sorted P5 CMs, we found that > 95% of the dip- d), we found an overall ordering of the CMs in pseudo- loid and 50% of the tetraploid CMs were mononucleated, time that corresponds to the relative developmental stage, whereas 40% of the tetraploid CMs were binucleated with E16.5 CMs mainly represented early in pseudotime, (Supplementary Fig. 1e, f). The CM purity was > 95% after which they mature through the P1 stage and end- and cell clumps were scarce (Supplementary Fig. 1e, f), ing as P5 CMs (Fig. 2c, d and Supplementary Fig. 2a). the latter minimizing the appearance of false positive Yet, a small fraction of E16.5 CMs, especially tetraploid intermediates in the subsequent scRNA-seq analysis. CMs, did also align late in pseudotime, while some P1 High-throughput scRNA-seq with NextSeq (266,839,970 and P5 CMs resided in the early branches according to reads in total for 6 samples) on sorted 2n- and 4n CMs pseudotime (Fig. 2c, d and Supplementary Fig. 2a). This from E16.5, P1, and P5 revealed a total of 31,156 cells may suggest that the process of CM maturation and likely passing quality control filters with an average of 5,193 cell cycle exit is dynamic and ongoing already at E16.5, cells/sample and 1,135 genes in average per cell identi- whereas a small fraction of CMs at P5 may exhibit an fied (Supplementary Fig. 1 g). The cells were sequenced immature phenotype similar to E16.5 CMs. This was fur- at a median depth of 9,278 reads per cell with a median ther confirmed by branchpoint analysis (Fig. 2e–h, Sup- alignment rate of 74% per cell. Subsequent preparation plementary Fig. 2b) showing that the two branches early of data for analysis was performed using the R package in pseudotime (Fig. 2e, Supplementary Fig. 2b) primarily Seurat [62]. The total number of genes identified in all differ by metabolism and cell cycling where the earliest samples was 14,222 prior to any filtering. Expression of trajectory encompasses less differentiated CMs (Fig. 2g). CM markers (Fig. 1d,e) confirmed a 96.85% CM purity Particularly, we observed a higher cell cycling activ- of analyzed cells, yet, to exclude the few contaminants, ity for the lower trajectory of 4n-CMs (Supplementary we prefiltered the data by defining CMs as those with a Fig. 2b). The later bifurcations from the main trajectory combined expression of Tnni3, Tnnt2, Tnnc1, and Actc1 all seem to exhibit lower cell cycle activity (Fig. 2e) but (Fig. 1e,f). This resulted in an average of 5136 CMs per higher level of CM differentiation (“Shape and adhesion”, sample (Fig. 1e). After filtering, the remaining data for “ECM maturation”, “Fatty acid metabolism”, “Sarcomere only CMs were log-normalized, variable genes were iden- assembly”, and “Muscle contraction”) as well as “Cell tified, and data were scaled according to the total detected Migration” (Fig. 2c). Cell cycle activity was, however, number of molecules in each cell (nCount_RNA). Thus, regained for 4n-CMs at the tip of the lower trajectory our new approach combining FACS of fixed CMs with (Supplementary Fig. 2b). Since the P5 cell cycle active scRNA analysis enabled us to increase scRNA-seq resolu- CMs remain after the proliferation stop at P3 [61], and tion with high numbers of CMs stratified by ploidy. also exhibit mature CM characteristics, the P5 cell cycle activity most likely represent binucleation/polyploidiza- tion. Moreover, the data suggest that already before birth (E16.5) a small portion of CMs escape from cell division and starts maturing rendering P1 hearts almost devoid of CMs exhibiting cell division properties. 1 3 Basic Research in Cardiology (2023) 118:8 Page 9 of 24 8 Fig. 2 CM cell cycle exit, and maturation are initiated before cell cycling from BP heatmaps (below) is marked by a dotted line e. birth. a UMAP clustering based on k-nearest neighbors of CMs f, g, h, Heatmaps showing differentially expressed genes during CM with representative GO terms and ratio composing each cluster for development along pseudotime with dominating GO terms for each each ploidy/developmental stage b with colors referring to different cluster in each BP indicated. Hierarchical clustering is based on clusters. c, d, e, Trajectory analysis by Monocle displaying original Ward.D2 and unrelated to the clustering in (a, b). Above each heat- identity of cells c, pseudotime d, and trajectory state including three map, the states of the branches (according to (e)) are designated branchpoints (BP); The overall expression pattern of genes related to 1 3 8 Page 10 of 24 Basic Research in Cardiology (2023) 118:8 were scarce at P1 (2n = 2.2%; 4n = 8.7%) (Fig. 3c). Inversely, The cell cycling machinery of cycling E16.5 tetraploid G2/M CMs is defined by a certain S-phase CMs were more pronounced in diploid CMs (E16.5: 2n = 29.3%; 4n = 17.2%, P1: 2n = 15.4%; 4n = 10.2%, P5: set of TFs different from that in P5 cycle active tetraploid G2/M CMs 2n = 38.7%; 4n = 9.2%) (Fig. 3c). This agrees with cell cycle active diploid CMs mainly being in S-phase, while tetraploid To further explore the cell cycle status of the identified CMs have progressed to the G2/M phases. In pseudotime, S-phase CMs manifested in the start of the trajectory, but CM clusters (Fig. 3a), we used gene signatures that have previously been shown to denote G1, S, and G2/M phases some did align broadly throughout pseudotime as did also G1 phase CMs (Figs. 2d and 3d, e; Supplementary Fig. 2a). [67]. Thus, whereas ploidy was assigned by FACS prior to scRNA-seq, separation according to cell cycle identity was In contrast, the large cluster of 4n E16.5-G2/M CMs con- fined to the two earliest branches in pseudotime, whereas performed bioinformatically. Yet, as the G2 and M phases cannot be readily distinguished based on gene expression P5-G2/M CMs mainly located to the downward bifurca- tion in branchpoint 3, which represented more mature CMs they are considered united as “G2/M”. As such, we found that the two “Cell division” clusters (Fig. 2a, b) were com- characterized by "Fatty acid metabolism” and “Muscle con- traction” (Fig. 2e). These data thus supported that E16.5- posed of CMs active in either the S-phase or G2/M-phases (Fig. 2a, b) whereas the CMs composing the “Fatty acid 4n-G2/M CMs likely represented dividing CMs whereas the more mature P5-4n-G2/M CMs were in the process of metabolic process”-cluster (Fig. 2a, b) were mainly ascribed to the G1-phase (Fig. 3a, b). The percentage of predicted polyploidization/binucleation, which agrees with CM prolif- eration being absent after P3 [61] and our combined FACS G1-phase CMs was fairly constant between ploidies at P1 (2n = 82.3%; 4n = 81.1%) and P5 (2n = 56.9%; 4n = 54.9%) and microscopic analyses described above (Fig. 1b, c and Supplementary Fig. 1e, f). but differed at the embryonic stage E16.5 (2n = 57%; 4n = 31.8%) (Fig. 3c). These percentages also imply that a Then to identify which TFs that regulate the G2/M phases in immature CMs (E16.5) as compared to those that halt relatively higher percentage of cells are in active cell cycle (not G1) at E16.5 and at P5 as compared to P1 in agreement before karyokinesis/cytokinesis (P5), we extracted CMs composing the two “Cell division” clusters (Figs. 2a and with the GO term analysis (Fig. 2b). In consensus with biol- ogy, the majority of G2/M CMs were mainly found in the 4a) and focused the analysis on tetraploid CMs ascribed to the G2/M-phases extracting a subset of the data for further tetraploid portion of CMs, particularly the E16.5-4n CMs (2n = 13.6%; 4n = 51%) and to some extent the P5-4n CMs analysis (Fig. 4b). Then, we obtained a list of marker genes with higher expression in E16.5-4n-G2/M-CMs as compared (2n = 4.4%; 4n = 35.9%) (Fig. 3c). In contrast, G2/M CMs Fig. 3 Distribution of CMs within cell cycle phases. a, b, UMAP each cell cycle phase (n* = number of animals). d, e, Trajectory anal- plots of CMs with visualization of the cell cycle status across devel- ysis of CMs (d 2n and 4n pooled; e divided into 2n and 4n)) across opmental stages and ploidy, c, Quantification of cells representing developmental stages with cell cycle phase identity marked 1 3 Basic Research in Cardiology (2023) 118:8 Page 11 of 24 8 P0 to P5-4n-G2/M-CMs (Fig. 2c) and performed subsequent cycle activity in CM was measured 72 h after AAV9-cTnT- GO term analysis. Several terms related to the cell cycle: TF transductions using flow cytometry for EdU/MYH1/ “Mitotic nuclear division”, “Cell division”, “Chromosome mCherry/Hoechst (Fig. 5a). Virus functionality for each condensation”, “Regulation of mitotic cell cycle”, and of our AAV9-cTnT-TFs was validated by mCherry and TF “Nuclear division” and associated genes were unraveled by mRNA expression (Supplementary Fig. 6a, c), whereas a gene concept network map (Fig. 4d). Using the oPOS- flow cytometry for mCherry CMs confirmed transduc- SUM platform, enriched TF binding sites were determined tion efficiencies (Supplementary Fig. 6b). Both mCherry for the gene expression enriched in E16.5-4n-G2/M CMs mRNA and reporter fluorescence showed that the efficiency (Fig. 4b, c), and allowed us to predict a list of TFs potentially of TF expression decreased with the increasing size of the regulating G2/M progression in dividing CMs (Fig. 4e). To TF (Supplementary Fig. 6a, c, d). To counterbalance we confirm this list of TFs and avoid the initial selection of increased the number of CMs analyzed by flow cytometry the “Cell division” clusters, which could bias the approach, for large-sized TFs. Moreover, to ensure a high biological we combined E16.5- and P5-CMs into a new dataset and diversity and account for a 12 h difference in birth deliv - P0 reanalyzed with specific identification of E16.5-4n-G2/M ery, each independent experiment embraced CM pooled CMs and P5-4n-G2/M CMs (Fig. 4f). This repeated analysis from one litter each constituting 6–9 mice. At the time of P0 resulted in a second list of TFs (Fig. 4g) very similar to that analysis, 72 h after transduction, CM cultures consisted obtained for the cluster-based approach (Fig. 4e). Moreover, of 58.7 ± 8.0% CMs (mean, SD, n = 8), and AAV9-cTnT- to exclude that the identified set of TFs (Fig. 4e, g) reflected TF expression was highly specific for CMs (Fig. 5b). The + P0 data of low sequence saturation, we performed NovaSeq percentage of EdU CM transduced with an empty AAV9- (mean reads per CM were amplified 4.5- and 8.3-fold for cTnT-mCherry vector did not differ between mCherry and E16.5-4n and P5-4n CMs, respectively) with an increase mCherry CMs (5.3 ± 3.3% versus 4.4 ± 2.3%; mean, SD, from 13,578 to 16,118 in the total number of genes detected n = 8; p = 0.9992; Adjusted p-value, Sidak test) (Fig. 5c, d), (Supplementary Fig. 3a–c). Together, these analyses sup- suggesting that transduction did not alter S-phase progres- ported that the identified TFs likely play a role in regulating sion in itself. This also verie fi d that cell cycle activity indeed P0 G2/M of dividing CMs. Based on the Fisher score (Fig. 2e, is relatively low per se in CM cultures. When compared + P0 g, i), we therefore selected NFYA, SRF, MYC::MAX, USF1, to empty control transduced mCher ry CM (5.3 ± 3.3%), MYCN, EGR1, ARNT, MYC, E2F1, and ZEB1 for further Mycn (16.5 ± 3.6%), Egr1 (9.3 ± 1.8%), Arnt (29.8 ± 5.9%), analysis. We also included SP1 for further analysis as this TF M yc (15.5 ± 5.3%), Zeb1 (26.8 ± 5.6%), and Sp1 has been associated with CM cell cycling previously [21]. (28.4 ± 4.6%) overexpression resulted in significant increases + + Since, MYC::MAX represents a complex of the two indi- in the percentage of EdU mCherry CMs (Fig. 5d). This + + vidual TFs, MYC and MAX, we therefore proceeded with was consistent when comparing EdU mCherry CMs with + − 11 TFs in total for further analysis. EdU mCherry CMs within each culture (Fig. 5d). Thus, overexpression of TFs corresponded to 3.1- (Mycn), 1.8- ScRNA‑seq identified TFs that affect CM cell cycling (Egr1), 5.6- (Arnt), 2.9- (Myc), 5.1- (Zeb1), and 5.4-fold (Sp1) inductions in the percentage of EdU CMs. The Since, expression of TFs often are relatively low and given remaining TFs did not significantly alter the percentage of + + their nature as being upstream regulators of their tar- EdU mCherry CMs neither as compared to empty control + − get genes, detection of TF expression in scRNA-seq data nor as compared to EdU mCherry CMs (Fig. 5d). Thus, are often complicated. Despite these challenges, our CM the six TFs Mycn, Egr1, Arnt, Myc, Zeb1, and Sp1 were able P0 scRNA-seq data did reveal several CMs expressing the iden- on their own to increase cell cycle activity of CM . How- tified TFs. Yet, the percentages, of CMs expressing the TFs ever, it is generally acknowledged that EdU incorporation were still quite small, and their levels were decreasing with only reflects whether a given CM has progressed through developmental stage (Supplementary Figs. 4 and 5). the S-phase and therefore does not reflect if the CM com- Thus, to evaluate the biological effect of the 11 TFs on pleted cell division [37, 55]. We therefore assessed ploidy CM cell cycle activity we overexpressed them in P0 cardiac in the transduced CMs and defined borders for a ploidy P0 cells (CM ). To mimic the in vivo heart environment, we of either 2n, 4n, and > 4n (Fig. 5e), and used those gates + + initially used mixed cardiac cell cultures rather than a pure for analyzing EdU mCherry CMs (Fig. 5f). Neither EdU CM population. Yet, to enable CM specific TF overexpres- incorporation nor AAV9-cTnT transduction affects CM sion and identification of transduced CMs, we generated ploidy in themselves (Fig. 5g) ensuring the validity of the CM specific (cTnT promoter) adeno associated viruses of analysis. Using this setup, we found a significant change in + + serotype 9 (AAV9) with a mCherry reporter and an inter- ploidy for EdU mCherry CMs overexpressing Mycn, Egr1, nal ribosome entry site (IRES) for each of the 11 selected Arnt, Myc, Zeb1, and Sp1, whereas no change in ploidy was TFs (denoted AAV9-cTnT-TF) (Fig. 5a). Accumulated cell observed for the TFs also not altering the percentage of 1 3 8 Page 12 of 24 Basic Research in Cardiology (2023) 118:8 1 3 Basic Research in Cardiology (2023) 118:8 Page 13 of 24 8 ◂Fig. 4 Enriched set of transcription factors (TFs) specific for ZEB1 identified as a novel regulator of G2/M CM G2/M progression. a UMAP plot of 2n- and 4n CMs at E16.5, progression and CM proliferation before birth P1, and P5 with cell cycle phase identity visualized. b UMAP plot of extracted G2/M, 4n CMs from E16.5 and P5 developmental stage By reassessing the literature, the role of ZEB1 appears from the two Cell division clusters as indicated in (a). c Heatmap of expression of top genes with increased expression in E16.5 CMs poorly described in the heart and CMs. In a study by compared to P5 CMs in (b). d cnetplot of selected cell-cycle related Riechert and coworkers, it was found that Zeb1 is involved GO terms enriched among the genes in (c). e Enriched TFs (oPOS- in CM hypertrophy [54] and Cencioni et al. showed that the SUM) for genes with increased expression in E16.5 CMs compared to Zeb1-Hdac2-eNOS circuity identifies early cardiovascular P5 CMs in (b). f UMAP plot of 2n- and 4n CMs at E16.5 and P5 sep- arately analyzed with cell cycle phase identity visualized. g Enriched precursors [9]. However, the role of ZEB1 in CM prolif- TFs (oPOSSUM) for genes with increased expression in E16.5 CMs eration and polyploidization remains elusive. Interestingly, compared to P5 CMs in (f). h UMAP plot of re-sequenced data with Zeb1 null mice do not survive postnatally due to respira- NovaSeq. Clustering is indicated and CMs separated according to tory failure and T-cell deficiency [64]. Reinvestigating our developmental stage (E16.5 and P4, both 4n). i Enriched TFs (oPOS- SUM) for genes with increased expression in E16.5 CMs compared to scRNA-seq data we found that ZEB1 was present in only a P5 CMs in (h) small fraction of CMs (Fig. 6a, Supplementary Fig. 4) pref- erably in the G2/M phases (Fig. 6b), but the level declined + + EdU mCherry CMs (Fig. 5h). Myc and Mycn overexpres- with developmental stage (Supplementary Fig. 5). To inves- + + sion clearly resulted in EdU mCherry CMs being diploid tigate the implications of Zeb1 in CM proliferation before suggesting completed cytokinesis and thus proliferation. In birth, ZEB1 knockdown was performed using adenoviral contrast, Egr1, Arnt, Zeb1, and Sp1 favored polyploidy at the transduction for administration of validated Zeb1 shRNA expense of diploid CMs (Fig. 5h) indicating karyokinesis/ in E16.5 primary heart cultures (Fig. 6c). A high transduc- cytokinesis failure. tion efficiency was confirmed in CMs (Fig. 6d) with no We next subjected the target genes (according to oPOS- significant difference between control Ad-GFP-shRNA- SUM 3.0) of our six identified TFs to GO term analysis (93.1 ± 4.7%; mean, SD, n = 5) and Ad-GFP-shRNA-Zeb1 (p < 0.05, Benjamini corrections) and found that 4- (MYC), treatments (94.2 ± 4.0%; mean, SD, n = 5) (Fig. 6e), and 5- (MYCN), 3- (EGR1), 3- (ARNT), 13- (ZEB1), and 4- overall ZEB1 was reduced by 82.3 ± 5.9% (mean, SD, n = 3) (SP1) out of 13 GO terms related to “Cell cycling/division” (Fig. 6f). Using this setup, we found that cell cycle activity (3), “Mitotic division/cytokinesis” (4), “Telomers” (3), and in CMs as reflected by EdU incorporation was decreased “Regulation of cell cycling”/”Positive regulation of cell pro- significantly in Ad-GFP-shRNA-Zeb1 treated CMs from liferation” (3) (Supplementary Fig. 7). When adjusting for 12.2 ± 3.3% to 2.8 ± 0.8% (mean, SD, n = 5)(Fig. 6g,h). gene overlap between the different cell cycle related GO Importantly, the diploid status of EdU /CMs decreased by terms, 53- (MYC), 60- (MYCN), 41- (EGR1), 45- (ARNT), Zeb1 knockdown (Fig. 6i) suggesting that not only S-phase 118- (ZEB1), and 72- (SP1) genes related to these cell cycle progression was inhibited, but also cytokinesis was reduced related GOs were affected by the given TF (Supplementary by Zeb1 knockdown. This was supported by a significant Fig. 7). downregulation, in Ad-GFP-shRNA-Zeb1 treated cells, of Overall, these data underscore the design and quality Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1) and Ccnd3 (Cyc- of our CM scRNA-seq data, and enabled us to identify at lin D3), while the levels of Ccne2 (Cyclin E2) and Ccng2 least six TFs regulating cell cycle activity of CMs, where (Cyclin G2) associated with endoreplication [48, 82] were two (Myc, Mycn) and four (Egr1, Arnt, Zeb1, and Sp1) TFs unchanged (Fig. 6j). Although, the level of the major cyclin resulted in cytokinesis or a lack hereof, respectively. Since dependent kinase, Cdk1 (Cyclin-dependent kinase 1), was the Zinc Finger E-Box Binding Homeobox 1 gene (Zeb1) slightly reduced when Zeb1 was knocked down (Fig. 6k), no was superior to the other TFs in regulating the number (118) difference was observed for Cdk4 (Cyclin-dependent kinase of target genes associated to cell cycle activity in CMs, we 4) and the cell cycle inhibitors Cdkn1a (Cyclin-dependent hypothesized that ZEB1 could be a novel key player in CM kinase inhibitor 1a, p21) and Cdkn1b (Cyclin-dependent proliferation. kinase inhibitor 1b, p27) (Fig. 6k). With a focus on factors known from the G2/M phase, where Zeb1 predominantly was observed (Fig. 6b), we found a significant increase in the expression of Cenpe (Centromere protein E), Cenpf (Cen- tromere protein F), Aurkb (Aurora kinase B), and Aurka 1 3 8 Page 14 of 24 Basic Research in Cardiology (2023) 118:8 Fig. 5 ScRNA-seq identified transcription factors (TFs) regu- duced) and mCherry (non-transduced of the same culture) CMs + + late CM cell cycle activity. a Schematic of the study design and quantified by flow cytometry. The percentage of EdU mCherry workflow. Neonatal heart cells were isolated at P0 and cultured for CMs was compared to that of empty vector transduced CMs 24 h, before transductions with AAV9-cTnT-TF. Insert depicts as (AAV9-cTnT) (ANOVA and Fisher’s LSD post test, N = 3–8, modified from Addgene. EdU incorporation into mCherry and *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001 and ****P ≤ 0.0001) or to the % − + + − mCherry CMs together with CM- (MYH1 ) and ploidy- (Hoe- EdU mCherry CMs within the culture (Data not shown). e–h Flow chst) identity were then assessed by flow cytometry. b Flow cyto- cytometric assessment of the percentage of diploid- (2n), tetraploid- metric dot plots of non-transduced and transduced heart cells cul- (4n) and polyploid- (> 4n) transduced and non-transduced CMs as tured for 3 days after viral transduction showing CM specificity for indicated (ANOVA and Fisher’s LSD post test, *P ≤ 0.05, **P ≤ 0.01, AAV9-cTnT-TF expression (two-way ANOVA with Sidak’s post test, ***P < 0.001 and ****P ≤ 0.0001). Gates were defined using all CMs ****P ≤ 0.0001). c-d EdU incorporation in mCherry (TF trans- in (e) (Aurora kinase A), but no change in the level of Gmnn ZEB1 overexpression leads to CM endoreplication (Geminin) (Fig. 6l). These data confirm the scRNA-seq and high ploidy after birth data that ZEB1 mediates the cell cycle program and seems required for CM proliferation before birth. With Zeb1 as a key player in CM proliferation before birth, we then investigated if reintroducing ZEB1 at high efficiency 1 3 Basic Research in Cardiology (2023) 118:8 Page 15 of 24 8 in postnatal CMs could override terminal CM differentiation Additionally, we found that the muscle size inhibitor Mstn and lead to CM proliferation after birth. To overcome the (Myostatin) were substantially increased in ZEB1 overex- observed challenge of AAV9-TF transduction effectivity of pressing CMs, while the Myh6 expression was substan- the large-sized Zeb1 (Supplementary Fig. 6d), we generated tially reduced (Fig. 8f), which may explain why the smaller adenovirus (Ad) with an eGFP reporter to be expressed sepa- size of CMs was observed early after ZEB1 overexpression rately from ZEB1 and used that for inducing ZEB1 expres- (Fig. 7i,j). Finally, we injected Ad-GFP and Ad-GFP-Zeb1 P0 sion in CM (Fig. 7a). As visualized by immunocytochem- into the superficial temporal vein of P0 mice to evaluate istry (Fig. 7b) and quantified by flow cytometry (Fig. 7c, ZEB1 overexpression directly in the heart, and analyzed d), Ad-GFP CM transduction efficiency was 98.22 ± 2.2% by EdU incorporation, its effect on CM cell cycling from at 72 h with a parallel and specific expression of ZEB1 P0 to P8 (Fig. 8g). Scattered transduced GFP CMs were (Fig. 7e, f, Supplementary Fig. 8a, b). In agreement, with found in all hearts of Ad injected pups (Fig. 8h), and as its nature as a TF, the overexpressed ZEB1 protein mainly estimated by flow cytometry 1.5–2.5% of CMs were GFP localized to the nucleus in CMs (Fig. 7e, Supplementary at P8 (Fig. 8i). EdU incorporation was visualized in Ad- Fig. 8a, b). We did, however, not observe ZEB1 associated GFP-Zeb1 transduced CMs (Fig. 8h), and upon quantifica- to the tubulin spindle apparatus (Supplementary Fig. 8b) as tion by flow cytometry a significant higher percentage of noted in cancer cell lines [16], but we did notice a few scat- EdU CMs were found in Ad-GFP-Zeb1 transduced CMs tered CMs with cytoplasmic ZEB1 (Fig. 7e and Supplemen- as compared to Ad-GFP transduced CMs (Fig. 8j) confirm- tary Fig. 8b). EdU pulse chase labelling with empty virus ing the in vitro results with increased cell cycle activity as Ad-GFP and Ad-GFP-Zeb1 supported the above experi- a result of Zeb1. Moreover, 99 ± 1% of the EdU /Ad-GFP- + + ments showing that the percentage of EdU CMs increased Zeb1 CMs exhibited a ploidy of > 4n, while the remaining + + significantly with ZEB1 overexpression (Fig. 7h). Moreo- 1% were 4n, which was different from EdU /Ad-GFP CMs ver, a massive reduction in CM size was apparent after 72 h showing lower ploidy (Fig. 8k). The “high ploidy” Ad-GFP- of ZEB1 overexpression, and did not occur in non-CMs Zeb1 transduced CMs were 30% larger than their Ad-GFP (Fig. 7i, j). No difference was observed in the percentage of CM counterparts at P8 (Fig. 8l). CMs between non-transduced, Ad-GFP, and Ad-GFP-Zeb1 These data thus demonstrate that Zeb1 also after birth cultures at 72 h after transduction (Fig. 7k), suggesting that facilitates CM cell cycle activity, but unlike before birth, CM death or apoptosis was minimal at this timepoint and favors CM endoreplication and not division leading to was not induced by virus treatment. ZEB1 overexpression increased CM ploidy. revealed both single-, bi-, and multinucleated CMs at 72 h after transduction (Fig. 8a and Supplementary Fig. 8b), sup- porting the observed ability to induce CM S-phase progres- Discussion sion (Figs. 5d and 7h) leading to polyploidy and not division (Fig. 5h). In contrast to our Ad-GFP-shRNA-Zeb1 studies While most studies focus on regeneration of CMs at either (Fig. 6j), we observed a downregulation of Ccnd1 with a fetal or adult stages, the understanding of the transcriptional concomitant upregulation of Ccne2 and Ccng2, while the changes leading to the transition from a fetal to a postnatal levels of Ccnb1 and Ccnd3 were unchanged (Fig. 8b). No CM phenotype is relatively poor [49]. The postnatal period change was observed for Cdk1 and Cdk4, whereas Cdkn1a for a CM is characterized by a change from hyperplastic to was dramatically reduced. Likewise, Cdkn1b was reduced, hypertrophic growth [38], a switch from glycolytic to fatty but to a lesser extent (Fig. 8c). We also evaluated the Hippo acid oxidation [50], and sarcomere maturation with adult pathway known from cardiac regeneration through CM contractile isoforms [14, 73]. Together, this leads to cell proliferation, and found its downstream targets, Axl (Axl cycle exit and terminal CM differentiation [49]. Through a receptor tyrosine kinase) and Ctgf (connective tissue growth systematic, high-resolution scRNA-seq approach, analyzing factor) to be reduced in Ad-GFP-Zeb1 transduced CMs, with mouse CMs around the time of birth, we identified distinct a slight increase in Tead1 (TEA domain family member 1) transcriptional profiles of CM subpopulations, and identified (Fig. 8d). Also, in contrast to Ad-GFP-shRNA-Zeb1 treat- multiple master TFs that control numerous cell cycling genes ments (Fig. 6), there was no change in expression of the in CMs. Specifically, detailed analysis of ZEB1, a previously G2/M phase factors Cenpe, Cenpf, Aurkb, and Gmnn besides unknown factor in CM cell cycling, showed that before birth, a minor increase in Aurka (Aurora kinase A) levels (Fig. 8e). ZEB1 works as a key regulator of cell cycle promoting genes 1 3 8 Page 16 of 24 Basic Research in Cardiology (2023) 118:8 and is required for CM proliferation. Yet, after birth ZEB1 and leads to polyploid CMs. In agreement with the literature mediated CM cell cycling occurs through endoreplication [11, 32, 61], the number of dividing CMs is indeed also very 1 3 Basic Research in Cardiology (2023) 118:8 Page 17 of 24 8 ◂Fig. 6 ZEB1 knockdown reduce CM proliferation before birth. a understand the underlying mechanisms of the switch from UMAP plot of E16.5-4n CMs extracted from UMAP plot of all six CM proliferation to polyploidy occurring around the time of conditions (as in Fig. 2a) with blue color indicating CMs express- birth in mammals. Whether Zeb1 re-expression also underly ing Zeb1 mRNA. b Dot plot of Zeb1 expression for all six conditions the cell cycle activity observed in discrete CMs after MI with size indicating percentage of CMs expressing Zeb1 and color indicating the average expressing level in Zeb1-expressing CMs. c remains to be determined but could provide a target to ena- Schematic of the study design and workflow. Embryonic hearts were ble CM proliferation or forced CM polyploidy at this stage to isolated at E16.5 and cells were cultured for 24 h before adenovi- increase CM mass and compensating the CM loss after MI. ral transduction with either Ad-GFP-shRNA (control; scrambled Several scRNA-seq studies on in vivo CMs have shRNA) or Ad-GFP-shRNA-Zeb1. The insert depicts the viral con- struct containing an eGFP reporter. Cells were fixed 96 h after trans- recently provided new knowledge on heart development duction and EdU pulsing, and analyzed by flow cytometry and immu- by identifying genes that are differentially expressed at nocytochemistry (ICC). d ICC of E16.5 primary cardiac cultures different stages of development. Yet, many of the studies transduced with either Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1 are limited in the number of CMs detected [5, 10, 30, 40] after 96 h (GFP , green; DAPI (cell nuclei), blue). e GFP expres- sion quantified by flow cytometry of Ad-GFP-shRNA and Ad-GFP- restricting subsequent detailed analysis such as TF binding shRNA-Zeb1 transduced cells 96 h after transduction (Paired t-test, site enrichment studies. Other studies fail to implement n = 5, NS). f Normalized mRNA level of ZEB1 in Ad-GFP-shRNA data on ploidy and cell cycle status [23, 39]. To our knowl- and Ad-GFP-shRNA-Zeb1 transduced cells (normalized against B2m edge analysis of TFs in CMs based on scRNA-seq has and Gapdh; Paired t-test, n = 3, *P ≤ 0.05). g Flow cytometry dot plot of EdU incorporation in MYH cells transduced with Ad-GFP- been described in only four settings for CM development, shRNA or Ad-GFP-shRNA-Zeb1. h EdU positive CMs transduced but none used ploidy stratification as performed herein with Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1 quantified by flow [10, 27, 30, 39]. However, in a recent study, Yekelchyk cytometry (Paired t-test, n = 5, **P ≤ 0.01). i CM ploidy of Ad-GFP- et al. showed transcriptional homogeneity by scRNA-seq shRNA and Ad-GFP-shRNA-Zeb1 transduced, EdU positive cells quantified by flow cytometry based on Hoechst intensity (Paired of adult rod-shaped mono- and multi-nucleated ventricular t-test, n = 5, **P ≤ 0.01; ****P ≤ 0.0001). j Normalized mRNA lev- CMs, although, not performing TF analysis [77]. With the els of Ccnd1, Ccne2, Ccng2, Ccnb1, and Ccnd3 (normalized against established protocol, we unravel the uniqueness of in vivo B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05; **P ≤ 0.01). k Nor- cycling CMs in the G2/M phases around the time of termi- malized mRNA levels of Cdk1, Cdk4, Cdkn1a, and Cdkn1b (normal- ized against B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05). l Nor- nal CM differentiation, and besides Zeb1 identified several malized mRNA levels of Cenpe, Cenpf, Aurkb, Gmnn, and Aurka TFs potentially involved in the process of G2/M phases (normalized against B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05; completion. Mycn and Myc were shown to enhance not **P ≤ 0.01) only S-phase progression, but also G2/M completion, in agreement with recent data [8, 60]. The more novel play- scarce after birth in our data, and it seems likely that CMs ers in CM cell cycling: Arnt, Zeb1, Sp1, and Egr1 spe- also in the mouse already initiates the final round of cell cifically promoted S-phase progression herein with high cycling before birth at which point ZEB1 seems to impact efficiency, yet all four seemed to leave the postnatal CMs CMs. This would be similar to what is observed in humans, in a polyploid state thus favoring karyo-/cytokinesis fail- where CMs starts the process of terminal differentiation with ure. Whereas Arnt has been associated with hypoxia [74], increased polyploidy already before birth [18]. Thus, it is Egr1 seems to be implicated in several pathologies of the possible that ZEB1 represents a mechanism where CM pro- cardiovascular system [31], and Sp1 is a well-known TF liferation is sustained by ZEB1 but at some point, mitotic in cell growth and peripherally related to CM cell cycling stress is reached forcing the self-limiting DNA damage [21]. Zeb1 is mainly described for its enhancing role in response to initiate terminal differentiation and avoid can - epithelial to mesenchymal transition during cancer and cer. By this, ZEB1 may ensure polyploidy to achieve high embryonic development [81], but has recently been linked production of RNA and proteins required for the high muscle also to Hematopoietic stem cell renewal and asymmetric work of fully differentiated CMs in adulthood, and its timed cell division [1]. Furthermore, ZEB1 has been suggested downregulation eventually caused by a self-limiting DNA to interact directly with the Hippo pathway in cancer response then enables p21 mediated CM differentiation. This cells through YAP [34]. The functional roles of the TFs, could be in line with the emerging theory that polyploidy is however, were only predicted bioinformatically and not an essential biological mechanism for tissue differentiation directly examined besides EdU incorporation and deter- and homeostasis [19]. mination of ploidy. Herein, we found Zeb1 to regulate Thus, our novel approach has allowed us to acquire valu- the highest number of genes related to CM cell cycling able new biological information that may be used further to in our dataset. Knockdown of Zeb1 in E16.5 CMs led to 1 3 8 Page 18 of 24 Basic Research in Cardiology (2023) 118:8 impaired S-phase progression with reduced expression of Cdk1 as well as a decrease in the level of Cenpe, Cenpf, the major cell cycle regulators Ccnd1, Ccnb1, Ccnd3, and Aurkb, and Aurka, which are all genes expressed in the 1 3 Basic Research in Cardiology (2023) 118:8 Page 19 of 24 8 ◂Fig. 7 ZEB1 increases ploidy and decreases cell size in vitro. a upon ZEB1 overexpression. Whether this in turn inhib- Schematic of the study design and workflow. Neonatal hearts were its G1/S phase transition and prevents non-cycling CMs isolated at P0 and cultured for 24 h before transduction with adeno- from further entering the cell cycle remains elusive but virus (Ad). The insert depicts the viral construct, note the eGFP could explain why only a proportion of CMs undergo reporter in both Ads. Cells were fixed after 72 h transduction and EdU subjection and analyzed by flow cytometry or immunocyto- S-phase progression despite expressing ZEB1. Yet, we did chemistry (ICC). b ICC of Ad-GFP transduced primary CMs after observe that all ZEB1 overexpressing CMs reduced their + + 24, 48, and 72 h of transduction (GFP , green; Mef-2c (CM nuclei), size in vitro, which therefore likely represents another red; DAPI (cell nuclei), blue). c Flow cytometric dot plots of GFP − + mechanism. The smaller size of ZEB1 expressing CMs and GFP MYH1 CMs in non-transduced and Ad-GFP transduced CMs. d GFP expression quantified by flow cytometry after 72 h of at early timepoints was accompanied by an increase in culturing (non-transduced) or Ad-GFP transduction (Paired t-test, the major muscle size inhibitors Mstn, Ccng2, and Tead1, n = 8, ****P ≤ 0.0001). e ICC of Ad-GFP (top panel) and Ad-GFP- and downregulation of the Yap targets Axl and Ctgf. All Zeb1 (lower panel) transduced CMs after 24, 48, and 72 h transduc- + + these genes are related to cell size regulation, and recently tion (actinin (CMs), green; ZEB1 , red; DAPI (cell nuclei), blue). f Zeb1 expression in CMs after 72 h Ad-GFP or Ad-GFP-Zeb1 trans- ZEB1 was demonstrated to inhibit skeletal muscle cell size duction as quantified by qRT-PCR (normalized against B2m and in mouse [59]. Thus, the reduced CM size and decrease Rpl_13A; Unpaired t-test, n = 6–7, **P ≤ 0.01). g Flow cytometric in Myh6 fit very well with ZEB1 preventing cell cycle dot plots of EdU incorporation in MYH1 CMs after 72 h Ad-GFP exit, while promoting S-phase progression in the early transduction (top panel) and Ad-GFP-Zeb1 transduction (lower panel) (forward side scatter (FSC)). h Incorporation of EdU was observed phase. At P8 in vivo, ZEB1-mediated endoreplication at both 50, 100, and 150 MOI after 72 h Ad-GFP and Ad-GFP-Zeb1 then results in polyploid CMs of increased size, which transduction (Unpaired t-test, n = 3, *P ≤ 0.05, ***P ≤ 0.001). i Flow agrees with the literature [82]. Interestingly, our studies cytometric contour plots depicting decreased MYH1 CM size after indicate that ZEB1 regulates a distinct set of genes before 72 h Ad-GFP-Zeb1 transduction. j Quantification of the geometric mean of CMs and non-myocytes (NMs) 72 h after Ad-GFP or Ad- and after birth, thereby promoting CM proliferation before GFP-Zeb1 transduction (Two-way ANOVA, n = 9, ****P ≤ 0.0001). birth, while favoring polyploidization when reintroduced k Percentages of CMs of the total cell number after 72 h in culture after birth. To our knowledge this clear molecular switch (non-transduced), or after 72 h Ad-GFP or Ad-GFP-Zeb1 transduc- from proliferation to endoreplication around birth has not tion (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 5) previously been shown for one TF. Thus, the decrease in Zeb1 expression occurring around birth may contribute to cell cycle arrest and terminal CM differentiation, as also G2/M phases of the cell cycle, confirming our bioinfor - observed for other TFs [17]. One example is YAP1, which matic prediction of ZEB1 as a regulator of the cell cycle as part of the Hippo pathway supports CM proliferation in CMs before birth. Ccnb1, Ccnd1, Cdk1, and Cdk4 are during embryonic development in combination with its known regulators of CM cell cycling and overexpression interaction partner TEAD1 [43]. Thus, in the postnatal of these four factors was recently found to promote prolif- period downregulation of YAP1 and TEAD1 is required eration of post-mitotic CMs [46]. Manipulating Zeb1 by for CM cell cycle arrest [22]. Postnatal upregulation of overexpression in postnatal CMs showed that ZEB1 after YAP1 retain CM proliferative capacity after birth causing birth maintains cell cycling in the form of endoreplica- cardiomegaly and heart failure [49]. MYC, which was also tion specifically governed by Cyclin E while likely inhib- detected in our TF analysis, induces CM proliferation dur- iting cell cycle exit through Cdkn1a (p21) regulation. It ing development, while expression of MYC in adult mice is known that p21 binds and inhibits CDK1/Cyclin B1 leads to an increase in polyploid cells [76]. However, in thereby blocking G1/S and G2/M phase transitions, and another study it was suggested that combinational overex- that p21 knockout increases CM ploidy significantly [66], pression of MYC and Cyclin T1 induce CM proliferation while p21 expression forces CM cell cycle exit [65]. In without any notable change in CM size and nucleation agreement, S-phase progression and ploidy was high in [8]. Thus, it is intriguing to speculate, whether overex- ZEB1 expressing CMs. Since, p21 blocks endoreplication pression of Zeb1 in combination with other genes or TFs [66] through co-repressing Cyclin E [24], the major cyc- also after birth could promote CM proliferation rather than lin of endoreplication [82], it is thus intriguing to specu- endoreplication, and by this may be a target for therapeutic late that ZEB1 herein also downregulates p21 and hereby perspectives in MI patients. increases Cyclin E2 expression resulting in endoreplica- In conclusion, we here provide new knowledge on Zeb1’s tion of cycling CMs and polyploidy as observed for a frac- cell cycle promoting actions in CMs as well as a comprehen- tion of CMs both in vitro and in vivo. In this regard, it is sive scRNA-seq of CMs before and after birth, which may important to note, that Cyclin D1 in parallel was decreased 1 3 8 Page 20 of 24 Basic Research in Cardiology (2023) 118:8 1 3 Basic Research in Cardiology (2023) 118:8 Page 21 of 24 8 manuscript editing, and final approval of manuscript. SBM: collection ◂Fig. 8 ZEB1 increase ploidy and cell size in vivo. a Confocal of in vitro and in vivo mouse data, cloning of plasmids, production of images of Ad-GFP-Zeb1 transduced CMs after 72 h transduction virus, manuscript writing, manuscript editing, and final approval of (phalloidin (F-actin) green; ZEB1 , red; DAPI (cell nuclei), blue). manuscript. TC: cloning of plasmids and production of virus, Collec- Binucleated CM (##), tetranucleated CM (###), cytoplasm localized tion of in vitro mouse data, manuscript writing, and final approval of ZEB1 (arrowheads). b Normalized mRNA levels of Ccnd1, Ccne2, manuscript. SF: cloning of plasmids and production of virus, collection Ccng2, Ccnb1, and Ccnd3 (normalized against B2m and Rpl_13A; of in vitro and in vivo mouse data, and final approval of manuscript. Unpaired t-test or Mann–Whitney based on normality, n = 6–7, KSA: collection of confocal data, final approval of manuscript. CDF: **P ≤ 0.01). c Normalized mRNA levels of Cdk1, Cdk4, Cdkn1a, ScRNA-seq method establishment, collection of scRNA-seq data, and Cdkn1b (normalized against B2m and Rpl_13A; Unpaired t-test manuscript writing, and final approval of manuscript. MB: ScRNA- or Mann–Whitney based on normality, n = 6–7, **P ≤ 0.01). d Nor- seq method establishment, scRNA-seq analysis, manuscript writing, malized mRNA levels of Axl, Ctgf, and Tead1 (normalized against and final approval of manuscript. MT: ScRNA-seq method establish- B2m and Rpl_13A; Unpaired t-test or Mann–Whitney based on nor- ment, Sequencing of cDNA libraries, manuscript editing, and Final mality, n = 6–7, *P ≤ 0.05, **P ≤ 0.01). e Normalized mRNA levels approval of manuscript. DCA: conception and design, data collection, of Cenpe, Cenpf, Aurkb, Gmnn, and Aurka (normalized against B2m data analysis and interpretation, manuscript writing and editing, final and Rpl_13A; Unpaired t-test or Mann–Whitney based on normality, approval of manuscript, and financial support. n = 6–7, *P ≤ 0.05). f Normalized mRNA levels of Mstn, and Myh6 (normalized against B2m and Rpl_13A; Unpaired t-test or Mann– Data availability ScRNA-seq data that support the findings of this Whitney based on normality, n = 6–7, **P ≤ 0.01). g Schematic of the study have been deposited in the Gene Expression Omnibus (GEO) study design and workflow. Neonatal pups (P0) were injected with under the accession code GSE162959. All other data supporting the Ad-GFP or Ad-GFP-Zeb1 through the superficial temporal vein, fol- findings of this study are available from the corresponding authors on lowed by two subcutaneous injections of EdU at P4 and P6. Hearts reasonable request. were collected at P8 for immunohistochemistry (IHC) or ventricu- lar dissociation for flow cytometry. h IHC of P8 heart ventricle tis- Code availability All codes central for the conclusions made in this sue from PBS (top panel, PBS injected pups were included in the manuscript are available from the corresponding author on reasonable study with the purpose to make this figure to show the GFP-signal request. in adenovirus-injected pups compared to PBS-injected pups) or Ad- + + GFP-Zeb1 (lower panel) P0 injected pups (GFP , green; EdU , red; DAPI (cell nuclei), blue). i Flow cytometric dot plots depicting the Declarations EdU incorporation in all MYH1 CMs (left panel) as well as in + + MYH1 GFP CMs (right panel) after 72 h Ad-GFP (top panel) or Conflict of interests DCA together with the University of Southern + + Ad-GFP-Zeb1 (lower panel) transduction. j Percentages EdU GFP Denmark and the Region of Southern Denmark have filed a patent CMs of GFP CMs at P8 after Ad-GFP injection at P0 compared to (#PCT/ EP23158550.6.) based on the data generated within this study. Ad-GFP-Zeb1 injection (Paired t-test, n = 7–9, **P ≤ 0.01). k Per- Otherwise, the authors declare no competing financial interests. centage-wise distribution of ploidy at P8 after Ad-GFP or Ad-GFP- Zeb1 injection at P0 (Paired t-test, n = 7–9, *P ≤ 0.05). l Quantifica- Open Access This article is licensed under a Creative Commons Attri- tion of the geometric mean of primary CMs at P8 after Ad-GFP or bution 4.0 International License, which permits use, sharing, adapta- Ad-GFP-Zeb1 transduction (Unpaired t-test, n = 7–9, **P ≤ 0.01) tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes be used to further dissect the switch from a proliferative to were made. The images or other third party material in this article are a terminally differentiated high power beating CM. included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in Supplementary Information The online version contains supplemen- the article's Creative Commons licence and your intended use is not tary material available at https://doi. or g/10. 1007/ s00395- 023- 00979-2 . permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a Acknowledgements We would like to thank Charlotte Nielsen, Tina copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . K. Andersen, and Tonja L. Jørgensen (Andersen group, Odense Uni- versity Hospital) for excellent technical assistance on this study, Lars Vitved (University of Southern Denmark) for assisting with FACS and References Professor Per Svenningsen (University of Southern Denmark) for help with AAV plasmids. The work was supported by research grants from The Novo Nordisk Foundation (#NNF17OC0028764), The Danish 1. Almotiri A, Alzahrani H, Menendez-Gonzalez JB, Abdelfattah A, National Research Council (Sapere Aude; # 8045-00019B), The Lun- Alotaibi B, Saleh L, Greene A, Georgiou M, Gibbs A, Alsayari A, dbeck Foundation (#R313-2019-573), The Program of China Scholar- Taha S, Thomas LA, Shah D, Edkins S, Giles P, Stemmler MP, ship Council (No. 201906320411) and financial support from the Dep. Brabletz S, Brabletz T, Boyd AS, Siebzehnrubl FA, Rodrigues NP of Clinical Biochemistry /Odense University Hospital. (2021) Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2023
ISSN: 0300-8428
eISSN: 1435-1803
DOI: 10.1007/s00395-023-00979-2
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Abstract

Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid prolifera- tive CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes. Keywords Heart development · Cardiomyocytes · Proliferation · Endoreplication · Zinc Finger E-Box Binding Homeobox 1 (Zeb1) Introduction Cardiovascular diseases are among the leading causes of death worldwide [68], and one major explanation is the inability to regenerate the heart after myocardial infarction (MI), leaving the affected subjects with impaired cardiac Sara Thornby Bak, Eva Bang Harvald and Ditte Gry Ellman have function [52]. Whereas, the mammalian heart forms through contributed equally to this work. cardiomyocyte (CM) proliferation during fetal development * Ditte Caroline Andersen [58], CMs enter cell cycle arrest around birth [4]. As in gen- dandersen@health.sdu.dk eral, CM proliferation requires the CM to go through the four cell cycle phases: G1, S, G2 and M [57]. Yet, around birth Andersen Group, Department of Clinical Biochemistry, Odense University Hospital, Odense, Denmark (P3 in mice) a portion of CMs exit the cell cycle and become quiescent in the G0 phase [28, 61, 71], whereas another CM Clinical Institute, University of Southern Denmark, Odense, Denmark fraction undergoes a final round of mitosis peaking at post- natal stage P5 (in mice), but indeed fails to complete karyo- Amplexa Genetics, Odense, Denmark 4 and/or cytokinesis. This is often referred to as the G2/M Department of Clinical Genetics, Odense University challenge which results in polyploid and binucleated CMs Hospital, Odense, Denmark Vol.:(0123456789) 1 3 8 Page 2 of 24 Basic Research in Cardiology (2023) 118:8 [72, 79, 80], and is considered a hindrance for heart regener- 130–098-373) according to the manufacturer’s recommen- ation through CM proliferation [20, 32]. The mechanism for dations. Following viable cell counting (NC-200, ChemoM- this switch is unclear, but has been associated with reduced etec), dissociated cells were stained with a fixable viability expression of cyclins and cyclin dependent kinases (CDKs) stain (Fixable viability stain 570; BD Biosciences, 564,995) in parallel with induced expression of several CDK inhibi- prior to fixing in methanol for 15 min followed by rehydra- tors [29, 44], changes in miRNA expression, extracellular tion to reverse the RNA to its original state. During rehydra- signaling pathways, centrosome integrity, telomere dysfunc- tion, the RNase inhibitor, RNasin Plus (Promega; N2615), tion, epigenetics as well as extracellular matrix compositions was added to prevent RNA degradation and included in all [51]. In the adult mouse and rat heart, ~ 80–95% [29] of CMs subsequent steps. After rehydration, samples were stored at are binucleated while ~ 5% CMs are mononucleated [6]. In -80 °C until analysis. All reagents were high grade, RNase adult humans, controversies remain regarding the ratio of free and the environment was kept strictly RNase free to binucleated CMs compared to mononucleated, and it has avoid degradation of the RNA. For comparison of fresh and been suggested that ~ 60% human CMs remains mononu- fixed scRNA-seq profiles, mouse myoblasts (C2C12; ATCC, cleated [47], whereas the percentages of binucleated range CRL-1772) were used and maintained as recommended. between 12 and 75% [35]. Even so, a large portion of the mononucleated human CMs is polyploidy [47]. The termi- Fluorescence‑activated cell sorting (FACS) nally differentiated and polyploid CMs are often referred to as dormant with respect to cell division [32, 38], although Fixed cardiac cells were stained for the CM marker MYH1 recent evidence indeed contradicts this [36]. As with CM (Mouse IgG2b,k; 1:300; MF20-c; DSHB) and visualized by proliferation, current mechanistic insight to understanding donkey anti-mouse IgG Alexa Fluor 488 (1:200; Invitro- polyploidy also remains deficient [32, 35]. More systematic gen, A21202), whereas Hoechst 33342 (Sigma) was added approaches are therefore required to identify factors mediat- 5 min before sorting (FACSAriaIII, BD Biosciences). Prior ing CM cell cycle activity, and which on a longer term may to FACS, cells were filtered (Falcon, 352235) to avoid cell be used to tackle the challenge of a non-regenerating heart. clumps. Strict RNase free conditions as described above Herein, we developed a new protocol combining fluores- including new tubing were prioritized throughout the pro- cence-activated cell sorting (FACS) of diploid and tetraploid cedure. Analysis and sorting gating strategy (Supplemen- murine CMs around birth with high-throughput single cell tary Fig. 1c) included hierarchical gating using the FACS- RNA sequencing (scRNA-seq) to uncover transcription fac- Diva software v8.0.1 (BD Biosciences) based on FSC/SSC, tors (TFs) that regulate the G2/M CM cell cycle process. viability Alexa 570, and MYH1-Alexa 488, and Hoechst 33342. For each developmental stage (E16.5, P1, P5), three independent sortings (n = 3, each consisting of cells from Methods one litter) were performed after carefully checking and vali- dating the FACS setup using FMO controls (Supplemen- Mice tary Fig. 1c). Prior to scRNA-seq analysis CM purity, nuclei number, and cell clumping of sorted cells were assessed C57BL/6 J mice were obtained from Taconic Europe, using immunofluorescence microscopy whereas the RNA housed with a 12/12 h light/dark cycle, and fed ad libitum. integrity number (RIN) was determined using an Agilent For scRNA-seq, mice were plug bred, and litters for each 2100 Bioanalyzer (Agilent Technologies) combined with the of the three timepoints were obtained from different breed- Agilent RNA 6000 Nano Kit (Agilent Technologies) [56]. ing pairs. Plug was checked in the morning and evening. Sorted cells were stored at -80 °C until scRNA-seq. For E16.5 primary cultures, mice were plug bred as well, whereas for primary P0 cultures continuous breeding was ScRNA‑seq used. All animal experiments were approved by the Dan- ish Council for Supervision with Experimental Animals For scRNA-seq, cells originating from three independent (#2016–15-0201–00,941 and #2022–15-0201–01119). FACS were pooled (14–20 pups/sample) to account for bio- logical diversity in the scRNA-seq analysis. Single Cell 3’ Preparation of CMs for scRNA‑seq RNA-Seq libraries were prepared using Chromium Single Cell 3′ Reagent Kits v2 (10 × Genomics) according to the For scRNA-seq, the left heart ventricle was dissected under user guide. In brief, cellular suspensions of approx. 1200 a stereomicroscope at E16.5, P1, and P5 (n = 3 litters, each cells/µl were mixed with master mix reagents and loaded counting 4–8 pups), and enzymatically dissociated using on a Single Cell A Chip (10 × Genomics) together with the semiautomatic GentleMACS tissue dissociator system Single Cell 3’ Gel Beads (10 × Genomics) and partitioning (MACS Miltenyi Biotec; Neonatal Heart Dissociation Kit oil to generate single cell gel beads-in-emulsion (GEMs). 1 3 Basic Research in Cardiology (2023) 118:8 Page 3 of 24 8 The GEM generation took place in a Chromium Control- visualization by Uniform Manifold Approximation and ler (10 × Genomics). Single cell reverse transcription was Projection (UMAP) clustering [69, 70]. Cell clustering by performed in a standard thermal cycler, and the GEMs were expression pattern was performed by first calculating the subsequently broken using Recovery Agent (10 × Genom- k-nearest neighbors and constructing the shared nearest ics). The resulting cDNA was cleaned up with DynaBeads neighbor (SNN) and next optimizing the modularity func- MyOne Silane Beads (Thermo Fisher Scientific) and tion to determine clusters. SPRIselect Reagent Beads (Beckman Coulter), and then Clustering and Heatmaps The Seurat v 2.3.0 and 3.1.5 [7, amplified by PCR using Single Cell 3′ Reagent Kit v2 (No. 62] were used for cluster visualization by UMAP and for dif- of cycles: 8). After another cDNA clean-up with SPRIselect ferential gene expression of marker genes between clusters. Beads, the fragment sizes and concentrations were measured For each of the samples, a Seurat object was created, and using QIAxcel DNA High Resolution Kit (1200) (Qiagen) the cells filtered based on whether they expressed a com- and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), bination of the CM-specific markers Tnni3, Tnnt2, Actc1, respectively. Enzymatic fragmentation, end-repair, and and Tnnc1. Each sample was then log-normalized, variable A-tailing were performed in one-step using the Single Cell features were identified using “vst” as selection method and 3′ Reagent Kit v2, and fragments of approx. 200 bp were 2000 nfeatures, and the data was scaled using nCount_RNA selected by double sided-size selection using SPRIselect for vars.to.regress. PCA was run and based on jackstraw- Beads. NGS libraries were then constructed by adapter liga- and PC elbow plots the optimal number of dimensions tion and PCR mediated sample indexing (No. of cycles: 13). was determined (range 9–14). Moreover, when applied, all After a final double-sided size selection, the NGS library samples were merged, integrated using FindIntegrationAn- concentrations were measured using Qubit dsDNA Assay chors and IntegrateData, filtered, normalized, and scaled as Kit. Libraries were sequenced on the Illumina NextSeq 500 described above with generated UMAP plots depicting cell platform using NextSeq 500/550, high output Reagent Car- cycle phases, clusters, and original data affiliation as for each tridge V2, Illumina Kit (Read 1 = 26 cycles, i7 Index = 8 individual sample. cycles, Read 2 = 130 cycles), and the second analysis was Visualization, clustering, and cell cycle analysis UMAP performed on a Illumina NovaSeq 6000. plots and clusters were generated as described above using PCA as reduction type and resolution = 0.6; based on the ScRNA‑seq data analysis top 30 marker genes for each cluster. Subsequent GO term enrichment was evaluated using clusterProfiler::enrichGO Read alignment and construction of gene expression matrix [75, 78] and the “org.Mm.eg.db” library with ont = “BP”, Base calls were converted to FASTQ format and demulti- pAdjustMethod = “BH”, and cutoff values = 0.01. Fea- plexed using the cellranger mkfastq function embedded in tures witg avg_log2FC > 0.5 were used, where each cluster the 10 × Genomics cellranger software package using default was named according to biological identity. Finally, each settings (https://suppo r t.10xg enomics. com/ sing le-cell- g ene- dataset was split into three groups (G1-, S-, or G2/M- expre ssion/ softw are/ overv iew/ welco me). Single cell gene phase) based on the expression of cell cycle markers [67] counts matrices were generated using the cellranger count and each cell was assigned with a cell cycle score using command. During this step, FASTQ files generated by the Seurat::CellCycleScoring. cellranger mkfastq step, were aligned to the Mus musculus Analysis across developmental stages After merging and genome (mm10/GRCm38) using the splice-aware aligner integration as described above, two clusters of cell cycle STAR [12]. Subsequently, STAR used the Mus musculus active E16.5 and P5 cells, respectively, were subtracted from transcriptome reference (GRCm38.84) to segregate the the data and compared using FindMarkers. The resulting list mapped reads into exonic, intronic and intergenic regions of features was used for generating cnetplot and TF analysis. and for assessment of how confidently the reads have been Mouse single site analysis was used for TFs (oPOSSUM mapped to these regions. Only non-duplicated reads which version 3.0) [25, 26, 33], all genes in current dataset as back- were confidently mapped to the transcriptome, and which ground, all vertebrate profiles with a minimum specificity of had barcodes and unique molecular identifiers (UMIs) were 8 bits, conservation cutoff 0.40, matrix score threshold 85%, used for UMI counting. The expression matrices were gen- up/downstream sequence 5000/5000). In addition, oPOS- erated by counting the number of strand-specific UMI for SUM results were supported by GSEA using the Molecu- each cell mapping to either the exonic or intronic regions lar Signature Database [41, 42, 63] and transcription factor of each gene. targets (TFT). Clustering and UMAP visualization Using the R pack- Trajectory analysis Data were prepared in Seurat (filtered age Seurat[62] v 2.3.0 and 3.1.5 dimensionality reduction and cell cycle assigned; since UMI data were used, nor- by principal component analysis (PCA) was performed; malization was avoided in agreement with recommendations subsequently the PCA data analysis was used as input for by the Monocle platform) in merged pools of either 2n-, 1 3 8 Page 4 of 24 Basic Research in Cardiology (2023) 118:8 4n-, or all samples, respectively. Subsequently, phenotype promoter; synthesized by GeneArt (Thermo Fisher); the data and feature data were extracted from the Seurat object DNA sequence was kindly provided by Professor Brent A. and converted to a Monocle CellDataSet (CDS) object. French, University of Virginia, USA) was inserted for CM Next, dispersion estimates for count dataset were obtained specificity by PCR amplification in two steps: first, plasmid using monocle::estimateDispersions and cells were sorted pAAV-EF1a-mCherry-IRES was PCR amplified by primers according to num_genes_expressed (500 < num_genes_ (F or w ard: GG A A TT CC A T A T GGG T A C CGG A TC CG T G A G expressed < 3000). A set of ordering genes was isolated C and R e v erse: GCT CT A G AA A TT CCC A CT CCT TT CAA G using differentialGeneTest and used to order the CDS by ACCT AG) containing the XbaI and NdeI restriction sites to the monocle::setOderingFilter. Next, the dimensions were excise the EF1a promoter. Secondly, the cTnT promoter was reduced and cells were ordered along the trajectory using inserted between the two restriction sites (Forward: GCT monocle::reduceDimension and monocle::orderCells, CT A G A G C A G T CTG and R e v erse: GG A A TT C C A T A T G A G respectively. The trajectory was plotted depicting origi- GTC ). The resulting pAcTnT-mCherry-IRES plasmid was nal identity, cell cycle phase, and pseudotime state. The then sequenced (Eurofins Genomics, Ebersberg, Germany) monocle::BEAM function was utilized in each branch point for validation (Data not shown). Genes (Origene plasmids of the trajectory plots to evaluate branch point dependent and Nfya) were inserted into the pAcTnT-mCherry-IRES gene expression. plasmid between the SgfI and MluI restriction sites. Since the SgfI restriction site was already included in the Egr1 Plasmids and AAV9 packaging sequence, Egr1 was amplified by the following primers For - war d: AA T GG T GG T T CT GG T GCG A T C GC A T GG C AG To determine the most efficient AAV serotype for CM trans- CGG CCA AG and Reverse: TT G A T A T CG AA T CTA ACG duction, pilot studies with both AAV6 and AAV9 transduc- CG T GC A AA T TT C AA T T G T C. N e xt t he Egr1sequence tion were performed, as these serotypes have previously was added to pAcTnT-mCherry-IRES by NEBuilder® HiFi shown efficient in CM transduction [53]. In our study design DNA Assembly Master Mix (NEB). Proper gene insertions we found the AAV9 serotype to be much more efficient in were validated by enzymatic digestion at the respective transducing CMs, as compared to the AAV6 serotype (data restriction sites and size determined by gel electrophoresis not shown). (Data not shown). Generation of plasmids Plasmids harboring the genes For plasmid packaging in an AAV9 serotype capsid we of interest were purchased from Origene (Mouse Tagged used the Rep/Cap plasmid, pAAV2/9n, a gift from James M. ORF Clones; Supplementary Table 1), except for Nfya, Wilson (Addgene plasmid # 112,865; http:// n2t. ne t/ addg ene: which were synthesized by GeneArt (Thermo Fisher; Sup- 112865; RRID:Addgene_112865) and the helper plasmid plementary Table 1). The AAV backbone transfer vector pHelper (a kind gift to our collaborator Per Svenningsen, was derived through modifications of the plasmid pAAV- University of Southern Denmark, from Ben Deverman, EF1a-mCherry-IRES-Cre (a gift from Karl Deisseroth; Caltech, Pasadena, USA). Addgene plasmid # 55,632; http:// n2t. net/ addge ne: 55632; Virus generation Large-scale AAV generation for in vitro RRID:Addgene_55632) [15], allowing simultaneous tran- use was performed in HEK293T cells (ATCC; CRL-3216) scription of mCherry and the gene of interest through the by co-transfection with pAcTnT-mCherry-IRES (empty vec- internal ribosome entry site (IRES). Thus, due to the IRES tor) or pAcTnT-mCherry-IRES harboring the gene of inter- site, transcription of the gene of interest correlates to the est, pAAV2/9n and pHelper. Transfection efficiency was level of mCherry. To unify the process of gene insertions, addressed by mCherry visualization using immunofluores- the restriction sites SgfI and MluI were inserted into the plas- cence microscopy. Five days after transfection, recombinant mid. Briefly, the already existing MluI restriction site was AAV was isolated by PEG 8000 precipitation and purified by removed by introducing a point mutation in the plasmid by iodixanol gradient ultracentrifugation followed by centrifu- PCR amplification using the following primers: Forward: gation through an Amicon Ultra Centrifugal filter (50 K). C GC A CG G G T A A G C TT T GC A AA G AT G G A TAA A G T Recombinant AAV yields were determined by quantitative TTTAAA C AGA GAGG A and Reverse: AAGCTT A CCCG T real-time PCR (qRT-PCR) through a titration of pAcTnT- GCG GCC GCA GGA ACC CCT AGT GAT . The Cre site was mCherry-IRES plasmid using the primers Forward: AGT then removed and the SgfI and MluI restriction sites were G TT GCA TT C CT C T CT GG and Reverse: AGC GCA T GA hereafter inserted by PCR amplification using the prim-ACT CCT TGA T. ers Forward: TCT GGT GCG ATC GCC TAG ACG CGT TAG Adenoviral constructs were generated by Vector Bio- ATT CGA TAT CAA GCT TAT CGA TAA TCA ACC TCT and labs (PA, USA) using Adenoviral Human Type 5 (dE1/ Reverse: CTAGGC G ATCGC A CCA GAA CCA CCA TTA TC E3) as backbone. For ZEB1 knockdown experiments, a U6 A TC G TG TTT TT C AAA GG A AAA CC A CG T CCC . Finall y , a promoter was driving ZEB1 short-hairpin RNA (shRNA) truncated chicken cardiac Troponin T promoter [53] (cTnT e xpr ession of t he seq uence 5´CCG G AT AG A GGC TAC 1 3 Basic Research in Cardiology (2023) 118:8 Page 5 of 24 8 AAG CGC TTT A-CTC GAG -TAA AGC GCT TGT AGC CTC were counted (NC-200; ChemoMetec). Cells were seeded in TA-TTT TTT G-3´ and a targeting sequence of ATA GAG 12-well plates pre-coated with ECM at a density of approx. GCT ACA AGC GCT TTA. An eGFP reporter was expressed 236,500 cells/cm and placed in an incubator (37 °C, 5% under a separate CMV promoter. Ad-GFP-U6-scrmb-shRNA CO ) or approx. 88,235 cells/cm on 4-well chamber slides (cat. no. 1122N) containing a scrambled shRNA and an (cat.no. 154917, Lab-Tek™ II) for confocal microscopy. eGFP reporter was used as control. For ZEB1 overexpres- Before deciding which concentration of virus to use, sion experiments, the backbone vector contained a CMV both for AAV9 and adenovirus transduction, titration tests promoter to drive expression of the gene of interest. Ad- were performed and the concentrations resulting in the most GFP-Zeb1 was generated using mouse cDNA (GenBank: efficient transduction without causing cytotoxicity were BC139768.1) and eGFP, and ZEB1 were expressed under used. After 24 h the number of cells in each experiment separate CMV promoters. Ad-GFP (cat.no. 1060) was used were estimated (NC-200; ChemoMetec), and cell cultures as empty control. were transduced with either 750,000 viral genomes (vg)/ cell of the desired AAV9 or 50 MOI of adenovirus. For E16.5 CM cultures and Zeb1 knockdown AAV9 experiments, six, 24, and 48 h after viral transduc- tion, medium was refreshed with medium containing 10 µM On embryonic day 16.5 (E16.5), the pregnant mice were EdU. For adenovirus, EdU was added together with the virus sacrificed by cervical dislocation and the hearts from the and replenished every 24 h. All cells for qRT-PCR were pups were quickly removed and placed in a cardioplegic replenished with medium without EdU. Cells were either buffer (MIB; 1.2 mM KH PO (pH 7.4); 0.25 g/l Na CO fixed in 2.5% Neutral Buffered Formalin (NBF) diluted in 2 4 2 3; 6.44 g/l NaCl; 2.6 mM KCl; 1.2 mM M g SO ; 11 mM HBSS/5%FBS/1%PS for flow cytometry analysis 72 h post 2 4 glucose) supplemented with 1% Bovine Serum Albumin transduction (see below), fixed in the wells in 10% NFB (BSA; MIB/1%BSA). The heart ventricles were dissected or 4% Paraformaldehyde (PFA), or the RNA was isolated under a stereomicroscope before enzymatically dissocia- for qRT-PCR 48 h post transduction for adenovirus or 72 h tion into a single cell suspension using the semiautomatic post transduction for AAV9 (see below). Transduction effi- GentleMACS tissue dissociator system as described by the ciency was addressed by immunofluorescence microscopy manufacturer. Dissociated cells were counted (NC-200; for mCherry during culture, and by qRT-PCR (mCherry and/ ChemoMetec), plated on extracellular matrix (ECM) at a or gene of interest) and flow cytometry for mCherry or GFP. density of approx. 118,500 cells/cm and cultured in growth Furthermore, we consistently observed lower levels of GFP medium (79.5% DMEM (supplemented with 1% PenStrep with the Ad-GFP-Zeb1 compared to Ad-GFP suggesting (PS)), 19.5% Medium 199 (supplemented with 1% PS), and correlation between GFP and ZEB1 expression. 1% newborn calf serum). After 24 h, the number of cells were counted in some wells to calculate the amount of virus Flow cytometry required. Optimal MOI was determined from titrating the virus and quantifying transduction efficiency as well as Fixed cells were permeabilized with phosphate buffered observing for immediate cytotoxicity. saline (PBS) containing 1% BSA and 0.1% Triton X-100 After 24 h of culturing, cells were transduced with 10 (TX100) and stained with primary antibodies in different MOI of either Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1. combinations (mouse anti-MYH1, 1:300, MF20-c, DSHB; In addition, 10 µM of 5-ethynyl-2′-deoxyuridine (EdU) was rat anti-mCherry, 1:500, M11217, Thermo Fisher; and rab- added to assess for cell cycle activity. The medium, with or bit anti-GFP, 1:500, ab290, Abcam) for 1 h in the dark on without EdU, was replenished every 24 h, and experiments ice while shaking. After washing, cells were incubated with were terminated as indicated at 96 h after transduction for EdU Click-it reaction cocktail according to the manufac- analysis. turer’s protocol (Invitrogen, C10419), and washed before incubation with secondary antibodies in different combina- Neonatal CM cultures and viral transduction tions (488-donkey anti-mouse, 1:200, A21202, Invitrogen; 555-donkey anti-rat, 1:200, Ab150154, Abcam; 555-don- Neonatal (P0) mouse pups from each litter were sacrificed key anti-mouse, 1:200, A31570, Invitrogen; and 488-donkey by decapitation, whereafter the hearts were quickly removed, anti-rabbit, 1:200, A21206, Invitrogen) for 30 min in the and the ventricles dissected under a stereomicroscope. Dis- dark on ice while shaking. Three final washes were per - sected ventricles were pooled in a tube with MIB/1%BSA formed in PBS/1% BSA/0.1% TX100 and Hoechst 33342 before enzymatic dissociation into a single cell suspension was added 5 min before flow cytometry using the LSRII using the semiautomatic GentleMACS tissue dissociator flow cytometer (BD Biosciences). Data was analyzed using system as described by the manufacturer. Dissociated cells the FACSDiva software v8.0.1, and initially gated according were resuspended in growth medium and the number of cells to the CM marker MYH1 and then sub-fractionated based 1 3 8 Page 6 of 24 Basic Research in Cardiology (2023) 118:8 on the antibody amplified mCherry or GFP signal. Cells and each heart was processed individually, or prepared for positive or negative for a reporter (mCherry or GFP) were paraffin embedding (see below). gated according to EdU incorporation to determine cell cycle activity. Ploidy was addressed in subpopulations by Hoechst Immunohisto‑ and immunocyto‑chemistry 33342 using gates (2n, 4, and > 4n) defined by the entire CM and microscopy population. Each analysis as indicated consisted of at least three to nine independent experiments designated n, each P8 hearts from virus injected pups were fixed in 10% NBF comprising cells from one litter. Within an experiment, 1–3 overnight, rinsed in PBS, dehydrated, and finally embed- replicates (n*) were performed and used as an average of the ded in paraffin. Embedded specimens were then cut into n for further statistical analysis as indicated. 10 µm sections before mounting on glass slides and stored at 4 °C. Sections were deparaffinized and rehydrated before staining. Immunostainings were performed on NBF or PFA RNA isolation, RNA integrity, and qRT‑PCR fixed cell cultures or paraffin embedded sections as previ- ously described in Andersen et al. [2] with the following RNA was isolated from each sample and qRT-PCR was per- primary antibodies: rabbit anti-GFP (1:500, ab290, Abcam), formed as described previously [3]. Briefly, the cells were mouse anti-MYH1 (1:300, MF20-c, DSHB), rabbit anti- lysed with TriReagent and the RNA was isolated using Poly- ZEB1 (1:500, PA5-28,221, Thermo Fisher), rabbit anti- acryl carrier, 1-Bromo-3-Chloro-Propane and 2-propanol. Mef-2c (1:500, 5030S, Cell Signaling Technology), mouse The RNA was rinsed using 75% ice cold ethanol. Finally, anti-actinin (1:200 A7811, Sigma), mouse anti-α-tubulin the RNA was dissolved in nuclease-free water and the RNA (1:250, 3873, Cell Signaling Technology), and 647-phalloi- concentration was determined using a nano-drop. For qRT- din (1:800, A30107, Thermo Fisher). Secondary antibodies PCR, cDNA was generated using the High-Capacity cDNA used were: 488-donkey anti-rabbit (1:200, A21206, Invitro- Reverse Transcriptase kit (Applied Biosystems; 4368814) gen), 555-donkey anti-rabbit (1:200, A31570, Invitrogen), according to the manufacturer’s recommendations. Each 647-donkey anti-mouse (1:200, A31571, Invitrogen). All sample for qRT-PCR contained 2–4 ng cDNA (Supplemen- sections were mounted with DAPI (Vectashield, Vector Lab., tary Table 2) in a total volume of 10 µl and were analyzed for Phalloidin and α-tubulin staining, Fluoroshield, Abcam in technical triplicates of qRT-PCR using a mixture of was used). Microscopy was performed on a Leica DMI 4000 Power SYBRGreen PCR Master Mix (Applied Biosystems, B microscope with a Leica CTR4000 illuminator and Leica 4367659) and appropriate forward and reverse primers (Sup- DFC300FX/DFC 340 FX cameras, and confocal micros- plementary Table 2). The qRT-PCR was run on a 7900HT copy was performed on an Olympus FV1000MPE confocal Fast Real-time PCR system (Applied Biosystems) under the laser scanning microscope equipped with an UPlanSApo following conditions: holding for 10 min at 95 °C, hereafter 60x/1.20 water objective. During analysis, all camera set- 40 cycles consisting of 15 s of denaturation at 94 °C, 30 s tings and picture processing were applied equally to samples of annealing at 57–60 °C (Supplementary Table 2) and 30 s and controls. of elongation at 72 °C. The obtained data was analyzed as previously described [3] by normalization to multiple stably Statistics and reproducibility expressed endogenous gene according to the qBase Plus 3.2 platform (Biogazelle). All statistics were performed using the GraphPad Prism (v 9.0.0) software and the appropriate tests, number of inde- Injection of adenovirus in P0 pups pendent experiments (n) and replicates (n*) are defined in the corresponding figure legends. We used the significance The litter was gently taken from their home cage. Pups level α = 0.05 for identifying significant results marked by were then anesthetized by induction of hypothermia before asterisks, yet have indicated throughout the exact p-value 7.60 × 10 PFU (in a total volume of 20 µl, diluted in sterile if between 0.05 and 0.1 to enable objective evaluation of PBS) of the desired adenovirus was injected into the super- trends. For scRNA-seq, each timepoint includes three bio- ficial temporal vein. Pups were reheated and placed together logically independent experiments each comprising mouse with their littermates before the litter was gently put back pups derived from three distinct litters. Cells were pooled into their home cage. Following the pups received a 50 µl just before library generation and scRNA-seq. ScRNA-seq subcutaneous injections of EdU (2.5 mg/ml) at P4 and P6 and subsequent gene expression analysis and transcription before they were sacrificed by decapitation at P8. Hearts factor binding site analysis was performed using two dif- were dissected and either dissociated for flow cytometry ferent sequencing platforms (NextSeq and NovaSeq) with as described above for P0 pups, except cells were strained similar results. All transduction experiments were success- (100 µm nylon cell strainer, 352360) prior to NBF fixation, fully reproduced with at least three biologically independent 1 3 Basic Research in Cardiology (2023) 118:8 Page 7 of 24 8 experiments obtaining similar results, thereby confirming of this method (Supplementary Fig. 1a) includes avoid- the design and robustness. ance of the long-term susceptibility of CMs to tissue dis- sociation, improvement of logistics with the ability to store scRNA-seq samples obtained at different timepoints, and Results application of FACS to enrich CMs using intracellular CM markers such as myosin heavy chain 1 (MYH1) and DNA FACS of fixed CMs combined with high‑throughput (Hoechst; Supplementary Fig. 1a–c). Based on a viability scRNA‑seq efficiently distinguishes diploid stain prior to fixation we excluded and reduced the num- and tetraploid CMs during heart development ber of dead cells (Supplementary Fig. 1c) and in general checked for clumps of cells (Supplementary Fig. 1e, f) To address the mechanisms underlying CM proliferation in prior to generating cDNA libraries for scRNA-seq. Over- the G2/M phases, our focus was fairly strict on tetraploid all, scRNA-seq data from fresh and fixed cells show a –16 CMs in the G2/M phases of the cell cycle (Fig. 1a). To that high degree of correlation (R = 0.98; p < 2.2 × 10 , Sup- end, we developed an approach that combines FACS of plementary Fig. 1d) confirming data consistency between fixed heart cells with scRNA-seq of the sorted CMs (Sup- the methods. plementary Fig. 1a), while preserving the RNA integrity We next aimed to use this approach for identifying TFs (RIN; Supplementary Fig. 1b) to increase resolution spe- specific for regulating the G2/M phases of CMs around cifically of CMs in the scRNA-seq analysis. Advantages birth. Accordingly, we isolated diploid (2n) and tetraploid Fig. 1 High scRNA-seq CM resolution of embryonic and neona- test; ****P ≤ 0.0001). d Heatmap visualizing differential expression tal CMs a Top: Schematic of the CM binucleation process during of known marker genes for different cell types (CMs, fibroblasts, heart development (E16.5, P1, and P5). Middle: Images of mouse endothelial cells, macrophages, and smooth muscle cells) in scRNA- hearts used for scRNA-seq with the left ventricle being dissected and seq analyzed heart cells (2n- and 4n, E16.5, P1 and P5) prior to ana- used (encircled by the dotted line). Bottom: FACS dot plots show- lytical CM-filtering. e Table displaying the number of cells prior ing the CM marker MYH1-488/Hoechst-stained cells (N = 3 litters/ to and after analytical CM filtering according to expression of the age; 14–20 pups/age). b–c Quantification of percentage of b non- CM marker genes Tnni3, Actc1, Tnnc1 and Tnnt2 at E16.5, P1 and myocytes (NMs) and CMs, c and 2n- and 4n-CMs, at E16.5, P1, and P5, and stratified by ploidy (2n and 4n). f UMAP-plots showing the P5 based on flow cytometric data and cell counts (mean, SD, N = 3, expression of the CM marker Tnni3 prior to filtering (blue: Tnni3 , Statistical analysis included two-way ANOVA with Tukey’s post grey: Tnni3 ) 1 3 8 Page 8 of 24 Basic Research in Cardiology (2023) 118:8 (4n) CMs from the left ventricle of mice before (E16.5 Gradual CM maturation and terminal differentiation and P1 samples) and after (P5 samples) (Fig. 1a) the is a dynamic process occurring already at E16.5, known proliferative stop at P3 [61]. At E16.5, the left leaving P1 hearts almost devoid of proliferating ventricle comprised of 80.8 ± 2.4% CMs and 17 ± 2.8% CMs non-myocytes (NMs) (mean, SD; n = 14–20). This CMs/ NMs ratio remained constant from E16.5 to P1 with To evaluate the biological activity of the sorted CMs, all 77.5 ± 5.8% CMs and 19.6 ± 5.3% NMs at P1, whereas scRNA-seq samples were integrated and CMs were clus- the percentage of NMs as expected increased rapidly from tered and visualized based on their gene expression pro- P1 to P5 (55.3 ± 6.9% NMs) (Fig. 1b). No difference was file, using Uniform Manifold Approximation and Projec- observed in the percentage of diploid CMs (2n-CMs) tion (UMAP) plots combined with Gene Ontology (GO) between E16.5 (59.7 ± 4.9%) and P1 (67.4 ± 7.5%), but term designation (Fig. 2a, b). In agreement with prior this percentage declined (36.2 ± 5.7%) at P5 (Fig. 1c). knowledge [13, 45], we observed that embryonic CMs Likewise, the percentage of tetraploid CMs (4n-CMs) switch from pyruvate metabolism to fatty acid metabo- remained constant from E16.5 (20.2 ± 0.8%) to P1 lism soon after birth (Fig. 2a, b), validating the scRNA- (15.5 ± 2.7%), but increased to 41 ± 7.3% at P5 (Fig. 1c). seq design. Moreover, we identified two major “Cell divi- Thus, as expected karyo-/cytokinesis failure in the G2/M sion” clusters (Fig. 2a, b), one mainly composed of E16.5 phases of P5 CMs were apparent. By examining the CMs and one dominated by P5 CMs, both embracing expression of MYH1 and the number of visible nuclei mainly tetraploid CMs. Using trajectory analysis (Fig. 2c, in FACS sorted P5 CMs, we found that > 95% of the dip- d), we found an overall ordering of the CMs in pseudo- loid and 50% of the tetraploid CMs were mononucleated, time that corresponds to the relative developmental stage, whereas 40% of the tetraploid CMs were binucleated with E16.5 CMs mainly represented early in pseudotime, (Supplementary Fig. 1e, f). The CM purity was > 95% after which they mature through the P1 stage and end- and cell clumps were scarce (Supplementary Fig. 1e, f), ing as P5 CMs (Fig. 2c, d and Supplementary Fig. 2a). the latter minimizing the appearance of false positive Yet, a small fraction of E16.5 CMs, especially tetraploid intermediates in the subsequent scRNA-seq analysis. CMs, did also align late in pseudotime, while some P1 High-throughput scRNA-seq with NextSeq (266,839,970 and P5 CMs resided in the early branches according to reads in total for 6 samples) on sorted 2n- and 4n CMs pseudotime (Fig. 2c, d and Supplementary Fig. 2a). This from E16.5, P1, and P5 revealed a total of 31,156 cells may suggest that the process of CM maturation and likely passing quality control filters with an average of 5,193 cell cycle exit is dynamic and ongoing already at E16.5, cells/sample and 1,135 genes in average per cell identi- whereas a small fraction of CMs at P5 may exhibit an fied (Supplementary Fig. 1 g). The cells were sequenced immature phenotype similar to E16.5 CMs. This was fur- at a median depth of 9,278 reads per cell with a median ther confirmed by branchpoint analysis (Fig. 2e–h, Sup- alignment rate of 74% per cell. Subsequent preparation plementary Fig. 2b) showing that the two branches early of data for analysis was performed using the R package in pseudotime (Fig. 2e, Supplementary Fig. 2b) primarily Seurat [62]. The total number of genes identified in all differ by metabolism and cell cycling where the earliest samples was 14,222 prior to any filtering. Expression of trajectory encompasses less differentiated CMs (Fig. 2g). CM markers (Fig. 1d,e) confirmed a 96.85% CM purity Particularly, we observed a higher cell cycling activ- of analyzed cells, yet, to exclude the few contaminants, ity for the lower trajectory of 4n-CMs (Supplementary we prefiltered the data by defining CMs as those with a Fig. 2b). The later bifurcations from the main trajectory combined expression of Tnni3, Tnnt2, Tnnc1, and Actc1 all seem to exhibit lower cell cycle activity (Fig. 2e) but (Fig. 1e,f). This resulted in an average of 5136 CMs per higher level of CM differentiation (“Shape and adhesion”, sample (Fig. 1e). After filtering, the remaining data for “ECM maturation”, “Fatty acid metabolism”, “Sarcomere only CMs were log-normalized, variable genes were iden- assembly”, and “Muscle contraction”) as well as “Cell tified, and data were scaled according to the total detected Migration” (Fig. 2c). Cell cycle activity was, however, number of molecules in each cell (nCount_RNA). Thus, regained for 4n-CMs at the tip of the lower trajectory our new approach combining FACS of fixed CMs with (Supplementary Fig. 2b). Since the P5 cell cycle active scRNA analysis enabled us to increase scRNA-seq resolu- CMs remain after the proliferation stop at P3 [61], and tion with high numbers of CMs stratified by ploidy. also exhibit mature CM characteristics, the P5 cell cycle activity most likely represent binucleation/polyploidiza- tion. Moreover, the data suggest that already before birth (E16.5) a small portion of CMs escape from cell division and starts maturing rendering P1 hearts almost devoid of CMs exhibiting cell division properties. 1 3 Basic Research in Cardiology (2023) 118:8 Page 9 of 24 8 Fig. 2 CM cell cycle exit, and maturation are initiated before cell cycling from BP heatmaps (below) is marked by a dotted line e. birth. a UMAP clustering based on k-nearest neighbors of CMs f, g, h, Heatmaps showing differentially expressed genes during CM with representative GO terms and ratio composing each cluster for development along pseudotime with dominating GO terms for each each ploidy/developmental stage b with colors referring to different cluster in each BP indicated. Hierarchical clustering is based on clusters. c, d, e, Trajectory analysis by Monocle displaying original Ward.D2 and unrelated to the clustering in (a, b). Above each heat- identity of cells c, pseudotime d, and trajectory state including three map, the states of the branches (according to (e)) are designated branchpoints (BP); The overall expression pattern of genes related to 1 3 8 Page 10 of 24 Basic Research in Cardiology (2023) 118:8 were scarce at P1 (2n = 2.2%; 4n = 8.7%) (Fig. 3c). Inversely, The cell cycling machinery of cycling E16.5 tetraploid G2/M CMs is defined by a certain S-phase CMs were more pronounced in diploid CMs (E16.5: 2n = 29.3%; 4n = 17.2%, P1: 2n = 15.4%; 4n = 10.2%, P5: set of TFs different from that in P5 cycle active tetraploid G2/M CMs 2n = 38.7%; 4n = 9.2%) (Fig. 3c). This agrees with cell cycle active diploid CMs mainly being in S-phase, while tetraploid To further explore the cell cycle status of the identified CMs have progressed to the G2/M phases. In pseudotime, S-phase CMs manifested in the start of the trajectory, but CM clusters (Fig. 3a), we used gene signatures that have previously been shown to denote G1, S, and G2/M phases some did align broadly throughout pseudotime as did also G1 phase CMs (Figs. 2d and 3d, e; Supplementary Fig. 2a). [67]. Thus, whereas ploidy was assigned by FACS prior to scRNA-seq, separation according to cell cycle identity was In contrast, the large cluster of 4n E16.5-G2/M CMs con- fined to the two earliest branches in pseudotime, whereas performed bioinformatically. Yet, as the G2 and M phases cannot be readily distinguished based on gene expression P5-G2/M CMs mainly located to the downward bifurca- tion in branchpoint 3, which represented more mature CMs they are considered united as “G2/M”. As such, we found that the two “Cell division” clusters (Fig. 2a, b) were com- characterized by "Fatty acid metabolism” and “Muscle con- traction” (Fig. 2e). These data thus supported that E16.5- posed of CMs active in either the S-phase or G2/M-phases (Fig. 2a, b) whereas the CMs composing the “Fatty acid 4n-G2/M CMs likely represented dividing CMs whereas the more mature P5-4n-G2/M CMs were in the process of metabolic process”-cluster (Fig. 2a, b) were mainly ascribed to the G1-phase (Fig. 3a, b). The percentage of predicted polyploidization/binucleation, which agrees with CM prolif- eration being absent after P3 [61] and our combined FACS G1-phase CMs was fairly constant between ploidies at P1 (2n = 82.3%; 4n = 81.1%) and P5 (2n = 56.9%; 4n = 54.9%) and microscopic analyses described above (Fig. 1b, c and Supplementary Fig. 1e, f). but differed at the embryonic stage E16.5 (2n = 57%; 4n = 31.8%) (Fig. 3c). These percentages also imply that a Then to identify which TFs that regulate the G2/M phases in immature CMs (E16.5) as compared to those that halt relatively higher percentage of cells are in active cell cycle (not G1) at E16.5 and at P5 as compared to P1 in agreement before karyokinesis/cytokinesis (P5), we extracted CMs composing the two “Cell division” clusters (Figs. 2a and with the GO term analysis (Fig. 2b). In consensus with biol- ogy, the majority of G2/M CMs were mainly found in the 4a) and focused the analysis on tetraploid CMs ascribed to the G2/M-phases extracting a subset of the data for further tetraploid portion of CMs, particularly the E16.5-4n CMs (2n = 13.6%; 4n = 51%) and to some extent the P5-4n CMs analysis (Fig. 4b). Then, we obtained a list of marker genes with higher expression in E16.5-4n-G2/M-CMs as compared (2n = 4.4%; 4n = 35.9%) (Fig. 3c). In contrast, G2/M CMs Fig. 3 Distribution of CMs within cell cycle phases. a, b, UMAP each cell cycle phase (n* = number of animals). d, e, Trajectory anal- plots of CMs with visualization of the cell cycle status across devel- ysis of CMs (d 2n and 4n pooled; e divided into 2n and 4n)) across opmental stages and ploidy, c, Quantification of cells representing developmental stages with cell cycle phase identity marked 1 3 Basic Research in Cardiology (2023) 118:8 Page 11 of 24 8 P0 to P5-4n-G2/M-CMs (Fig. 2c) and performed subsequent cycle activity in CM was measured 72 h after AAV9-cTnT- GO term analysis. Several terms related to the cell cycle: TF transductions using flow cytometry for EdU/MYH1/ “Mitotic nuclear division”, “Cell division”, “Chromosome mCherry/Hoechst (Fig. 5a). Virus functionality for each condensation”, “Regulation of mitotic cell cycle”, and of our AAV9-cTnT-TFs was validated by mCherry and TF “Nuclear division” and associated genes were unraveled by mRNA expression (Supplementary Fig. 6a, c), whereas a gene concept network map (Fig. 4d). Using the oPOS- flow cytometry for mCherry CMs confirmed transduc- SUM platform, enriched TF binding sites were determined tion efficiencies (Supplementary Fig. 6b). Both mCherry for the gene expression enriched in E16.5-4n-G2/M CMs mRNA and reporter fluorescence showed that the efficiency (Fig. 4b, c), and allowed us to predict a list of TFs potentially of TF expression decreased with the increasing size of the regulating G2/M progression in dividing CMs (Fig. 4e). To TF (Supplementary Fig. 6a, c, d). To counterbalance we confirm this list of TFs and avoid the initial selection of increased the number of CMs analyzed by flow cytometry the “Cell division” clusters, which could bias the approach, for large-sized TFs. Moreover, to ensure a high biological we combined E16.5- and P5-CMs into a new dataset and diversity and account for a 12 h difference in birth deliv - P0 reanalyzed with specific identification of E16.5-4n-G2/M ery, each independent experiment embraced CM pooled CMs and P5-4n-G2/M CMs (Fig. 4f). This repeated analysis from one litter each constituting 6–9 mice. At the time of P0 resulted in a second list of TFs (Fig. 4g) very similar to that analysis, 72 h after transduction, CM cultures consisted obtained for the cluster-based approach (Fig. 4e). Moreover, of 58.7 ± 8.0% CMs (mean, SD, n = 8), and AAV9-cTnT- to exclude that the identified set of TFs (Fig. 4e, g) reflected TF expression was highly specific for CMs (Fig. 5b). The + P0 data of low sequence saturation, we performed NovaSeq percentage of EdU CM transduced with an empty AAV9- (mean reads per CM were amplified 4.5- and 8.3-fold for cTnT-mCherry vector did not differ between mCherry and E16.5-4n and P5-4n CMs, respectively) with an increase mCherry CMs (5.3 ± 3.3% versus 4.4 ± 2.3%; mean, SD, from 13,578 to 16,118 in the total number of genes detected n = 8; p = 0.9992; Adjusted p-value, Sidak test) (Fig. 5c, d), (Supplementary Fig. 3a–c). Together, these analyses sup- suggesting that transduction did not alter S-phase progres- ported that the identified TFs likely play a role in regulating sion in itself. This also verie fi d that cell cycle activity indeed P0 G2/M of dividing CMs. Based on the Fisher score (Fig. 2e, is relatively low per se in CM cultures. When compared + P0 g, i), we therefore selected NFYA, SRF, MYC::MAX, USF1, to empty control transduced mCher ry CM (5.3 ± 3.3%), MYCN, EGR1, ARNT, MYC, E2F1, and ZEB1 for further Mycn (16.5 ± 3.6%), Egr1 (9.3 ± 1.8%), Arnt (29.8 ± 5.9%), analysis. We also included SP1 for further analysis as this TF M yc (15.5 ± 5.3%), Zeb1 (26.8 ± 5.6%), and Sp1 has been associated with CM cell cycling previously [21]. (28.4 ± 4.6%) overexpression resulted in significant increases + + Since, MYC::MAX represents a complex of the two indi- in the percentage of EdU mCherry CMs (Fig. 5d). This + + vidual TFs, MYC and MAX, we therefore proceeded with was consistent when comparing EdU mCherry CMs with + − 11 TFs in total for further analysis. EdU mCherry CMs within each culture (Fig. 5d). Thus, overexpression of TFs corresponded to 3.1- (Mycn), 1.8- ScRNA‑seq identified TFs that affect CM cell cycling (Egr1), 5.6- (Arnt), 2.9- (Myc), 5.1- (Zeb1), and 5.4-fold (Sp1) inductions in the percentage of EdU CMs. The Since, expression of TFs often are relatively low and given remaining TFs did not significantly alter the percentage of + + their nature as being upstream regulators of their tar- EdU mCherry CMs neither as compared to empty control + − get genes, detection of TF expression in scRNA-seq data nor as compared to EdU mCherry CMs (Fig. 5d). Thus, are often complicated. Despite these challenges, our CM the six TFs Mycn, Egr1, Arnt, Myc, Zeb1, and Sp1 were able P0 scRNA-seq data did reveal several CMs expressing the iden- on their own to increase cell cycle activity of CM . How- tified TFs. Yet, the percentages, of CMs expressing the TFs ever, it is generally acknowledged that EdU incorporation were still quite small, and their levels were decreasing with only reflects whether a given CM has progressed through developmental stage (Supplementary Figs. 4 and 5). the S-phase and therefore does not reflect if the CM com- Thus, to evaluate the biological effect of the 11 TFs on pleted cell division [37, 55]. We therefore assessed ploidy CM cell cycle activity we overexpressed them in P0 cardiac in the transduced CMs and defined borders for a ploidy P0 cells (CM ). To mimic the in vivo heart environment, we of either 2n, 4n, and > 4n (Fig. 5e), and used those gates + + initially used mixed cardiac cell cultures rather than a pure for analyzing EdU mCherry CMs (Fig. 5f). Neither EdU CM population. Yet, to enable CM specific TF overexpres- incorporation nor AAV9-cTnT transduction affects CM sion and identification of transduced CMs, we generated ploidy in themselves (Fig. 5g) ensuring the validity of the CM specific (cTnT promoter) adeno associated viruses of analysis. Using this setup, we found a significant change in + + serotype 9 (AAV9) with a mCherry reporter and an inter- ploidy for EdU mCherry CMs overexpressing Mycn, Egr1, nal ribosome entry site (IRES) for each of the 11 selected Arnt, Myc, Zeb1, and Sp1, whereas no change in ploidy was TFs (denoted AAV9-cTnT-TF) (Fig. 5a). Accumulated cell observed for the TFs also not altering the percentage of 1 3 8 Page 12 of 24 Basic Research in Cardiology (2023) 118:8 1 3 Basic Research in Cardiology (2023) 118:8 Page 13 of 24 8 ◂Fig. 4 Enriched set of transcription factors (TFs) specific for ZEB1 identified as a novel regulator of G2/M CM G2/M progression. a UMAP plot of 2n- and 4n CMs at E16.5, progression and CM proliferation before birth P1, and P5 with cell cycle phase identity visualized. b UMAP plot of extracted G2/M, 4n CMs from E16.5 and P5 developmental stage By reassessing the literature, the role of ZEB1 appears from the two Cell division clusters as indicated in (a). c Heatmap of expression of top genes with increased expression in E16.5 CMs poorly described in the heart and CMs. In a study by compared to P5 CMs in (b). d cnetplot of selected cell-cycle related Riechert and coworkers, it was found that Zeb1 is involved GO terms enriched among the genes in (c). e Enriched TFs (oPOS- in CM hypertrophy [54] and Cencioni et al. showed that the SUM) for genes with increased expression in E16.5 CMs compared to Zeb1-Hdac2-eNOS circuity identifies early cardiovascular P5 CMs in (b). f UMAP plot of 2n- and 4n CMs at E16.5 and P5 sep- arately analyzed with cell cycle phase identity visualized. g Enriched precursors [9]. However, the role of ZEB1 in CM prolif- TFs (oPOSSUM) for genes with increased expression in E16.5 CMs eration and polyploidization remains elusive. Interestingly, compared to P5 CMs in (f). h UMAP plot of re-sequenced data with Zeb1 null mice do not survive postnatally due to respira- NovaSeq. Clustering is indicated and CMs separated according to tory failure and T-cell deficiency [64]. Reinvestigating our developmental stage (E16.5 and P4, both 4n). i Enriched TFs (oPOS- SUM) for genes with increased expression in E16.5 CMs compared to scRNA-seq data we found that ZEB1 was present in only a P5 CMs in (h) small fraction of CMs (Fig. 6a, Supplementary Fig. 4) pref- erably in the G2/M phases (Fig. 6b), but the level declined + + EdU mCherry CMs (Fig. 5h). Myc and Mycn overexpres- with developmental stage (Supplementary Fig. 5). To inves- + + sion clearly resulted in EdU mCherry CMs being diploid tigate the implications of Zeb1 in CM proliferation before suggesting completed cytokinesis and thus proliferation. In birth, ZEB1 knockdown was performed using adenoviral contrast, Egr1, Arnt, Zeb1, and Sp1 favored polyploidy at the transduction for administration of validated Zeb1 shRNA expense of diploid CMs (Fig. 5h) indicating karyokinesis/ in E16.5 primary heart cultures (Fig. 6c). A high transduc- cytokinesis failure. tion efficiency was confirmed in CMs (Fig. 6d) with no We next subjected the target genes (according to oPOS- significant difference between control Ad-GFP-shRNA- SUM 3.0) of our six identified TFs to GO term analysis (93.1 ± 4.7%; mean, SD, n = 5) and Ad-GFP-shRNA-Zeb1 (p < 0.05, Benjamini corrections) and found that 4- (MYC), treatments (94.2 ± 4.0%; mean, SD, n = 5) (Fig. 6e), and 5- (MYCN), 3- (EGR1), 3- (ARNT), 13- (ZEB1), and 4- overall ZEB1 was reduced by 82.3 ± 5.9% (mean, SD, n = 3) (SP1) out of 13 GO terms related to “Cell cycling/division” (Fig. 6f). Using this setup, we found that cell cycle activity (3), “Mitotic division/cytokinesis” (4), “Telomers” (3), and in CMs as reflected by EdU incorporation was decreased “Regulation of cell cycling”/”Positive regulation of cell pro- significantly in Ad-GFP-shRNA-Zeb1 treated CMs from liferation” (3) (Supplementary Fig. 7). When adjusting for 12.2 ± 3.3% to 2.8 ± 0.8% (mean, SD, n = 5)(Fig. 6g,h). gene overlap between the different cell cycle related GO Importantly, the diploid status of EdU /CMs decreased by terms, 53- (MYC), 60- (MYCN), 41- (EGR1), 45- (ARNT), Zeb1 knockdown (Fig. 6i) suggesting that not only S-phase 118- (ZEB1), and 72- (SP1) genes related to these cell cycle progression was inhibited, but also cytokinesis was reduced related GOs were affected by the given TF (Supplementary by Zeb1 knockdown. This was supported by a significant Fig. 7). downregulation, in Ad-GFP-shRNA-Zeb1 treated cells, of Overall, these data underscore the design and quality Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1) and Ccnd3 (Cyc- of our CM scRNA-seq data, and enabled us to identify at lin D3), while the levels of Ccne2 (Cyclin E2) and Ccng2 least six TFs regulating cell cycle activity of CMs, where (Cyclin G2) associated with endoreplication [48, 82] were two (Myc, Mycn) and four (Egr1, Arnt, Zeb1, and Sp1) TFs unchanged (Fig. 6j). Although, the level of the major cyclin resulted in cytokinesis or a lack hereof, respectively. Since dependent kinase, Cdk1 (Cyclin-dependent kinase 1), was the Zinc Finger E-Box Binding Homeobox 1 gene (Zeb1) slightly reduced when Zeb1 was knocked down (Fig. 6k), no was superior to the other TFs in regulating the number (118) difference was observed for Cdk4 (Cyclin-dependent kinase of target genes associated to cell cycle activity in CMs, we 4) and the cell cycle inhibitors Cdkn1a (Cyclin-dependent hypothesized that ZEB1 could be a novel key player in CM kinase inhibitor 1a, p21) and Cdkn1b (Cyclin-dependent proliferation. kinase inhibitor 1b, p27) (Fig. 6k). With a focus on factors known from the G2/M phase, where Zeb1 predominantly was observed (Fig. 6b), we found a significant increase in the expression of Cenpe (Centromere protein E), Cenpf (Cen- tromere protein F), Aurkb (Aurora kinase B), and Aurka 1 3 8 Page 14 of 24 Basic Research in Cardiology (2023) 118:8 Fig. 5 ScRNA-seq identified transcription factors (TFs) regu- duced) and mCherry (non-transduced of the same culture) CMs + + late CM cell cycle activity. a Schematic of the study design and quantified by flow cytometry. The percentage of EdU mCherry workflow. Neonatal heart cells were isolated at P0 and cultured for CMs was compared to that of empty vector transduced CMs 24 h, before transductions with AAV9-cTnT-TF. Insert depicts as (AAV9-cTnT) (ANOVA and Fisher’s LSD post test, N = 3–8, modified from Addgene. EdU incorporation into mCherry and *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001 and ****P ≤ 0.0001) or to the % − + + − mCherry CMs together with CM- (MYH1 ) and ploidy- (Hoe- EdU mCherry CMs within the culture (Data not shown). e–h Flow chst) identity were then assessed by flow cytometry. b Flow cyto- cytometric assessment of the percentage of diploid- (2n), tetraploid- metric dot plots of non-transduced and transduced heart cells cul- (4n) and polyploid- (> 4n) transduced and non-transduced CMs as tured for 3 days after viral transduction showing CM specificity for indicated (ANOVA and Fisher’s LSD post test, *P ≤ 0.05, **P ≤ 0.01, AAV9-cTnT-TF expression (two-way ANOVA with Sidak’s post test, ***P < 0.001 and ****P ≤ 0.0001). Gates were defined using all CMs ****P ≤ 0.0001). c-d EdU incorporation in mCherry (TF trans- in (e) (Aurora kinase A), but no change in the level of Gmnn ZEB1 overexpression leads to CM endoreplication (Geminin) (Fig. 6l). These data confirm the scRNA-seq and high ploidy after birth data that ZEB1 mediates the cell cycle program and seems required for CM proliferation before birth. With Zeb1 as a key player in CM proliferation before birth, we then investigated if reintroducing ZEB1 at high efficiency 1 3 Basic Research in Cardiology (2023) 118:8 Page 15 of 24 8 in postnatal CMs could override terminal CM differentiation Additionally, we found that the muscle size inhibitor Mstn and lead to CM proliferation after birth. To overcome the (Myostatin) were substantially increased in ZEB1 overex- observed challenge of AAV9-TF transduction effectivity of pressing CMs, while the Myh6 expression was substan- the large-sized Zeb1 (Supplementary Fig. 6d), we generated tially reduced (Fig. 8f), which may explain why the smaller adenovirus (Ad) with an eGFP reporter to be expressed sepa- size of CMs was observed early after ZEB1 overexpression rately from ZEB1 and used that for inducing ZEB1 expres- (Fig. 7i,j). Finally, we injected Ad-GFP and Ad-GFP-Zeb1 P0 sion in CM (Fig. 7a). As visualized by immunocytochem- into the superficial temporal vein of P0 mice to evaluate istry (Fig. 7b) and quantified by flow cytometry (Fig. 7c, ZEB1 overexpression directly in the heart, and analyzed d), Ad-GFP CM transduction efficiency was 98.22 ± 2.2% by EdU incorporation, its effect on CM cell cycling from at 72 h with a parallel and specific expression of ZEB1 P0 to P8 (Fig. 8g). Scattered transduced GFP CMs were (Fig. 7e, f, Supplementary Fig. 8a, b). In agreement, with found in all hearts of Ad injected pups (Fig. 8h), and as its nature as a TF, the overexpressed ZEB1 protein mainly estimated by flow cytometry 1.5–2.5% of CMs were GFP localized to the nucleus in CMs (Fig. 7e, Supplementary at P8 (Fig. 8i). EdU incorporation was visualized in Ad- Fig. 8a, b). We did, however, not observe ZEB1 associated GFP-Zeb1 transduced CMs (Fig. 8h), and upon quantifica- to the tubulin spindle apparatus (Supplementary Fig. 8b) as tion by flow cytometry a significant higher percentage of noted in cancer cell lines [16], but we did notice a few scat- EdU CMs were found in Ad-GFP-Zeb1 transduced CMs tered CMs with cytoplasmic ZEB1 (Fig. 7e and Supplemen- as compared to Ad-GFP transduced CMs (Fig. 8j) confirm- tary Fig. 8b). EdU pulse chase labelling with empty virus ing the in vitro results with increased cell cycle activity as Ad-GFP and Ad-GFP-Zeb1 supported the above experi- a result of Zeb1. Moreover, 99 ± 1% of the EdU /Ad-GFP- + + ments showing that the percentage of EdU CMs increased Zeb1 CMs exhibited a ploidy of > 4n, while the remaining + + significantly with ZEB1 overexpression (Fig. 7h). Moreo- 1% were 4n, which was different from EdU /Ad-GFP CMs ver, a massive reduction in CM size was apparent after 72 h showing lower ploidy (Fig. 8k). The “high ploidy” Ad-GFP- of ZEB1 overexpression, and did not occur in non-CMs Zeb1 transduced CMs were 30% larger than their Ad-GFP (Fig. 7i, j). No difference was observed in the percentage of CM counterparts at P8 (Fig. 8l). CMs between non-transduced, Ad-GFP, and Ad-GFP-Zeb1 These data thus demonstrate that Zeb1 also after birth cultures at 72 h after transduction (Fig. 7k), suggesting that facilitates CM cell cycle activity, but unlike before birth, CM death or apoptosis was minimal at this timepoint and favors CM endoreplication and not division leading to was not induced by virus treatment. ZEB1 overexpression increased CM ploidy. revealed both single-, bi-, and multinucleated CMs at 72 h after transduction (Fig. 8a and Supplementary Fig. 8b), sup- porting the observed ability to induce CM S-phase progres- Discussion sion (Figs. 5d and 7h) leading to polyploidy and not division (Fig. 5h). In contrast to our Ad-GFP-shRNA-Zeb1 studies While most studies focus on regeneration of CMs at either (Fig. 6j), we observed a downregulation of Ccnd1 with a fetal or adult stages, the understanding of the transcriptional concomitant upregulation of Ccne2 and Ccng2, while the changes leading to the transition from a fetal to a postnatal levels of Ccnb1 and Ccnd3 were unchanged (Fig. 8b). No CM phenotype is relatively poor [49]. The postnatal period change was observed for Cdk1 and Cdk4, whereas Cdkn1a for a CM is characterized by a change from hyperplastic to was dramatically reduced. Likewise, Cdkn1b was reduced, hypertrophic growth [38], a switch from glycolytic to fatty but to a lesser extent (Fig. 8c). We also evaluated the Hippo acid oxidation [50], and sarcomere maturation with adult pathway known from cardiac regeneration through CM contractile isoforms [14, 73]. Together, this leads to cell proliferation, and found its downstream targets, Axl (Axl cycle exit and terminal CM differentiation [49]. Through a receptor tyrosine kinase) and Ctgf (connective tissue growth systematic, high-resolution scRNA-seq approach, analyzing factor) to be reduced in Ad-GFP-Zeb1 transduced CMs, with mouse CMs around the time of birth, we identified distinct a slight increase in Tead1 (TEA domain family member 1) transcriptional profiles of CM subpopulations, and identified (Fig. 8d). Also, in contrast to Ad-GFP-shRNA-Zeb1 treat- multiple master TFs that control numerous cell cycling genes ments (Fig. 6), there was no change in expression of the in CMs. Specifically, detailed analysis of ZEB1, a previously G2/M phase factors Cenpe, Cenpf, Aurkb, and Gmnn besides unknown factor in CM cell cycling, showed that before birth, a minor increase in Aurka (Aurora kinase A) levels (Fig. 8e). ZEB1 works as a key regulator of cell cycle promoting genes 1 3 8 Page 16 of 24 Basic Research in Cardiology (2023) 118:8 and is required for CM proliferation. Yet, after birth ZEB1 and leads to polyploid CMs. In agreement with the literature mediated CM cell cycling occurs through endoreplication [11, 32, 61], the number of dividing CMs is indeed also very 1 3 Basic Research in Cardiology (2023) 118:8 Page 17 of 24 8 ◂Fig. 6 ZEB1 knockdown reduce CM proliferation before birth. a understand the underlying mechanisms of the switch from UMAP plot of E16.5-4n CMs extracted from UMAP plot of all six CM proliferation to polyploidy occurring around the time of conditions (as in Fig. 2a) with blue color indicating CMs express- birth in mammals. Whether Zeb1 re-expression also underly ing Zeb1 mRNA. b Dot plot of Zeb1 expression for all six conditions the cell cycle activity observed in discrete CMs after MI with size indicating percentage of CMs expressing Zeb1 and color indicating the average expressing level in Zeb1-expressing CMs. c remains to be determined but could provide a target to ena- Schematic of the study design and workflow. Embryonic hearts were ble CM proliferation or forced CM polyploidy at this stage to isolated at E16.5 and cells were cultured for 24 h before adenovi- increase CM mass and compensating the CM loss after MI. ral transduction with either Ad-GFP-shRNA (control; scrambled Several scRNA-seq studies on in vivo CMs have shRNA) or Ad-GFP-shRNA-Zeb1. The insert depicts the viral con- struct containing an eGFP reporter. Cells were fixed 96 h after trans- recently provided new knowledge on heart development duction and EdU pulsing, and analyzed by flow cytometry and immu- by identifying genes that are differentially expressed at nocytochemistry (ICC). d ICC of E16.5 primary cardiac cultures different stages of development. Yet, many of the studies transduced with either Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1 are limited in the number of CMs detected [5, 10, 30, 40] after 96 h (GFP , green; DAPI (cell nuclei), blue). e GFP expres- sion quantified by flow cytometry of Ad-GFP-shRNA and Ad-GFP- restricting subsequent detailed analysis such as TF binding shRNA-Zeb1 transduced cells 96 h after transduction (Paired t-test, site enrichment studies. Other studies fail to implement n = 5, NS). f Normalized mRNA level of ZEB1 in Ad-GFP-shRNA data on ploidy and cell cycle status [23, 39]. To our knowl- and Ad-GFP-shRNA-Zeb1 transduced cells (normalized against B2m edge analysis of TFs in CMs based on scRNA-seq has and Gapdh; Paired t-test, n = 3, *P ≤ 0.05). g Flow cytometry dot plot of EdU incorporation in MYH cells transduced with Ad-GFP- been described in only four settings for CM development, shRNA or Ad-GFP-shRNA-Zeb1. h EdU positive CMs transduced but none used ploidy stratification as performed herein with Ad-GFP-shRNA or Ad-GFP-shRNA-Zeb1 quantified by flow [10, 27, 30, 39]. However, in a recent study, Yekelchyk cytometry (Paired t-test, n = 5, **P ≤ 0.01). i CM ploidy of Ad-GFP- et al. showed transcriptional homogeneity by scRNA-seq shRNA and Ad-GFP-shRNA-Zeb1 transduced, EdU positive cells quantified by flow cytometry based on Hoechst intensity (Paired of adult rod-shaped mono- and multi-nucleated ventricular t-test, n = 5, **P ≤ 0.01; ****P ≤ 0.0001). j Normalized mRNA lev- CMs, although, not performing TF analysis [77]. With the els of Ccnd1, Ccne2, Ccng2, Ccnb1, and Ccnd3 (normalized against established protocol, we unravel the uniqueness of in vivo B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05; **P ≤ 0.01). k Nor- cycling CMs in the G2/M phases around the time of termi- malized mRNA levels of Cdk1, Cdk4, Cdkn1a, and Cdkn1b (normal- ized against B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05). l Nor- nal CM differentiation, and besides Zeb1 identified several malized mRNA levels of Cenpe, Cenpf, Aurkb, Gmnn, and Aurka TFs potentially involved in the process of G2/M phases (normalized against B2m and Gapdh; Paired t-test, n = 3, *P ≤ 0.05; completion. Mycn and Myc were shown to enhance not **P ≤ 0.01) only S-phase progression, but also G2/M completion, in agreement with recent data [8, 60]. The more novel play- scarce after birth in our data, and it seems likely that CMs ers in CM cell cycling: Arnt, Zeb1, Sp1, and Egr1 spe- also in the mouse already initiates the final round of cell cifically promoted S-phase progression herein with high cycling before birth at which point ZEB1 seems to impact efficiency, yet all four seemed to leave the postnatal CMs CMs. This would be similar to what is observed in humans, in a polyploid state thus favoring karyo-/cytokinesis fail- where CMs starts the process of terminal differentiation with ure. Whereas Arnt has been associated with hypoxia [74], increased polyploidy already before birth [18]. Thus, it is Egr1 seems to be implicated in several pathologies of the possible that ZEB1 represents a mechanism where CM pro- cardiovascular system [31], and Sp1 is a well-known TF liferation is sustained by ZEB1 but at some point, mitotic in cell growth and peripherally related to CM cell cycling stress is reached forcing the self-limiting DNA damage [21]. Zeb1 is mainly described for its enhancing role in response to initiate terminal differentiation and avoid can - epithelial to mesenchymal transition during cancer and cer. By this, ZEB1 may ensure polyploidy to achieve high embryonic development [81], but has recently been linked production of RNA and proteins required for the high muscle also to Hematopoietic stem cell renewal and asymmetric work of fully differentiated CMs in adulthood, and its timed cell division [1]. Furthermore, ZEB1 has been suggested downregulation eventually caused by a self-limiting DNA to interact directly with the Hippo pathway in cancer response then enables p21 mediated CM differentiation. This cells through YAP [34]. The functional roles of the TFs, could be in line with the emerging theory that polyploidy is however, were only predicted bioinformatically and not an essential biological mechanism for tissue differentiation directly examined besides EdU incorporation and deter- and homeostasis [19]. mination of ploidy. Herein, we found Zeb1 to regulate Thus, our novel approach has allowed us to acquire valu- the highest number of genes related to CM cell cycling able new biological information that may be used further to in our dataset. Knockdown of Zeb1 in E16.5 CMs led to 1 3 8 Page 18 of 24 Basic Research in Cardiology (2023) 118:8 impaired S-phase progression with reduced expression of Cdk1 as well as a decrease in the level of Cenpe, Cenpf, the major cell cycle regulators Ccnd1, Ccnb1, Ccnd3, and Aurkb, and Aurka, which are all genes expressed in the 1 3 Basic Research in Cardiology (2023) 118:8 Page 19 of 24 8 ◂Fig. 7 ZEB1 increases ploidy and decreases cell size in vitro. a upon ZEB1 overexpression. Whether this in turn inhib- Schematic of the study design and workflow. Neonatal hearts were its G1/S phase transition and prevents non-cycling CMs isolated at P0 and cultured for 24 h before transduction with adeno- from further entering the cell cycle remains elusive but virus (Ad). The insert depicts the viral construct, note the eGFP could explain why only a proportion of CMs undergo reporter in both Ads. Cells were fixed after 72 h transduction and EdU subjection and analyzed by flow cytometry or immunocyto- S-phase progression despite expressing ZEB1. Yet, we did chemistry (ICC). b ICC of Ad-GFP transduced primary CMs after observe that all ZEB1 overexpressing CMs reduced their + + 24, 48, and 72 h of transduction (GFP , green; Mef-2c (CM nuclei), size in vitro, which therefore likely represents another red; DAPI (cell nuclei), blue). c Flow cytometric dot plots of GFP − + mechanism. The smaller size of ZEB1 expressing CMs and GFP MYH1 CMs in non-transduced and Ad-GFP transduced CMs. d GFP expression quantified by flow cytometry after 72 h of at early timepoints was accompanied by an increase in culturing (non-transduced) or Ad-GFP transduction (Paired t-test, the major muscle size inhibitors Mstn, Ccng2, and Tead1, n = 8, ****P ≤ 0.0001). e ICC of Ad-GFP (top panel) and Ad-GFP- and downregulation of the Yap targets Axl and Ctgf. All Zeb1 (lower panel) transduced CMs after 24, 48, and 72 h transduc- + + these genes are related to cell size regulation, and recently tion (actinin (CMs), green; ZEB1 , red; DAPI (cell nuclei), blue). f Zeb1 expression in CMs after 72 h Ad-GFP or Ad-GFP-Zeb1 trans- ZEB1 was demonstrated to inhibit skeletal muscle cell size duction as quantified by qRT-PCR (normalized against B2m and in mouse [59]. Thus, the reduced CM size and decrease Rpl_13A; Unpaired t-test, n = 6–7, **P ≤ 0.01). g Flow cytometric in Myh6 fit very well with ZEB1 preventing cell cycle dot plots of EdU incorporation in MYH1 CMs after 72 h Ad-GFP exit, while promoting S-phase progression in the early transduction (top panel) and Ad-GFP-Zeb1 transduction (lower panel) (forward side scatter (FSC)). h Incorporation of EdU was observed phase. At P8 in vivo, ZEB1-mediated endoreplication at both 50, 100, and 150 MOI after 72 h Ad-GFP and Ad-GFP-Zeb1 then results in polyploid CMs of increased size, which transduction (Unpaired t-test, n = 3, *P ≤ 0.05, ***P ≤ 0.001). i Flow agrees with the literature [82]. Interestingly, our studies cytometric contour plots depicting decreased MYH1 CM size after indicate that ZEB1 regulates a distinct set of genes before 72 h Ad-GFP-Zeb1 transduction. j Quantification of the geometric mean of CMs and non-myocytes (NMs) 72 h after Ad-GFP or Ad- and after birth, thereby promoting CM proliferation before GFP-Zeb1 transduction (Two-way ANOVA, n = 9, ****P ≤ 0.0001). birth, while favoring polyploidization when reintroduced k Percentages of CMs of the total cell number after 72 h in culture after birth. To our knowledge this clear molecular switch (non-transduced), or after 72 h Ad-GFP or Ad-GFP-Zeb1 transduc- from proliferation to endoreplication around birth has not tion (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 5) previously been shown for one TF. Thus, the decrease in Zeb1 expression occurring around birth may contribute to cell cycle arrest and terminal CM differentiation, as also G2/M phases of the cell cycle, confirming our bioinfor - observed for other TFs [17]. One example is YAP1, which matic prediction of ZEB1 as a regulator of the cell cycle as part of the Hippo pathway supports CM proliferation in CMs before birth. Ccnb1, Ccnd1, Cdk1, and Cdk4 are during embryonic development in combination with its known regulators of CM cell cycling and overexpression interaction partner TEAD1 [43]. Thus, in the postnatal of these four factors was recently found to promote prolif- period downregulation of YAP1 and TEAD1 is required eration of post-mitotic CMs [46]. Manipulating Zeb1 by for CM cell cycle arrest [22]. Postnatal upregulation of overexpression in postnatal CMs showed that ZEB1 after YAP1 retain CM proliferative capacity after birth causing birth maintains cell cycling in the form of endoreplica- cardiomegaly and heart failure [49]. MYC, which was also tion specifically governed by Cyclin E while likely inhib- detected in our TF analysis, induces CM proliferation dur- iting cell cycle exit through Cdkn1a (p21) regulation. It ing development, while expression of MYC in adult mice is known that p21 binds and inhibits CDK1/Cyclin B1 leads to an increase in polyploid cells [76]. However, in thereby blocking G1/S and G2/M phase transitions, and another study it was suggested that combinational overex- that p21 knockout increases CM ploidy significantly [66], pression of MYC and Cyclin T1 induce CM proliferation while p21 expression forces CM cell cycle exit [65]. In without any notable change in CM size and nucleation agreement, S-phase progression and ploidy was high in [8]. Thus, it is intriguing to speculate, whether overex- ZEB1 expressing CMs. Since, p21 blocks endoreplication pression of Zeb1 in combination with other genes or TFs [66] through co-repressing Cyclin E [24], the major cyc- also after birth could promote CM proliferation rather than lin of endoreplication [82], it is thus intriguing to specu- endoreplication, and by this may be a target for therapeutic late that ZEB1 herein also downregulates p21 and hereby perspectives in MI patients. increases Cyclin E2 expression resulting in endoreplica- In conclusion, we here provide new knowledge on Zeb1’s tion of cycling CMs and polyploidy as observed for a frac- cell cycle promoting actions in CMs as well as a comprehen- tion of CMs both in vitro and in vivo. In this regard, it is sive scRNA-seq of CMs before and after birth, which may important to note, that Cyclin D1 in parallel was decreased 1 3 8 Page 20 of 24 Basic Research in Cardiology (2023) 118:8 1 3 Basic Research in Cardiology (2023) 118:8 Page 21 of 24 8 manuscript editing, and final approval of manuscript. SBM: collection ◂Fig. 8 ZEB1 increase ploidy and cell size in vivo. a Confocal of in vitro and in vivo mouse data, cloning of plasmids, production of images of Ad-GFP-Zeb1 transduced CMs after 72 h transduction virus, manuscript writing, manuscript editing, and final approval of (phalloidin (F-actin) green; ZEB1 , red; DAPI (cell nuclei), blue). manuscript. TC: cloning of plasmids and production of virus, Collec- Binucleated CM (##), tetranucleated CM (###), cytoplasm localized tion of in vitro mouse data, manuscript writing, and final approval of ZEB1 (arrowheads). b Normalized mRNA levels of Ccnd1, Ccne2, manuscript. SF: cloning of plasmids and production of virus, collection Ccng2, Ccnb1, and Ccnd3 (normalized against B2m and Rpl_13A; of in vitro and in vivo mouse data, and final approval of manuscript. Unpaired t-test or Mann–Whitney based on normality, n = 6–7, KSA: collection of confocal data, final approval of manuscript. CDF: **P ≤ 0.01). c Normalized mRNA levels of Cdk1, Cdk4, Cdkn1a, ScRNA-seq method establishment, collection of scRNA-seq data, and Cdkn1b (normalized against B2m and Rpl_13A; Unpaired t-test manuscript writing, and final approval of manuscript. MB: ScRNA- or Mann–Whitney based on normality, n = 6–7, **P ≤ 0.01). d Nor- seq method establishment, scRNA-seq analysis, manuscript writing, malized mRNA levels of Axl, Ctgf, and Tead1 (normalized against and final approval of manuscript. MT: ScRNA-seq method establish- B2m and Rpl_13A; Unpaired t-test or Mann–Whitney based on nor- ment, Sequencing of cDNA libraries, manuscript editing, and Final mality, n = 6–7, *P ≤ 0.05, **P ≤ 0.01). e Normalized mRNA levels approval of manuscript. DCA: conception and design, data collection, of Cenpe, Cenpf, Aurkb, Gmnn, and Aurka (normalized against B2m data analysis and interpretation, manuscript writing and editing, final and Rpl_13A; Unpaired t-test or Mann–Whitney based on normality, approval of manuscript, and financial support. n = 6–7, *P ≤ 0.05). f Normalized mRNA levels of Mstn, and Myh6 (normalized against B2m and Rpl_13A; Unpaired t-test or Mann– Data availability ScRNA-seq data that support the findings of this Whitney based on normality, n = 6–7, **P ≤ 0.01). g Schematic of the study have been deposited in the Gene Expression Omnibus (GEO) study design and workflow. Neonatal pups (P0) were injected with under the accession code GSE162959. All other data supporting the Ad-GFP or Ad-GFP-Zeb1 through the superficial temporal vein, fol- findings of this study are available from the corresponding authors on lowed by two subcutaneous injections of EdU at P4 and P6. Hearts reasonable request. were collected at P8 for immunohistochemistry (IHC) or ventricu- lar dissociation for flow cytometry. h IHC of P8 heart ventricle tis- Code availability All codes central for the conclusions made in this sue from PBS (top panel, PBS injected pups were included in the manuscript are available from the corresponding author on reasonable study with the purpose to make this figure to show the GFP-signal request. in adenovirus-injected pups compared to PBS-injected pups) or Ad- + + GFP-Zeb1 (lower panel) P0 injected pups (GFP , green; EdU , red; DAPI (cell nuclei), blue). i Flow cytometric dot plots depicting the Declarations EdU incorporation in all MYH1 CMs (left panel) as well as in + + MYH1 GFP CMs (right panel) after 72 h Ad-GFP (top panel) or Conflict of interests DCA together with the University of Southern + + Ad-GFP-Zeb1 (lower panel) transduction. j Percentages EdU GFP Denmark and the Region of Southern Denmark have filed a patent CMs of GFP CMs at P8 after Ad-GFP injection at P0 compared to (#PCT/ EP23158550.6.) based on the data generated within this study. Ad-GFP-Zeb1 injection (Paired t-test, n = 7–9, **P ≤ 0.01). k Per- Otherwise, the authors declare no competing financial interests. centage-wise distribution of ploidy at P8 after Ad-GFP or Ad-GFP- Zeb1 injection at P0 (Paired t-test, n = 7–9, *P ≤ 0.05). l Quantifica- Open Access This article is licensed under a Creative Commons Attri- tion of the geometric mean of primary CMs at P8 after Ad-GFP or bution 4.0 International License, which permits use, sharing, adapta- Ad-GFP-Zeb1 transduction (Unpaired t-test, n = 7–9, **P ≤ 0.01) tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes be used to further dissect the switch from a proliferative to were made. The images or other third party material in this article are a terminally differentiated high power beating CM. included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in Supplementary Information The online version contains supplemen- the article's Creative Commons licence and your intended use is not tary material available at https://doi. or g/10. 1007/ s00395- 023- 00979-2 . permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a Acknowledgements We would like to thank Charlotte Nielsen, Tina copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . K. Andersen, and Tonja L. Jørgensen (Andersen group, Odense Uni- versity Hospital) for excellent technical assistance on this study, Lars Vitved (University of Southern Denmark) for assisting with FACS and References Professor Per Svenningsen (University of Southern Denmark) for help with AAV plasmids. The work was supported by research grants from The Novo Nordisk Foundation (#NNF17OC0028764), The Danish 1. Almotiri A, Alzahrani H, Menendez-Gonzalez JB, Abdelfattah A, National Research Council (Sapere Aude; # 8045-00019B), The Lun- Alotaibi B, Saleh L, Greene A, Georgiou M, Gibbs A, Alsayari A, dbeck Foundation (#R313-2019-573), The Program of China Scholar- Taha S, Thomas LA, Shah D, Edkins S, Giles P, Stemmler MP, ship Council (No. 201906320411) and financial support from the Dep. Brabletz S, Brabletz T, Boyd AS, Siebzehnrubl FA, Rodrigues NP of Clinical Biochemistry /Odense University Hospital. (2021) Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia. 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Pediatr 48 Cardiol 40:1345–1358. https://doi. or g/10. 1007/ s00246- 019- 02163-7 82. Zielke N, Edgar BA, DePamphilis ML (2013) Endoreplication. Cold 72. Walsh S, Ponten A, Fleischmann BK, Jovinge S (2010) Cardiomyo- Spring Harb Perspect Biol 5:a012948 doi:https:// doi. org/ 10. 1101/ cyte cell cycle control and growth estimation in vivo–an analysis cshpe rspect. a0129 48 1 3

Journal

Basic Research in Cardiology – Springer Journals

Published: Mar 2, 2023

Keywords: Heart development; Cardiomyocytes; Proliferation; Endoreplication; Zinc Finger E-Box Binding Homeobox 1 (Zeb1)

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Pseudo-bipolar spindle formation and cell division in postnatal binucleated cardiomyocytes

Leone, M; Engel, FB
Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations

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Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development

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Transcriptomic profiling maps anatomically patterned subpopulations among single embryonic cardiac cells

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Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

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Cardiomyocyte proliferation contributes to heart growth in young humans

Mollova, M; Bersell, K; Walsh, S; Savla, J; Das, LT; Park, SY; Silberstein, LE; Dos Remedios, CG; Graham, D; Colan, S; Kuhn, B
Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2

Ohno, S; Ikeda, JI; Naito, Y; Okuzaki, D; Sasakura, T; Fukushima, K; Nishikawa, Y; Ota, K; Kato, Y; Wang, M; Torigata, K; Kasama, T; Uchihashi, T; Miura, D; Yabuta, N; Morii, E; Nojima, H
Maturation of cardiac energy metabolism during perinatal development

Piquereau, J; Ventura-Clapier, R
Understanding cardiomyocyte proliferation: an insight into cell cycle activity

Ponnusamy, M; Li, PF; Wang, K
The biological basis for cardiac repair after myocardial infarction: from inflammation to fibrosis

Prabhu, SD; Frangogiannis, NG
Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution

Prasad, KMR; Xu, Y; Yang, Z; Acton, ST; French, BA
A chemical method for fast and sensitive detection of DNA synthesis in vivo

Salic, A; Mitchison, TJ
The RIN: an RNA integrity number for assigning integrity values to RNA measurements

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Cardiac regeneration based on mechanisms of cardiomyocyte proliferation and differentiation

Senyo, SE; Lee, RT; Kuhn, B
Mammalian heart renewal by pre-existing cardiomyocytes

Senyo, SE; Steinhauser, ML; Pizzimenti, CL; Yang, VK; Cai, L; Wang, M; Wu, TD; Guerquin-Kern, JL; Lechene, CP; Lee, RT
ZEB1 protects skeletal muscle from damage and is required for its regeneration

Siles, L; Ninfali, C; Cortes, M; Darling, DS; Postigo, A
A conserved HH-Gli1-Mycn network regulates heart regeneration from newt to human

Singh, BN; Koyano-Nakagawa, N; Gong, W; Moskowitz, IP; Weaver, CV; Braunlin, E; Das, S; van Berlo, JH; Garry, MG; Garry, DJ
Cardiomyocyte DNA synthesis and binucleation during murine development

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Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

Subramanian, A; Tamayo, P; Mootha, VK; Mukherjee, S; Ebert, BL; Gillette, MA; Paulovich, A; Pomeroy, SL; Golub, TR; Lander, ES; Mesirov, JP
DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages

Takagi, T; Moribe, H; Kondoh, H; Higashi, Y
CDK inhibitors, p21(Cip1) and p27(Kip1), participate in cell cycle exit of mammalian cardiomyocytes

Tane, S; Ikenishi, A; Okayama, H; Iwamoto, N; Nakayama, KI; Takeuchi, T
Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth

Tane, S; Okayama, H; Ikenishi, A; Amemiya, Y; Nakayama, KI; Takeuchi, T
Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma

Tirosh, I; Venteicher, AS; Hebert, C; Escalante, LE; Patel, AP; Yizhak, K; Fisher, JM; Rodman, C; Mount, C; Filbin, MG; Neftel, C; Desai, N; Nyman, J; Izar, B; Luo, CC; Francis, JM; Patel, AA; Onozato, ML; Riggi, N; Livak, KJ; Gennert, D; Satija, R; Nahed, BV; Curry, WT; Martuza, RL; Mylvaganam, R; Iafrate, AJ; Frosch, MP; Golub, TR; Rivera, MN; Getz, G; Rozenblatt-Rosen, O; Cahill, DP; Monje, M; Bernstein, BE; Louis, DN; Regev, A; Suva, ML
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Visualizing Data using t-SNE

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Postnatal cardiac development and regenerative potential in large mammals

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Cardiomyocyte cell cycle control and growth estimation in vivo–an analysis based on cardiomyocyte nuclei

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Cardiomyocyte growth and sarcomerogenesis at the intercalated disc

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Inducible activation of c-Myc in adult myocardium in vivo provokes cardiac myocyte hypertrophy and reactivation of DNA synthesis

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Mono- and multi-nucleated ventricular cardiomyocytes constitute a transcriptionally homogenous cell population

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Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

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Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

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