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During embryonic development, cardiomyocytes undergo differentiation and maturation, processes that are tightly regulated by tissue-specific signaling cascades. Although redox signaling pathways involved in cardiomyogenesis are established, the exact sources responsible for reactive oxygen species (ROS) formation remain elusive. The present study investigates whether ROS produced by the mitochondrial flavoenzyme monoamine oxidase A (MAO-A) play a role in cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs). Wild type (WT) and MAO-A knock out (KO) hiPSCs were generated by CRISPR/Cas9 genome editing and subjected to cardiomyocyte differentiation. Mitochondrial ROS levels were lower in MAO-A KO compared to the WT cells throughout the differentiation process. MAO-A KO hiPSC-derived cardiomyocytes (hiPSC-CMs) displayed sarcomere disarray, reduced α- to β-myosin heavy chain ratio, GATA4 upregula- 2+ tion and lower macroautophagy levels. Functionally, genetic ablation of MAO-A negatively affected intracellular Ca homeostasis in hiPSC-CMs. Mechanistically, MAO-A generated ROS contributed to the activation of AKT signaling that was considerably attenuated in KO cells. In addition, MAO-A ablation caused a reduction in WNT pathway gene expression consistent with its reported stimulation by ROS. As a result of WNT downregulation, expression of MESP1 and NKX2.5 was significantly decreased in MAO-A KO cells. Finally, MAO-A re-expression during differentiation rescued expression 2+ levels of cardiac transcription factors, contractile structure, and intracellular Ca homeostasis. Taken together, these results suggest that MAO-A mediated ROS generation is necessary for the activation of AKT and WNT signaling pathways during cardiac lineage commitment and for the differentiation of fully functional human cardiomyocytes. Keywords Reactive oxygen species · hiPSCs · Cardiomyocyte differentiation · Development · Cell fate AbbreviationsCMs Cardiomyocytes 1P/2P 1 Pulse/2 pulses ERMICC ER–mitochondria contact coefficient AKT AKT serine/threonine kinaseER/SR Endoplasmic/sarcoplasmic reticulum ESCs Embryonic stem cells FCCP Carbonyl cyanide 4-(trifluoromethoxy) * Fabio Di Lisa phenylhydrazone email@example.com GATA4 GATA binding protein 4 * Nina Kaludercic GSK3β Glycogen synthase kinase 3ß firstname.lastname@example.org hiPSCs Human induced pluripotent stem cells Department of Biomedical Sciences, University of Padova, KO/KD Knock out/knock down Via Ugo Bassi 58/B, 35131 Padua, Italy LC3B Microtubule-associated protein 1 light chain Max Planck Institute for Biology of Ageing, 50931 Cologne, 3ß Germany MAO-A/B Monoamine oxidase A/B Neuroscience Institute, National Research Council of Italy MCU Mitochondrial calcium uniporter (CNR), Via Ugo Bassi 58/B, 35131 Padua, Italy MESP1 Mesoderm posterior BHLH transcription Center for Neuroscience and Cell Biology, University factor 1 of Coimbra, 3004–504 Coimbra, Portugal MM Maturation medium Fondazione Istituto di Ricerca Pediatrica Città della Speranza MMTV Integration site family protein (IRP), 35127 Padua, Italy Vol.:(0123456789) 1 3 4 Page 2 of 19 Basic Research in Cardiology (2023) 118:4 MPG N-(2-mercaptopropionyl)-glycine [60, 67]. Both enzymes are expressed in the heart, although MTR Mitotracker Red CM-H XRos MAO-A appears to be the predominant isoform . The MYHC Myosin heavy chain pathological role of MAOs in the heart has been exten- + 2+ NCX1 Na /Ca -exchange protein 1 sively studied in the last decade. Excessive MAOs activa- NKX2.5 NK2 homeobox 5 tion causes oxidative stress-mediated mitochondrial damage p38 Mitogen-activated protein kinase and cardiomyocyte necrosis [1, 16, 30, 33, 74], while lower/ p62 Sequestosome 1 moderate levels of MAO-dependent H O can trigger signal- 2 2 PLN Phospholamban ing cascades regulating cell growth . Previous studies qRT-PCR Quantit ative real time PCR suggested a physiological role for MAO-A in embryonic RCAN1 Regulator of calcineurin 1 brain development . However, the involvement of MAO- ROS Reactive oxygen species dependent ROS as a mechanism underlying developmental RYR2 R yanodine receptor 2 processes has never been investigated. SERCA2A Sarco-endoplasmic reticulum calcium Here, we tested the hypothesis that ROS deriving from ATPase 2A MAOs directly participate in the regulation of human car- TEM Transmission electron microscopy diac specification by activating ROS-dependent cardiomy - TMRM Tetramethylrhodamine ogenic pathways. Specifically, we investigated whether (i) WNT Wingless-related integration site MAO-A is involved in mitochondrial ROS formation during Wnt-C59 Porcupine (PORCN) inhibitor human cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs); (ii) MAO-A-dependent ROS generation can trigger the activation of ROS-sensitive sign- Introduction aling pathways that control fate commitment; (iii) MAO-A ablation affects cardiomyogenesis. Lineage specification is driven by a tight regulation of organ- and tissue-specific signaling cascades . Among many factors, intracellular reactive oxygen species (ROS) play Methods a crucial role during organogenesis, integrating cell redox state with differentiation [39, 71]. The nature of intracellular A detailed, expanded Methods section is available in the ROS and their spatiotemporal modulation is finely tuned Online Supplement. and can regulate stem cell self-renewal, pluripotency, and differentiation [2 , 45, 54]. Notably, H O is directly involved hiPSCs culture and treatments 2 2 in the physiological regulation of different signal transduc- tion pathways by inducing post-translational modifications hiPSCs (SCVI15) were kindly provided by Professor Joseph . Mitochondrial ROS play a pivotal role during cardiac C. Wu (Stanford Cardiovascular Institute, USA). ATCC- lineage commitment [11, 12], directly communicating with BYS0112 hiPSCs were purchased by ATCC (ATCC, ACS- the cytosol, initiating and amplifying ROS-dependent sign- 1026). hiPSCs line UST000013 was purchased from uSTEM aling pathways that determine cell fate [21, 50, 76, 79]. Yet, s.r.l. Cells were maintained in Essential 8 Flex Medium mitochondrial enzymes responsible for ROS formation con- (Thermo Fisher Scientific, USA) on Geltrex Matrix (Thermo tributing to cardiomyogenesis remain unknown. Fisher Scientific) and passaged every 4 days using 0.5 mM Mitochondrial electron transport chain is considered one ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher 2+ 2+ of the main sites for mitochondrial ROS production . Scientific) in Dulbecco’s PBS without Ca or Mg (Life However, other enzymes generate ROS within mitochon- Technologies). Cells were expanded in 6-well plates by pas- dria . In particular, monoamine oxidases (MAOs) are saging 1:8. Where indicated, MAO-A knock out (KO) cells an important source of mitochondrial ROS in the heart and were treated at day 4 of cardiac differentiation with 1 µM other tissues [1, 16, 25, 30, 32]. MAOs are a class of flavoen- H O for 2 h. To inhibit AKT activity, hiPSCs were incu- 2 2 zymes that reside in the outer mitochondrial membrane and bated with 5 µM MK-2206 (SYNkinase) for 4 days during exist in two isoforms, MAO-A and B [4, 68], distinguished differentiation. by different substrate specificity and inhibitor sensitivity. MAOs catalyze the degradation of endogenous monoamine Differentiation of hiPSCs into cardiomyocytes neurotransmitters and biogenic amines relevant for cardiac function . Importantly, the oxidative breakdown of sub- hiPSCs were differentiated into cardiomyocytes using the strates generates H O , thus representing a significant source STEMdiff™ cardiomyocyte differentiation kit (Stemcell 2 2 of mitochondrial ROS . The relative expression level Technologies, Canada) according to the manufacturer’s of the two isoforms significantly differs in human tissues instructions. On day 14, beating cardiomyocytes were 1 3 Basic Research in Cardiology (2023) 118:4 Page 3 of 19 4 selected as described before . hiPSCs-derived cardio- fluorescence intensity ratio F /F , in which λ (~ 410 nm) λ1 λ2 1 myocytes (hiPSC-CMs) were used for experiments 4 days and λ (~ 480 nm), respectively. after applying the selection protocol. The maturation of hiPSC-CMs was achieved by treating cells with maturation Mitophagy detection analysis medium (MM) for 3 days as described before . To monitor mitophagy levels in hiPSC-CMs, cells were plated on µ-Plate 96-well black plates (Ibidi) and the Design and construction of CRISPRs targeting mitophagy detection Kit (Dojindo) was used. Briefly, cells human MAOA and len ‑ tiviral production were washed twice with PBS and incubated with 100 nmol/l Mtphagy Dye working solution at 37 °C for 30 min. Sub- Lentiviral CRISPR/Cas9 expression constructs contain- sequently, the supernatant was discarded and wells washed ing the guides for human MAO-A were generated using twice with PBS. To induce mitophagy, positive control the lentiCRISPRv2 one vector system [59, 63]. Briefly, groups were incubated with FCCP (Sigma-Aldrich) 10 µM oligos were synthetized using Cas9 target design tool at 37 °C for 120 min. At the end of the induction time, (ThermoFisher Scientific). Tested oligos are listed in medium was removed and cells washed twice with PBS. To Supplementary Methods. Lentiviral particles have been observe the co-localization of Mtphagy Dye and lysosome, produced by transfecting HEK293T cells with four differ - cardiomyocytes were incubated at 37 °C for 30 min with ent plasmids . To determine the efficacy of gene KO 1 μmol/l Lyso Dye working solution. Images were collected by lentiCRISPR transduction, single guide RNAs (sgR- in live imaging solution (Thermo) using Crest X-light V3 NAs) targeting MAO-A locus were tested in HEK293T confocal system and quantification of co-localization was cells (Supplementary Methods). EGFP sgRNA was used as determined using ImageJ FIJI. Three independent experi- control (Addgene, cat# 51,760) . MAO-A and MAO-B ments were performed and at least ten fields of view per expression levels were evaluated by western blot analysis experiment were quantified. after puromycin selection. Wells showing undetectable levels of MAO-A protein were chosen for knock-out cell Citrate synthase activity assay line expansion and experiments. The citrate synthase activity in hiPSC-CMs was measured using the citrate synthase activity assay kit (Sigma-Aldrich) Live cell imaging according to the manufacturer's instructions. Briefly, sam- ples were diluted with citrate synthase assay buffer. After hiPSC-CMs were differentiated on µ-Plate 96-well black the dilution step, samples, positive controls and reduced plates (Ibidi), experiments were carried out in an extra- glutathione (GSH) standard solutions were loaded into a cellular medium, images were acquired and quantified as 96-well plate. To start the reaction, 50 µL of mix containing previously described [1, 61]. citrate synthase developer and substrate were added to each To monitor mitochondrial ROS and mitochondrial well. Absorbance at 412 nm was measured every 5 min for membrane potential, cells were incubated with 25 nM 45 min at 25 °C. After background subtraction, citrate syn- MitoTracker Red CM-H XRos, λ 580 nm and λ thase activity was calculated according to the GSH amount 2 exc em 600 nm (MTR, Thermo Fisher Scientific), or 25 nM tetra- (calculated using a standard curve), the reaction time and methylrhodamine, λ 550 nm and λ 580 nm (TMRM, normalized to the total amount of protein loaded. exc em Thermo Fisher Scientific), respectively. Images were col- lected at four different time points. Amplex red assay 2+ To monitor cytosolic Ca transients, cells were loaded with Fluo-4 acetoxymethyl ester, λ 490 nm and λ Hydrogen peroxide formation was determined using Amplex exc em 515 nm (Thermo Fisher Scientific), 0.01% w/v pluronic Red assay as previously described . Briefly, Amplex F-127 (Sigma), and 250 µM sulfinpyrazone (Sigma). To Red is a substrate of horseradish peroxidase (HRP), which 2+ evaluate the release of Ca from the sarcoplasmic reticu- in presence of H O oxidizes 10-acetyl-3,7-dihyrdoxy- 2 2 lum (SR), a pulse of 10 mM caffeine was added to the phenoxazine (Amplex Red), resulting in the production cells. of a red fluorescent compound resorufin (excitation/emis - 2+ To monitor mitochondrial Ca , hiPSC-CMs were sion: 560/590 nm). hiPSCs were differentiated in 12-well infected with an adenovirus containing mito-GCaMP5G plates and cells at D0, D2, and D4 were dissociated using  and 48 h later imaged both at baseline and follow- StemPro™ Accutase™ Cell Dissociation Reagent (Thermo ing a pulse of 10 mM caffeine. Results are expressed as Fisher Scientific) for 5 min at room temperature. hiPSC- CMs (D20) were dissociated using Trypsin–EDTA 0.25% 1 3 4 Page 4 of 19 Basic Research in Cardiology (2023) 118:4 1 3 Basic Research in Cardiology (2023) 118:4 Page 5 of 19 4 ◂Fig. 1 MAO-A and -B levels during cardiomyocyte differentiation and phalloidin TRITC conjugated (Sigma-Aldrich, 1:500) 2+ and effect of MAO-A deletion on hiPSC-CMs structure and Ca for actin staining. DAPI was used to stain nuclei (Invitro- homeostasis. A MAO-A and MAO-B protein expression during dif- gen). Images were collected using Zeiss LSM 700 confocal ferent stages of cardiomyocyte differentiation. Densitometry analy - system equipped with a PlanApo 40x/1.2 oil objective at sis is shown on the right. MAO-A expression at day 0 (D0) in WT cells was arbitrarily considered as a unit. Values were normalized 2048 × 2048 pixels per image with a 100 Hz acquisition rate, to GAPDH. *p < 0.05 vs D0, **p < 0.005 vs D0, #p < 0.05 vs D2 by and analyzed as previously described . one-way ANOVA, Dunn’s post hoc pairwise comparison. B MAO-A and MAO-B protein abundance during cardiomyocyte matura- Western blot analysis tion. Densitometry analyses are shown in the lower panel. MAO-A or MAO-B expression at day 20 (D20) was arbitrarily considered as a unit. Values were normalized to GAPDH. *p < 0.05, **p < 0.01 Cells were homogenized and protein concentration was by two-tailed Student’s t test. C Sarcomere organization in WT and determined using BCA protein assay (Pierce). Proteins were MAO-A KO hiPSC-CMs assessed by immunofluorescent labeling separated using SDS–PAGE (Invitrogen) and transferred to of α-sarcomeric actinin (green). Phalloidin is shown in red, while nuclei were stained with DAPI (blue). The patterning of α-sarcomeric nitrocellulose membrane (Bio-Rad). Following incubation actinin fluorescence intensity is plotted on the right, denoting sar - with primary and secondary HRP-conjugated antibodies comere organization within the cell. Approximately 20/30 cells were (Bio-Rad), bands were detected and analyzed as previously analyzed in each experiment. Scale bar 10 µm. D Representative 2+ described [1, 61]. Antibodies used in this study are listed in traces of spontaneous cytosolic Ca oscillations in WT and MAO-A 2+ KO hiPSC-CMs (left panel) and of cytosolic Ca peak induced by Supplementary Methods. caffeine stimulation (right panel). E Quantification of spontaneous 2+ cytosolic Ca oscillations frequency and peak amplitude. *p < 0.05, Quantitative real time PCR analysis (qRT‑PCR) **p < 0.001 by two-tailed Student’s t test. Five regions of interest (ROIs) were selected in each field of view and at least three differ - ent fields of view were analyzed in each experiment. F Quantifica- Total RNA was extracted using TRIzol (Invitrogen), and 2+ tion of cytosolic Ca peak induced by caffeine stimulation in terms reverse transcription was performed using reverse Super- of peak amplitude, influx and efflux rate. *p < 0.05, **p < 0.001 by Script IV (Thermo Fisher Scientific). qRT-PCR was per - two-tailed Student’s t test. Kruskal–Wallis test was applied to not formed using Power SYBR Green PCR Master Mix (Applied normally distributed data. All experiments were performed at least three times using three different preparations. Results are expressed Biosystems). Relative amounts of analyzed genes were cal- as mean ± S.E.M culated by the comparative ∆∆C(t) method. Primers used in this study are listed in Supplementary Methods. (Thermo Fisher Scientific) for 8 min at 37 °C. Cells were permeabilized with 50 µM digitonin (Sigma-Aldrich) in Data analysis PBS for 3–4 min at 37 °C. Permeabilized cells were then incubated in PBS in the presence of 5 µM Amplex Red rea- All values are expressed as mean ± S.E.M. Comparisons gent and 4 µg/ml HRP, and dispensed into a black 96-well between groups were performed by either one-way or two- plate at different densities, ranging from of 2 × 10 cells/well way ANOVA, followed by either Tukey’s or Dunn’s post hoc (D20) to 5 × 10 cells/well (D0). The reaction was started pairwise comparison when data were normally distributed. adding 50 µM tyramine, and the resorufin fluorescence was Data that did not follow normal distribution were analyzed monitored for 1 h at 37 °C using the Infinite 200 microplate by Kruskal–Wallis test, followed by Bonferroni post hoc plate reader (Tecan). Results are shown as pmol H O /min/ multiple comparison. Comparisons between two groups 2 2 million of cells. Since the production of H O can derive were performed using two-tailed Student’s t-test. A value 2 2 from other cellular sources different than MAO-A, resoru- of p < 0.05 was considered significant. fin fluorescence was monitored in cells treated with 50 µM tyramine and 100 µM MAO inhibitor pargyline; the relative production of H O was used as background and subtracted Results 2 2 from the one induced by tyramine alone. MAOA is the only isof ‑ orm expressed in hiPSCs Immunocytochemistry and during main stages of cardiac differentiation Cells were prepared for staining using Cardiomyocyte To investigate MAOs expression profile during human car - Immunocytochemistry Kit (Thermo Fisher Scientific) diomyogenesis, hiPSCs were differentiated into cardiomyo- following manufacturer’s instructions. Sarcomeres were cytes in vitro. During this process, MAO-A was the only iso- stained using anti-α-sarcomeric actinin antibody (Sigma; form expressed and its expression increased during the first 1:500, mouse) overnight at 4 °C. The day after, samples 20 days of cardiomyogenesis (Fig. 1A, Suppl. Figure 1A-B). were incubated for 1 h at room temperature with Alexa The expression level of MAO-A was 5 times higher at the Fluor 488 conjugated anti-mouse (Life Technologies, 1:250) stage of mesoderm-cardiac specification (day 4) compared 1 3 4 Page 6 of 19 Basic Research in Cardiology (2023) 118:4 1 3 Basic Research in Cardiology (2023) 118:4 Page 7 of 19 4 ◂Fig. 2 Effect of MAO-A deletion on cardiomyocyte-specific protein a stable isogenic MAO-A KO hiPSCs line by means of expression and autophagy/mitophagy in hiPSC-CMs. A MYHC6, CRISPR/Cas9-mediated genome editing (Suppl. Figure 1C, MYHC7 and GATA4 protein levels in WT and MAO-A KO hiPSC- D) . MAO-A ablation did not impinge on the pluripo- CMs. Densitometry analyses are shown on the right. Values were tency potential, as revealed by the mRNA expression level of normalized to GAPDH. Protein levels in WT cells were arbitrar- ily considered as a unit. *p < 0.01 by two-tailed Student’s t test. B the pluripotency marker NANOG (Suppl. Figure 1E) and by Representative western blots (left panel) and densitometry analyses immunostaining for NANOG and SSEA4 proteins (Suppl. (right panel) of p62, LC3B in WT and MAO-A KO hiPSC-CMs at Figure 1F). In both isogenic control and MAO-A KO cells baseline or after treatment with inhibitors of lysosomal degradation the first spontaneously contracting cardiomyocyte clusters NH Cl and leupeptin (N/L). Values were normalized to actin. LC3-II abundance in WT N/L was arbitrarily considered as a unit. p62 abun- were observed in approximately 8–10 days. Noteworthy, the dance in WT-vehicle was arbitrarily considered as a unit. *p < 0.05, loss of the MAO-A isoform in hiPSCs and hiPSC-CMs was **p < 0.001 by two-way ANOVA with post hoc Tukey’s multiple not accompanied by a compensatory expression of MAO-B comparison test. C Representative images of Mtphagy Dye and Lyso (Suppl. Figure 1C, D). Dye in hiPSC-CMs WT (upper panel) and MAO-A KO hiPSC-CMs (lower panel) stained cells in the absence or in the presence of FCCP; WT hiPSC-CMs displayed a fully organized sarcomere examples of co-localization regions are indicated by arrowheads. D structure after 20 days of differentiation, with a regu- Fluorescence intensity quantification in WT and MAO-A KO hiPSC- lar α-sarcomeric actinin striation pattern that was lost in CMs at the basal level or with the induction of mitophagy by FCCP. MAO-A KO cells (Fig. 1C). To test if this structural altera- Scale bar 10 µm. ***p < 0.001 by Kruskal–Wallis with post hoc 2+ Tukey’s multiple comparison test. All experiments were performed tion was paralleled by an impairment in Ca homestasis, 2+ at least three times using three different preparations. Results are spontaneous cytosolic Ca oscillations of WT and MAO-A expressed as mean ± S.E.M KO hiPSC-CMs were monitored. The ablation of MAO-A 2+ led to a significant increase in Ca oscillations frequency to undifferentiated cells (Fig. 1A). Importantly, the increase and a reduction in peak amplitude (Fig. 1D-E). In addition, 2+ 2+ in MAO-A protein expression occurred independently from the cytosolic Ca peak induced by the release of Ca from changes in mitochondrial mass (Suppl. Figure 1A). To rule the sarcoplasmic reticulum (SR) following caffeine appli- out the possibility that these results were related to one spe- cation, was reduced in mutant cardiomyocytes in terms of cific genetic background, we assessed MAO-A expression peak amplitude, influx and efflux rate (Fig. 1D and F). On levels in two additional hiPSCs lines obtained from healthy the other hand, there were no significant differences in the 2+ donors and subjected to differentiation. In accordance mitochondrial Ca content between WT and MAO-A KO with the findings shown in Fig. 1A, the expression level of hiPSC-CMs, either at baseline or after caffeine stimulation MAO-A protein increased during cardiomyocyte differentia- (Suppl. Figure 1G). Taken together, these data demonstrate tion in a similar manner (Suppl. Figure 1B). On the contrary, that MAO-A ablation induces structural derangements and 2+ MAO-B protein was undetectable during the entire pro- significant alterations in intracellular Ca homeostasis in cess and became expressed after 20 days of culture, with a hiPSC-CMs. delayed but remarkable increase during the latter stages (day Deterioration of cardiac performance during cardiac 40, Fig. 1A). Interestingly, MAO-A expression peaked at the remodeling arises from adaptive cardiomyocyte modifica- mesoderm-cardiac specification and subsequently decreased tions, characterized by changes in gene expression , over time by 40 days (Fig. 1B). Furthermore, when hiPSC- energy metabolism , sarcomeric protein composition CMs were exposed to the MM to improve their metabolic , and autophagic response . Several pathologic maturation, MAO-B level significantly increased, whereas stimuli can cause a shift in the myosin heavy chain (MYHC) no major changes in MAO-A were detected (Fig. 1B). composition (i.e., MYHC6/MYHC7 ratio) [36, 48]. We thus These data suggest that MAO-A could be important dur- explored the possibility that levels of these proteins could ing the early stages of cardiac commitment in developing be altered in cells lacking MAO-A. MYHC6 levels were human myocardium. Moreover, hiPSC-CMs represent a significantly reduced in MAO-A KO hiPSC-CMs, while no suitable in vitro model for the study of MAOs role during substantial differences were observed for MYHC7 (Fig. 2A). human cardiomyogenesis, since they express comparable In addition, a marked increase in GATA4 was observed in levels of MAO isoforms found in the human heart . MAO-A KO hiPSC-CMs when compared to their isogenic control (Fig. 2A), a condition that resembles data obtained MAOA abla ‑ tion negatively affects hiPSC‑CMs in dysfunctional heart . On the other hand, proteins 2+ 2+ sarcomere structure and intracellular Ca involved in the regulation of Ca homeostasis were unal- homeostasis leading to adaptive cardiomyocyte tered at the transcriptional level between WT and MAO-A changes KO hiPSC-CMs (Suppl. Figure 2A). In addition, the phos- phorylation status of phospholamban (PLN) appeared To determine the role of MAO-A during cardiac specifi - unchanged (Suppl. Figure 2B), indicating that in MAO- cation, a loss-of-function approach was used to generate KO hiPSC-CMs the regulation of SERCA2A by PLN is 1 3 4 Page 8 of 19 Basic Research in Cardiology (2023) 118:4 1 3 Basic Research in Cardiology (2023) 118:4 Page 9 of 19 4 ◂Fig. 3 Effect of MAO-A deletion on mitochondrial ROS formation compared to their isogenic control (Fig. 3A) whereas during cardiomyocyte differentiation. A Representative images of mitochondrial membrane potential remained unaltered MitoTracker Red CM-H XRos (MTR) stained cells (left panel) and (Fig. 3B), suggesting that MAO-A is prominently involved fluorescence intensity quantification (right panel) in WT and MAO-A in mitochondrial ROS generation throughout the differ - KO cells throughout different stages of differentiation. Values were normalized and expressed as % vs WT D0. *p < 0.05, by two-way entiation process. To better correlate MAO-A expression ANOVA with post hoc Tukey’s multiple comparison test. At least 100 and enzyme activity during differentiation, we measured cells were analyzed per condition in each experiment. Scale bar 5 µm. MAO-A dependent ROS formation during different stages of B Mitochondrial membrane potential in WT and MAO-A KO cells at differentiation in the presence of exogenously added MAO day 0 (D0), day 2 (D2), day 4 (D4) and day 20 (D20). Quantification of TMRM fluorescence intensity evaluated before and after the addi- substrate tyramine. Results shown in Fig. 3C show that there tion of FCCP is shown on the right. Results are expressed as F/F , FCCP is a progressive increase in ROS formation over time in WT normalized to WT D0 and statistically analyzed by two-way ANOVA. cells that reaches a peak at days 4 and 20, in accordance with At least 30 cells were analyzed per condition in each experiment. the increase in MAO-A protein expression over time. This Scale bar 5 µm. C MAO activity was measured fluorometrically in permeabilized cells at different time points during cardiac commit- indicates that the potential for MAO-dependent ROS for- ment (D0, D2, D4 and D20). Amplex Red fluorescence was measured mation increases during cardiomyocyte differentiation and kinetically after the administration of the MAOs substrate tyramine. depends on the substrate availability. Moreover, consider- Data were compared to MAO activity at D0 and statistically analyzed ing that MAO-B genetic locus is still functional in MAO-A by Kruskal–Wallis with post hoc Bonferroni test. All experiments were performed at least three times using three different preparations. deleted cells, this result suggests that there is no compensa- Results are expressed as mean ± S.E.M tory effect elicited by MAO-B activity. To test for potential consequences of indiscriminate ROS preserved. To assess whether the genetic ablation of an outer scavenging on human cardiomyocyte differentiation, hiPSCs mitochondrial membrane protein (i.e., MAO-A) could have were differentiated in the presence of either MAO-A inhibi - potentially altered the interaction between mitochondria and tor pargyline, a general ROS scavenger N-2-mercaptoprop- endoplasmic/sarcoplasmic reticulum (ER/SR), we exam- ionylglycine (MPG), or a specific mitochondrial ROS buffer ined subcellular structures in MAO-A KO cells by means (mitoTEMPO). Either mitochondrial or total ROS scaveng- of transmission electron microscopy (TEM). The distance ing severely impaired cardiac differentiation, with null or between ER and mitochondria the ER–mitochondria con- few spare contracting foci observed in treated wells (Suppl. tact coefficient (ERMICC) appeared unchanged in WT and Figure 3A) . In line with our previous results (Fig. 1 MAO-A KO hiPSCs or hiPSC-CMs (Suppl. Figure 2C–D). C-F), treatment with MAO inhibitor pargyline did not block Alterations in autophagy levels in the heart frequently the formation of beating foci (Suppl. Figure 3A) and it par- occur in response to stress . Here we tested whether tially reduced NKX2.5 gene expression (Suppl. Figure 3B). structural and functional alterations observed in MAO-A This supports the evidence that, although impaired, differen- KO hiPSC-CMs could be accompanied by an alteration tiation of beating cardiomyocytes is still able to occur when of autophagy flux. Interestingly, macroautophagy levels MAO-A is downregulated/inhibited. In contrast, mitochon- were reduced in MAO-A KO hiPSC-CMs, as evidenced by drial ROS scavenging with mitoTEMPO was sufficient to reduced accumulation of LC3B-II and p62 in the presence of impair cardiac differentiation, as demonstrated by the drastic inhibitors of lysosomal degradation (i.e., leupeptin/NH Cl) reduction of NKX2.5 gene expression, a marker of cardiac (Fig. 2B). However, mitophagy levels remained unchanged commitment (Suppl. Figure 3B). Altogether, these results in MAO-A-KO hiPSC-CMs either at basal level or after the confirm the pivotal role of ROS during cardiac differentia- induction of mitophagy (Fig. 2. C–D). In line with this evi- tion process and highlight the role of MAO-A generated dence, mitochondrial content in cardiomyocytes was equally ROS in cardiac lineage commitment. balanced in both cell types (Suppl. Figure 2E) confirming that the mitochondrial mass was not affected. MAOA dependen ‑ t ROS formation modulates AKT and WNT signaling pathways MAO‑dependent ROS generation contributes substantially to mitochondrial ROS levels Next, we tested whether MAO-A ablation impairs differen- during cardiac commitment tiation by altering ROS-dependent signaling pathways. AKT and p38 MAPKs, master regulators of cardiac lineage com- A constant rise in mitochondrial H O formation has been mitment, can be modulated by ROS [3, 35, 51, 55, 78]. Thus, 2 2 observed during cardiac commitment, from hiPSCs to we sought to determine whether differences in AKT and p38 hiPSC-CMs . We examined to what extent MAO-A phosphorylation/activation between WT and MAO-A KO generated H O was contributing to mitochondrial ROS cells were detectable during mesoderm/cardiac specifica- 2 2 levels during this process. MAO-A KO hiPSCs displayed tion (from days 1 to 4) and during cardiac differentiation remarkably lower levels of mitochondrial ROS in all stages (from days 6–20). AKT phosphorylation showed a biphasic 1 3 4 Page 10 of 19 Basic Research in Cardiology (2023) 118:4 response during the initial stages of cardiac specification cholinesterase . In addition, MAO inhibition has been and was significantly reduced in MAO-A KO vs WT cells proposed to control the proliferative potential of lymphoma at day 4 (Fig. 4A). A reduction in phosphorylation levels cells . However, no information is available on MAO- of GSK3β at serine 9 (S9) was also observed in MAO-A induced ROS formation and WNT signaling in the context of KO cells (Fig. 4A), indicating an overall decrease of the cardiomyocyte lineage commitment. Expression level of key AKT signaling cascade. In addition, a reduction in AKT genes belonging to the WNT family was unchanged between and GSK3β phosphorylation persisted during formation of WT and KO cells at day 4 (Suppl. Figure 3E). Nevertheless, cardiac precursors (day 6) and in beating cardiomyocytes a drastic downregulation in WNT3A gene expression was (day 20, Fig. 4B). Conversely, no significant alterations observed in KO cells at day 6, with concomitant reduction in p38 MAPK phosphorylation were detected in MAO-A in WNT3 and WNT11 gene expression levels (Fig. 4D). Of KO hiPSC-CMs (Fig. 4A–B). Of note, we found that phos- note, mitochondrial ROS levels were reduced in MAO-A KO phatase INPP4A was significantly upregulated in MAO-A cells also at day 6, while mitochondrial membrane potential KO hiPSC-CMs (Suppl. Figure 3C). INPP4A is a lipid remained unaffected (Suppl. Figure 3F-G). phosphatase that dephosphorylates PtdIns(3,4)P to form To rule out a possible involvement of AKT in WNT gene PtdIns(4)P and PtdIns(3)P, acting as a negative regulator expression regulation during cardiac differentiation of hiP - of the PI3K/AKT pathway. Recently, we showed that MAO SCs, we used a highly selective AKT inhibitor MK-2206 activity is able to regulate miR-27a-3p levels that, in turn, (Suppl. Figure 3H) during the first 4 days of differentiation binds Inpp4a mRNA and modulates its levels . Taken and measured the expression level of WNT3, WNT3A and together, these findings suggest that the activity of INPP4A NKX2.5 genes at day 6. Pharmacological AKT inhibition is likely increased in MAO-A KO cells, thereby resulting in reduced NKX2.5 expression level thereby confirming its impaired signal transduction and AKT activation. role in cardiomyogenesis , but it did not affect WNT3 or To further corroborate the significance of these findings, WNT3A expression (Suppl. Figure 3I), suggesting that tran- we tested whether dysregulation of the AKT/GSK3β path- scription of these genes is not directly controlled by AKT way in MAO-A KO cells could alter the expression of genes activity. related to myocardial commitment such as MESP1, the pri- Taken together, our data show that persistent alteration mary cardiac mesoderm regulator, GATA4 and NKX2.5, two of the mitochondrial redox balance in MAO-A KO cells key factors required for cardiac specification. MAO-A KO negatively affects AKT/GSK3β pathway at day 4 and WNT cells displayed a significant down regulation of both MESP1 axis later on. Impaired activity of these signaling cascades and NKX2.5 genes at day 6, but no statistically significant in MAO-A KO cells causes a reduction in the expression of changes in GATA4 levels have been detected (Fig. 4C). cardiac transcription factors that impinges on cardiomyocyte Activation of the AKT cascade alone is necessary but not commitment. sufficient to support cardiac differentiation. Indeed, this pro- cess involves also WNT pathway that with AKT signaling Re‑expression of MAOA during lineage ‑ converges in causing GSK3β inactivation . Temporally commitment restores AKT/GSK3β and WNT controlled canonical/non-canonical WNT axes are strongly pathways signaling and partially rescues phenotype alterations implicated in the specification of mesoderm cells toward car - diac progenitors and subsequent cardiomyocyte formation [37, 42]. Notably, ROS have been reported to stimulate the To further corroborate the hypothesis that cells are particu- WNT pathway through the dissociation of disheveled from larly sensitive to ROS oscillations at day 4 of differentia- oxidized nucleoredoxin . Interestingly, ROS appear to be tion, we exposed MAO-A KO cells to a pulse of H O at 2 2 linked to WNT in an amplification pathway whereby stimu- that time point. A significant increase in AKT and GSK3β lation of NOX1 activity downstream of the WNT receptor phosphorylation was observed after 2 h (Fig. 5A). In addi- causes an increase in ROS levels that eventually promote tion, this single pulse of H O led to an increase in NKX2.5, 2 2 WNT-related gene expression [18, 29]. In our experimental WNT3 and WNT3A gene expression at day 6 (Fig. 5B). model, pharmacological inhibition of NOX activity with Conversely, treatment of WT cells with mitoTEMPO for apocynin or treatment of the cells with Wnt-C59 (a potent 4 days drastically reduced the expression level of WNT3A WNT signaling pathway inhibitor) during cardiac differen- at day 6 (Suppl. Figure 3 J). This WNT3A reduction upon tiation drastically reduced the percentage of beating foci mitoTEMPO treatment is in line with the results obtained (Suppl. Figure 3D). On the other hand, mitochondrial ROS for NKX2.5 gene expression (Suppl. Figure 3B), confirm- formation has rarely been considered [57, 73]. A correla- ing that buffering mitochondrial ROS impinges on cardiac tion between MAO inhibition and WNT signaling regulation differentiation. was explored in the treatment of Alzheimer’s disease with Finally, we hypothesized that reactivation of MAO-A a new hybrid compound able to inhibit both MAO and at day 4 could restore the activity of AKT and WNT 1 3 Basic Research in Cardiology (2023) 118:4 Page 11 of 19 4 Fig. 4 Effect of MAO-A deletion on AKT and WNT signaling path- protein levels. *p < 0.05, **p < 0.01 by two-tailed Student’s t test. C ways during cardiomyocyte differentiation. A AKT, GSK3β and p38 MESP1, NKX2.5, and GATA4 mRNA expression levels in WT and phosphorylation was assessed at day 0 (D0), day 2 (D2) and day 4 MAO-A KO cells assessed at day 6 (D6). For each gene, WT val- (D4) during mesoderm/cardiac specification in WT and MAO-A ues were arbitrarily considered as a unit. Values were normalized to KO cells. Densitometry analyses are shown on the right. For each GAPDH. *p < 0.01 by two-tailed Student’s t test. D WNT3, WNT3A group, WT values were arbitrarily considered as a unit. Phospho- and WNT11 mRNA expression levels in WT and MAO-A KO cells rylation levels were normalized to total protein levels. *p < 0.001 by assessed at day 6 (D6). For each gene, WT values were arbitrarily two-tailed Student’s t test. B AKT, GSK3β and p38 phosphorylation considered as a unit. Values were normalized to GAPDH. *p < 0.05, was assessed in WT and MAO-A KO cells at day 6 (D6) and day 20 **p < 0.01, ***p < 0.001 by two-tailed Student’s t test. All experi- (D20) during cardiomyocyte differentiation. Densitometry analyses ments were performed at least three times using three different prepa- are shown on the right. For each group, WT values were arbitrarily rations. Results are expressed as mean ± S.E.M considered as a unit. Phosphorylation levels were normalized to total 1 3 4 Page 12 of 19 Basic Research in Cardiology (2023) 118:4 1 3 Basic Research in Cardiology (2023) 118:4 Page 13 of 19 4 ◂Fig. 5 Effect of H O bolus treatment and MAO-A re-expression the restoration of mitochondrial ROS levels at day 4 and 2 2 on AKT and WNT pathways during cardiac differentiation. A AKT WNT3A and NKX2.5 expression at day 6 in 1P- compared and GSK3β phosphorylation following a bolus addition of H O to 2 2 to 2P-treated cells (Fig. 5D–E). MAO-A KO cells at day 4 (D4) of cardiac differentiation. Densi- In line with results obtained in MAO-A KO cells, the tometry analyses are shown on the right. AKT and GSK3β phospho- rylation levels in MAO-A KO untreated (UNT) cells were arbitrarily majority of cardiomyocytes derived from hiPSCs subjected considered as a unit. Phosphorylation levels were normalized to total to the 2P protocol exhibited myofilament disarray that was protein levels. *p < 0.01 by two-tailed Student’s t test. B NKX2.5, not detected in cells treated with 1P protocol (Fig. 6A). WNT3 and WNT3A mRNA expression level in MAO-A KO cells at Moreover, 2P MAO-A KD cells displayed a significant day 6 following H O bolus treatment. For each gene, MAO-A KO 2 2 2+ untreated (UNT) values were arbitrarily considered as a unit. Val- increase in the frequency of spontaneous Ca oscillations ues were normalized to GAPDH. *p < 0.05 by two-tailed Student’s t in the cytosol and a significant decrease in the peak ampli- test. C MAO-A protein levels and AKT/GSK3β phosphorylation fol- tude (Fig. 6B–C). The 2P protocol induced an impairment in lowing treatment with scramble (SC) RNA, 1 pulse (1P) or 2 pulses response to caffeine in KD cells, with a significant decrease (2P) of siRNA against MAO-A. Densitometry analyses for MAO-A expression levels, and AKT and GSK3β phosphorylation at day 4 are in the influx rate (Fig. 6B–D). Notably, these alterations in 2+ shown in the lower panel. For each group, SC values were arbitrar- spontaneous Ca oscillations were partially recovered in ily considered as a unit. Phosphorylation levels were normalized to cells treated with the 1P protocol. To further strengthen our total protein levels. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way findings, siRNA experiments were carried out in two addi- ANOVA with post hoc Tukey’s multiple comparison test. D Mito- chondrial ROS levels at day 4 following treatment with scramble tional hiPSCs cell lines that also showed MAO-A upregu- (SC) RNA, 1 pulse (1P) or 2 pulses (2P) of siRNA against MAO- lation during mesoderm-cardiac specification and cardio- A. Values were normalized and expressed as % vs SC. *p < 0.01, myocyte formation (Suppl. Figure 1B). Also in this case, **p < 0.001 by one-way ANOVA with post hoc Tukey’s multiple the reduction in AKT/GSK3β phosphorylation levels and comparison test. At least 100 cells were analyzed per condition in each experiment. E WNT3A and NKX2.5 mRNA expression lev- sarcomere disarray were causally related to MAO-A silenc- els at day 6 in cells treated with scramble (SC) RNA, 1 pulse (1P) ing (Suppl. Figure 4D and Suppl. Figure 5A–B). Moreover, or 2 pulses (2P) of MAO-A siRNA. For each gene, SC values were in cells treated with MAO-A siRNA (2P) we observed a arbitrarily considered as a unit. Values were normalized to GAPDH. significant increase in the frequency of spontaneous oscil- *p < 0.05, **p < 0.01 by one-way ANOVA with post hoc Tukey’s 2+ multiple comparison test. All experiments were performed at least lations in the cytosolic Ca and a significant decrease in three times using three different preparations. Results are expressed the peak amplitude (Suppl. Figure 5C–D). The 2P protocol as mean ± S.E.M induced an impairment in response to caffeine in KD cells, 2+ with a significant decrease in the Ca influx rate (Suppl. pathways, rescuing the altered phenotype observed in Figure 5C–E). Altogether, these results strongly support MAO-A KO hiPSC-CMs. Given that MAO-A expression MAO-dependent ROS generation as a regulator of AKT and is tightly regulated during cardiac commitment (Fig. 1A) WNT pathways during cardiac differentiation. In particular, and its excessive activation results in oxidative stress , the increase in MAO-A-dependent ROS formation during we took advantage of the siRNA strategy to transiently the transition from cardio-mesoderm to cardiomyocytes is reduce MAO-A protein levels only in the first phase of required for the correct differentiation of hiPSCs into cardiac differentiation (Suppl. Figure 4A). Cells were treated with cells. MAO-A siRNA at two different time points (2 pulses (2P) st protocol), specifically at the stage of hiPSCs (1 pulse, day 0) and between mesoderm and cardiac specification (2nd Discussion pulse, day 2), leading to the generation of MAO-A knock down (KD) cells that display 50% reduction in MAO-A This study identifies MAO-A dependent ROS formation as protein level at days 2 and 4 (Suppl. Figure 4B, Fig. 5C). an important process contributing to cardiomyocyte lineage In parallel, a single siRNA administration was used to commitment. MAO-A KO/KD hiPSC-CMs exhibit impaired silence MAO-A only in the first 2 days of differentiation sarcomere structure and function, along with lower mito- (1 pulse (1P) protocol, day 0, Suppl. Figure 4A–B), allow- chondrial ROS levels. Lack of MAO-A dependent ROS for- ing the cells to naturally re-express MAO-A to the physi- mation leads to a decrease in AKT/GSK3β phosphorylation, ological levels by day 4 (Fig. 5C). Regardless of the pro- WNT expression and downstream activation of cardiac tran- tocol used, MAO-A protein levels were restored by day 6 scription factors MESP1 and NKX2.5. This causal relation- (Suppl. Figure 4C). Similarly to MAO-A KO hiPSC-CMs ship was further supported by showing that either exogenous (Fig. 4A), MAO-A KD by the 2P protocol significantly H O administration to MAO-A KO cells or MAO-A re- 2 2 reduced AKT and GSK3β phosphorylation (Fig. 5C). expression improved AKT/GSK3β phosphorylation, NKX2.5 Notably, MAO-A re-expression by means of 1P proto- and WNT3A transcript abundance, and rescued structural 2+ col partially but significantly restored AKT and GSK3β disarray and alterations in Ca homeostasis. phosphorylation (Fig. 5C). This result was paralleled by 1 3 4 Page 14 of 19 Basic Research in Cardiology (2023) 118:4 1 3 Basic Research in Cardiology (2023) 118:4 Page 15 of 19 4 ◂Fig. 6 Effect of MAO-A re-expression on hiPSC-CMs sarcomere early embryo is promoted by ROS [21, 26]. Our experiments 2+ organization and Ca homeostasis. A α-sarcomeric actinin (green) suggest that the potential for MAO-dependent ROS forma- immunofluorescent labeling in cells treated with scramble (SC) RNA, tion greatly depends on the substrate availability. Further 1 pulse (1P) or 2 pulses (2P) of MAO-A siRNA. Phalloidin is shown studies will elucidate whether baseline MAO-A activity is in red, while nuclei were stained with DAPI (blue). The patterning of α-sarcomeric actinin fluorescence intensity is plotted on the right, sufficient to drive cardiomyocyte differentiation or whether denoting sarcomere organization within the cell. Approximately induction of MAO-A expression is the key element regulat- 20/30 cells were analyzed in each experiment. Scale bar 10 µm. B ing this process. 2+ Representative traces of spontaneous cytosolic Ca oscillations in Reduced autophagy, alteration in GATA4 activity and scramble (SC) RNA, 1 pulse (1P) or 2 pulse (2P) MAO-A siRNA 2+ treated hiPSC-CMs (left panel) and of cytosolic Ca peak induced myofibrillar protein expression are frequently signs of a by caffeine stimulation (right panel). C Quantification of spontaneous maladaptive response to the underlying cardiac dysfunction 2+ cytosolic Ca oscillations frequency and peak amplitude. *p < 0.01, in several cardiac pathologies [13, 19, 52, 72]. Such altera- **p < 0.001 by one-way ANOVA with post hoc Tukey’s multi- tions were also observed in MAO-A KO hiPSC-CMs and are ple comparison test. Five regions of interest (ROIs) were selected in each field of view and at least three different fields of view were likely the result of an adaptive response to an impairment in analyzed in each experiment. D Quantification of caffeine-induced differentiation process. A compensatory increase in GATA4 2+ cytosolic Ca peak in terms of peak amplitude, influx and efflux activity is required for the maintenance of cardiac function rate. *p < 0.01 by one-way ANOVA with post hoc Tukey’s multiple in the postnatal heart or following stress stimuli [5, 53]. comparison test. Where data were not normally distributed, Kruskal– Wallis test was applied. All experiments were performed at least three Indeed, here we observed GATA4 upregulation in MAO-A times using three different preparations. Results are expressed as KO hiPSC-CMs as compared to control cells. These findings mean ± S.E.M are in line with another study showing that cardiomyogen- esis was inhibited and GATA4 levels increased following ROS are key players in cardiomyogenesis and differentia- administration of ROS scavengers to embryonic stem cells tion of stem cells into cardiomyocytes in vitro [7, 9, 21, 40]. (ESCs) during cardiac differentiation . In addition, mitochondrial ROS formation has been linked NKX2.5 is directly involved in the regulation of several to the activation of the NOX4 gene and p38 phosphorylation processes that collectively contribute to cardiomyogenesis . Indeed, mitochondria-targeted antioxidants blocked and morphogenesis of the mature heart . Moreover, cardiac die ff rentiation [ 12], suggesting that alterations in the expression of MESP1, a master regulator of multipotent mitochondrial redox equilibrium during cardiac specification cardiovascular progenitor specification [6 ], is promoted by compromise this process. Here we identify the mitochon- GSK3β activation . In our experimental setting, AKT drial flavoenzyme MAO-A as a prominent source of mito- and GSK3β phosphorylation were reduced in MAO-A KO chondrial ROS that play an important role in cardiomyocyte or KD cells starting from day 4. AKT phosphorylation levels lineage commitment. Genetic or pharmacological inhibition change dynamically during cardiomyogenesis, suggesting of MAO-A did not block the formation of contracting car- that AKT inhibition occurs during mesoderm specification diomyocytes, unlike ROS scavengers, pointing to the pos- (day 2) and partial AKT activation is required for cardiac sibility that a cross-talk between MAO-A and other ROS lineage commitment (from day 4 on). Our results suggest sources might exist. Nevertheless, myofibrillogenesis was that ROS/MAO-A dependent AKT activation plays a piv- impaired in MAO-A mutant cardiomyocytes, showing sar- otal role during cardiac mesoderm formation (day 4). Given comere structure disarray, reduction of the myofibrillar pro- that MAO-A dependent ROS formation is down-regulated 2+ tein MYHC6, as well as aberrant intracellular Ca cycling. throughout differentiation, present results imply that AKT 2+ While cytosolic Ca transients were dramatically reduced activation starts relying on ROS produced by MAO-A during in MAO-A KO hiPSC-CMs, no differences were observed cardiac precursors formation. This decreased activation of 2+ in the mitochondrial Ca levels. In addition, gene expres- AKT/GSK3β signaling in the absence of MAO-A resulted 2+ sion of Ca handling proteins, the phosphorylation status in lower levels of both MESP1 and NKX2.5, suggesting an of PLN and the distance/interaction between mitochondria altered cardiac commitment. Furthermore, we observed a and SR all remained unaffected by MAO-A deletion. This remarkable down regulation in WNT3A expression levels at 2+ suggests that mitochondrial Ca uptake does not impact on day 6 in KO cells, in parallel with a significant reduction of 2+ cytosolic Ca levels, at least in this model. Moreover, we both WNT3 and WNT11. unequivocally demonstrated that MAOA-dependent ROS Endogenous canonical WNT (WNT3 and 3A) levels dur- formation is important between days 4 and 6 of cardiomyo- ing cell fate determination are causally related to MESP1 cyte differentiation, i.e., during cardiac specification. These expression, mesodermal commitment and patterning findings are in line with previous studies suggesting that toward cardiac mesoderm [41, 46]. On the other hand, redox signaling during cardiomyogenesis might be both up-regulation of non-canonical WNT expression (WNT11) stage- and dose-dependent and that the commitment of in the later phase of differentiation plays an important cardiac progenitor cells toward the cardiac lineage in the role in cardiac development . Notably, stimulation of 1 3 4 Page 16 of 19 Basic Research in Cardiology (2023) 118:4 COST Action EU-CARDIOPROTECTION CA16225; MDS, SA, and canonical WNT pathway between days 4–6 with the solu- NK are fellows of the Leducq Transatlantic Networks of Excellence ble ligand WNT3A augments cardiac differentiation from (16CVD04 and 15CVD04). ESCs . Accordingly, inhibition of WNT pathway with dickkopf-related protein 1 during this window drastically Data availability The analyzed datasets are available from the corre- sponding author upon reasonable request. compromises cardiomyocyte differentiation , suggest- ing that timely canonical WNT signaling is required for Declarations cardiomyocyte formation. Moreover, cardiac progenitors are formed at this stage, and the following maturation into Conflict of interest The authors declare that they have no conflict of cardiomyocytes is prompted by specific cardiac regula - interest. tors. In human fetal cardiovascular progenitor cells derived Open Access This article is licensed under a Creative Commons Attri- from ESC lines, the self-renewal capacity and expansion of bution 4.0 International License, which permits use, sharing, adapta- the cells is promoted and sustained by WNT3A . In our tion, distribution and reproduction in any medium or format, as long model, pharmacological AKT inhibition with MK-2206 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 did not alter expression levels of WNT3 and WNT3A, indi- were made. The images or other third party material in this article are cating that downregulation of the canonical WNT genes included in the article's Creative Commons licence, unless indicated in MAO-A KO cells was not under the direct control of otherwise in a credit line to the material. If material is not included in AKT. However, when we re-expressed MAO-A protein the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will or when MAO-A KO cells have been exposed to a pulse need to obtain permission directly from the copyright holder. 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Basic Research in Cardiology – Springer Journals
Published: Jan 20, 2023
Keywords: Reactive oxygen species; hiPSCs; Cardiomyocyte differentiation; Development; Cell fate
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