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www.nature.com/cddiscovery ARTICLE OPEN Stomatin-like protein 2 deﬁciency exacerbates adverse cardiac remodeling 1,4 1,4 1 1 2 1 1 1 2 1 Yuntao Hu , Hongwei Jiang , Yueyue Xu , Ganyi Chen , Rui Fan , Yifei Zhou , Yafeng Liu , Yiwei Yao , Renjie Liu , Wen Chen , 3 1 1✉ 1✉ Ke Zhang , Xin Chen , Rui Wang and Zhibing Qiu © The Author(s) 2023 Myocardial ﬁbrosis, oxidative stress, and autophagy both play key roles in the progression of adverse cardiac remodeling. Stomatin- like protein 2 (SLP-2) is closely related to mitochondrial function, but little is known about its role and mechanism in cardiac remodeling. We developed doxorubicin (Dox), angiotensin (Ang) II, and myocardial ischemia-reperfusion (I/R) injury induced cardiac -/- remodeling model and Dox treated H9C2 cell injury model using SLP-2 knockout (SLP-2 ) mice and H9C2 cells with low SLP-2 expression. We ﬁrst examined cardiac functional and structural changes as well as levels of oxidative stress, apoptosis and autophagy. We found that SLP-2 deﬁciency leads to decreased cardiac function and promotes myocardial ﬁbrosis. After Dox and Ang II treatment, SLP-2 deﬁciency further aggravated myocardial ﬁbrosis, increased myocardial oxidative stress and apoptosis, and activated autophagy by inhibiting PI3K-Akt-mTOR signaling pathway, ultimately exacerbating adverse cardiac remodeling. Similarly, SLP-2 deﬁciency further exacerbates adverse cardiac remodeling after myocardial I/R injury. Moreover, we extracted cardiomyocyte mitochondria for proteomic analysis, suggesting that SLP-2 deﬁciency may be involved in myocardial I/R injury induced adverse cardiac remodeling by inﬂuencing ubiquitination of intramitochondrial proteins. In addition, the oxidative stress, apoptosis and autophagy levels of H9C2 cells with low SLP-2 expression were further enhanced, and the PI3K-Akt-mTOR signaling pathway was further inhibited under Dox stimulation. Our results suggest that SLP-2 deﬁciency promotes myocardial ﬁbrosis, disrupts normal mitochondrial function, overactivates autophagy via PI3K-Akt-mTOR signaling pathway, affects the level of ubiquitination, leads to irreversible myocardial damage, and ultimately exacerbates adverse cardiac remodeling. Cell Death Discovery (2023) 9:63 ; https://doi.org/10.1038/s41420-023-01350-z INTRODUCTION resulting in cardiomyocyte injury and death, and eventually Cardiovascular disease is the greatest threat to human health develop into impaired cardiac function . around the world and is the leading cause of death in China [1, 2]. Autophagy is a classical form of cell death resulting in Cardiac remodeling is closely associated with a variety of cardiac degradation of cytoplasmic contents after stimulation and injury, diseases, characterized by progressive ventricular dilatation, responsible for the removal of potentially toxic cytoplasmic protein myocardial hypertrophy, myocardial ﬁbrosis, and deterioration of aggregates and damaged organelles [12–14]. Autophagy is a strictly cardiac function . The mechanisms of cardiac remodeling regulated mechanism of lysosome degradation, which is crucial for include changes in cardiomyocyte, such as redox injury, endo- cell survival, homeostasis, and function . Autophagy is activated plasmic reticulum stress, and cardiomyocyte autophagy, as well as excessively under some conditions, causing uncontrolled cell phenotypic changes in other non-cardiomyocytes such as degradation and death . Mitochondria, as the center of energy ﬁbroblasts, endothelial cells, and inﬂammatory cells [4–6]. metabolism, play an important role in autophagy . Abnormal Myocardial ﬁbrosis, as one of the key factors in cardiac activation of mitophagy will lead to mitochondrial depletion, remodeling, has received extensive attention in recent years. resulting in energy metabolism disorder and reactive oxygen Myocardial ﬁbrosis is characterized by activation of cardiac species (ROS) accumulation, and eventually lead to cell senescence ﬁbroblasts , which is mainly manifested by disordered arrange- and death . Overactivation of autophagy in cardiomyocyte leads ment and excessive deposition of myocardial interstitial and to excessive digestion and degradation of proteins and organelles perivascular collagen ﬁbers, resulting in cardiac systolic and . Previous studies have shown that ROS can aggravates diastolic dysfunction, arrhythmia, and heart failure [8–10]. Cardiac myocardial I/R injury by activating autophagy . Inhibition of ﬁbroblasts are highly plastic and can be converted into cardiac autophagic initiation or ﬂux prevents cell death and alleviates myoﬁbroblasts under various injury or stress conditions . cardiac injury or dysfunction in some conditions [20, 21]. A study Cardiac myoﬁbroblasts can cause myocardial ﬁbrosis by synthesiz- shows that autophagy can convert cardiac ﬁbroblasts into cardiac ing excessive extracellular matrix, such as collagen I and III, myoﬁbroblasts, suggesting that autophagy may be involved in the 1 2 Department of Thoracic and Cardiovascular Surgery, Nanjing First Hospital, Nanjing Medical University, Jiangsu, China. School of Medicine, Southeast University, Jiangsu, China. 3 4 Department of Thoracic and Cardiovascular Surgery, Changzhou Second People’s Hospital, Nanjing Medical University, Jiangsu, China. These authors contributed equally: Yuntao Hu, Hongwei Jiang. email: email@example.com; firstname.lastname@example.org Received: 23 October 2022 Revised: 27 January 2023 Accepted: 30 January 2023 Ofﬁcial journal of CDDpress 1234567890();,: Y. Hu et al. occurrence and development of myocardial ﬁbrosis . Therefore, hepatic and renal function injury in mice under saline or Dox autophagy may be an important target for improving myocardial treatment (Fig. S2A, B). We could rule out the adverse cardiac ﬁbrosis, alleviating myocardial oxidative stress injury and reversing remodeling secondary to damaged hepatic and renal function adverse cardiac remodeling. caused by SLP-2 deﬁciency. HE staining and echocardiography SLP-2 is a mitochondrial inner membrane protein, which is showed that compared with the saline group, the heart volume was abundant in skeletal muscle and heart , and up-regulated in signiﬁcantly reduced, the ventricular wall and septum were most tumor cells [24, 25]. Previous studies have shown that SLP-2 signiﬁcantly thinner, the inner diameter of the ventricle was plays a critical role in protecting mitochondrial function. Up- signiﬁcantly increased, the ventricular cavity was signiﬁcantly regulation of SLP-2 expression increases mitochondrial oxygen expanded, and the cardiac function was signiﬁcantly decreased in -/- consumption and ATP production, as well as mitochondrial the Dox group. The DCM phenotype of SLP-2 Dox group was cardiolipin synthesis, which increases mitochondrial membrane more obvious than WT Dox group (Fig. 2A, B, Fig. S3A, B). WGA formation and biogenesis, and promotes the assembly of staining showed no signiﬁcant difference in cardiomyocyte area respiratory supercomplexes . Our previous research suggests between the two saline groups, while SLP-2 deﬁciency exacerbated that overexpression of SLP-2 reduces cardiomyocyte apoptosis, cardiomyocyte hypertrophy under Dox treatment (Fig. 2C, D). mitochondrial injury, and myocardial I/R injury . A study on Masson and PSR staining indicated that the degree of myocardial gastric cancer shows that silencing SLP-2 expression can induce perivascular and intercellular ﬁbrosis was signiﬁcantly aggravated in apoptosis and autophagy in cancer cells . At present, the Dox group (Fig. 2C, D), and IHC staining indicated that the mechanism of SLP-2 in myocardial ﬁbrosis and cardiomyocyte expression of myocardial ﬁbrosis markers (a-SMA and collagen III) autophagy remains unclear. was signiﬁcantly increased in Dox group (Fig. S3C). Similarly, Therefore, we explored for the ﬁrst time the mechanism that Western blot analysis showed that the expression of a-SMA, SLP-2 deﬁciency can mediate myocardial ﬁbrosis and adverse collagen I, collagen III and Fibronectin in Dox group were cardiac remodeling by regulating the PI3K-Akt-mTOR signaling signiﬁcantly higher than saline group (Fig. 2E, F). More importantly, pathway to enhance autophagy, which will play a vital role in SLP-2 deﬁciency signiﬁcantly aggravated the above-mentioned alleviating myocardial ﬁbrosis, cardiac remodeling and improving levels of myocardial ﬁbrosis under Dox treatment (Fig. 2C–Fand the prognosis of patients with end-stage heart disease. S3C). These results indicate that SLP-2 deﬁciency exacerbates Dox- induced DCM phenotype. RESULTS SLP-2 deﬁciency increases the levels of oxidative stress and SLP-2 deﬁciency induces cardiac function decline and apoptosis in Dox-induced DCM promotes myocardial ﬁbrosis SLP-2 is located in mitochondrial inner membrane. To further To comprehend the function of SLP-2 in the heart, we constructed explore the effect of SLP-2 deﬁciency on mitochondrial damage SLP-2 whole-body knockout mice. We utilized male mice aged and oxidative stress levels in DCM, we measured ROS production 6–24 weeks to examine the role of SLP-2 in cardiac remodeling. in cardiomyocytes and MDA and SOD levels in serum of mice. The –/– The expression of SLP-2 in the heart of SLP-2 mice decreased by results showed that compared with saline group, ROS, MDA and –/– nearly 90% compared with WT mice. We found that when SLP-2 apoptosis levels (TUNEL, Bax, Cleaved-caspase-3) were signiﬁ- mice aged, heart gradually expanded larger and heavier, and cantly increased, SOD level and the expression of anti-apoptotic abnormal cardiac remodeling eventually developed into heart protein (Bcl-2) were signiﬁcantly decreased in Dox group. More- failure (Fig. S1). There was no signiﬁcant difference in heart over, SLP-2 deﬁciency further increases the level of oxidative stress –/– weight/tibia length (HW/TL) between SLP-2 mice and WT mice and apoptosis in Dox group (Fig. 3A–F). These results indicate that at 6 weeks old, but increased by 18.4%, 18.3%, 21.1% and 19.94% SLP-2 deﬁciency aggravates Dox-induced DCM by causing compared with WT mice at 8 weeks, 10 weeks, 16 weeks and mitochondrial function impairment, myocardial oxidative stress 24 weeks, respectively (Fig. S1B). Similarly, echocardiography injury and apoptosis. showed that there was no difference in EF and FS at 6 weeks, but –/– EF and FS in SLP-2 mice began to decrease at 8 weeks, and at SLP-2 deﬁciency exacerbates DCM progression by regulating 24 weeks, EF decreased by 25%, FS decreased by 23% (Fig. S1C, D). PI3K-Akt-mTOR signaling pathway –/– The LV mass, IVS thickness, and LVPW thickness of SLP-2 mice We further explored the effect of SLP-2 deﬁciency on autophagy in were also signiﬁcantly increased compared with WT mice (Fig. DCM. Western blot analysis showed that the expression of S1D), which was further demonstrated by HE staining (Fig. S1E). By autophagy-associated proteins (LC3B II, Beclin1 and ATG5) in –/– –/– WGA staining, we found that the cardiomyocyte area in SLP-2 SLP-2 Dox group was signiﬁcantly higher than WT Dox (Fig. 4A, group was signiﬁcantly larger than WT group at 8w or 24w (Fig. B). The area of LC3B positive region in IF staining veriﬁed the above 1A, B). Masson and PSR staining showed that the degree of ﬁbrosis result (Fig. 4C). The expressions of mitophagy-associated proteins –/– in SLP-2 mice increased at 8 weeks and signiﬁcantly aggravated (PINK1 and Parkin) were increased after Dox treatment, and even at 24 weeks (Fig. 1C, D). The main feature of myocardial ﬁbrosis is more signiﬁcantly after SLP-2 deﬁciency (Fig. 4D, F). The IF staining the deposition of extracellular matrix proteins, such as collagen I results veriﬁed the above result (Fig. 4H). Mitochondrial fusion and and III. The results of IHC staining showed that the expression of ﬁssion are closely related to mitophagy. Our results showed that the –/– collagen I in SLP-2 group was signiﬁcantly higher than WT expression of mitochondrial fusion protein Mfn2 decreased, ﬁssion group (Fig. 1E, F). Cardiac ﬁbroblasts play a key role in the protein Drp1 increased after Dox treatment, and SLP-2 deﬁciency occurrence and development of cardiac ﬁbrosis. We found that exacerbated the change trend (Fig. 4E, G). In addition, there was no the number of cardiac ﬁbroblasts in signiﬁcant difference in all the above indexes between the two –/– SLP-2 mice increased signiﬁcantly by IF staining (Fig. 1G). These saline groups. We then detected the expression of PI3K-Akt-mTOR results suggest that SLP-2 deﬁciency leads to cardiomyocyte signaling pathway, western blot analysis showed that the protein hypertrophy and myocardial ﬁbrosis. ratios of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR in Dox group were signiﬁcantly lower than saline group, and SLP-2 deﬁciency further SLP-2 deﬁciency exacerbates Dox-induced DCM phenotype reduced all ratios, suggesting that SLP-2 deﬁciency inhibited PI3K- We further investigated the role of SLP-2 in the progression of Dox- Akt-mTOR signaling pathway, leading to excessive activation of induced dilated cardiomyopathy (DCM). We ﬁrst tested the serum autophagy (Fig. 4I, J). These results indicate that SLP-2 deﬁciency biochemical indexes of hepatic and renal function in mice, and the exacerbates the DCM progression by regulating PI3K-Akt-mTOR results indicated that SLP-2 deﬁciency did not exacerbate the signaling pathway to promote autophagy. Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 1 SLP-2 deﬁciency leads to cardiac hypertrophy and promotes myocardial ﬁbrosis. A–G Heart representative images of WGA staining, mean cardiomyocyte area, Masson and PSR staining, percentage of ﬁbrosis, IHC staining of collagen I, and IF staining of Vimentin in WT and –/– SLP-2 mice at 8w and 24w. (n = 4 per group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice 8w, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT mice 24w, student’s t test). SLP-2 deﬁciency exacerbates Ang II-induced cardiac hypertrophy (Fig. 5A–C). Masson and PSR staining showed that the –/– remodeling degree of myocardial ﬁbrosis in SLP-2 Ang II group was We further investigated whether SLP-2 affects Ang II-induced signiﬁcantly higher than WT Ang II group (Fig. 5B, C). Consistent cardiac remodeling. HE and WGA staining showed that SLP-2 with the results after Dox stimulation, SLP-2 deﬁciency further deﬁciency signiﬁcantly aggravated Ang II-induced cardiomyocyte aggravated the increase of ROS production and TUNEL positive Cell Death Discovery (2023) 9:63 Y. Hu et al. –/– Fig. 2 SLP-2 deﬁciency exacerbates Dox-induced DCM phenotype. A Representative images of HE staining in WT and SLP-2 mice hearts –/– after saline or dox treatment. B HW/TL and heart weight/body weight in WT and SLP-2 mice after saline or Dox treatment. C–F Representative images of WGA, Masson and PSR staining, cardiomyocyte area and percentage of ﬁbrosis, and expression of α-SMA, –/– collagen I, collagen III and Fibronectin, and quantitative analysis in WT and SLP-2 mice hearts after saline or Dox treatment. (n = 4 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. WT Saline group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. WT Dox group, 1-way ANOVA, Tukey test). Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 3 SLP-2 deﬁciency increases the levels of oxidative stress and apoptosis in Dox-induced DCM. A–C Heart representative images of ROS -/- staining and relative ROS production, and TUNEL staining in WT and SLP-2 mice after saline or Dox treatment. D Concentration of MDA and SOD –/– in serum, and TUNEL positive rate in WT and SLP-2 mice after saline or Dox treatment. E, F Representative images of expression of Bcl-2, Bax and –/– C-caspase-3, and quantitative analysis in WT and SLP-2 mice hearts after saline or Dox treatment. (n= 4 per group, *P <0.05, **P <0.01, ***P < 0.001, ****P < 0.0001 vs. WT Saline group, #P <0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. WT Dox group, 1-way ANOVA, Tukey test). rate in cardiomyocytes under Ang II treatment (Fig. 5D, E). Then are the procession of long-term chronic injury. Then we construct a model of acute myocardial I/R injury to verify whether SLP-2 is we detected the expression of autophagy-associated proteins and PI3K-Akt-mTOR signaling pathway. The results showed that the involved in cardiac remodeling caused by acute injury. By expression of LC3B further increased in SLP-2 deﬁciency, while the echocardiography, we found that the cardiac function of –/– ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR decreased SLP-2 mice decreased signiﬁcantly (Fig. 6A, C). We measured signiﬁcantly under Ang II treatment (Fig. S4A, B). These results the area at risk (AAR) and LV areas in mice by Evans blue/TTC indicate that SLP-2 deﬁciency promotes autophagy by regulating staining. The result showed that SLP-2 deﬁciency further increased PI3K-Akt-mTOR signaling pathway and plays a vital role in Ang II- the AAR/LV ratio and aggravated the myocardial I/R injury (Fig. 6B, induced cardiac remodeling. D). HE staining showed that the accumulation of inﬂammatory cells was more obvious, the inﬁltration depth was deeper, and the –/– SLP-2 deﬁciency exacerbates myocardial I/R injury by myocardial infarction area was larger in SLP-2 mice after affecting the level of ubiquitination of intramitochondrial myocardial I/R injury (Fig. 6E). Masson staining showed that proteins myocardial ﬁbrosis was signiﬁcantly aggravated after myocardial I/ –/– Previously, we conﬁrmed that SLP-2 deﬁciency exacerbates R injury in SLP-2 mice (Fig. 6E). These results suggest that SLP-2 adverse cardiac remodeling, but both Dox and Ang II stimulation deﬁciency exacerbates the adverse cardiac remodeling caused by Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 4 SLP-2 deﬁciency exacerbates DCM progression by regulating PI3K-Akt-mTOR signaling pathway. A–C Representative images of –/– expression of LC3B I/II, Beclin1 and ATG5, and quantitative analysis, and IF staining of LC3B in WT and SLP-2 mice hearts after saline or Dox treatment. D, F and H, Representative images of expression of PINK1 and Parkin, and quantitative analysis, and IF staining of PINK1 and Parkin in –/– WT and SLP-2 mice hearts after saline or Dox treatment. E, G Representative images of expression of Mfn2 and Drp1, and quantitative analysis in –/– WT and SLP-2 mice hearts after saline or Dox treatment. I, J Representative images of expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR, –/– and quantitative analysis in WT and SLP-2 mice hearts after saline or Dox treatment. (n= 4 per group, *P <0.05, **P < 0.01, ***P <0.001, ****P < 0.0001 vs. WT Saline group, #P <0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. WT Dox group, 1-way ANOVA, Tukey test). Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 5 SLP-2 deﬁciency exacerbates Ang II-induced cardiac remodeling. A–E Representative images of HE, WGA, Masson and PSR staining, and cardiomyocyte area and percentage of ﬁbrosis, and ROS and TUNEL staining, and relative ROS production and TUNEL positive rate in WT –/– and SLP-2 mice hearts after saline or Ang II treatment. (n = 4 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. WT Saline group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. WT Ang II group, 1-way ANOVA, Tukey test). Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 6 SLP-2 deﬁciency exacerbates myocardial I/R injury by affecting the level of ubiquitination of intramitochondrial proteins. –/– A, C Representative echocardiographs and echocardiographic parameters in WT and SLP-2 mice after sham or myocardial I/R injury. B, D Cross-sections of left ventricles (LV) stained with Evans Blue/TTC and statistical graph of AAR/LV. E Heart representative images of HE and –/– Masson staining in WT and SLP-2 mice after sham or myocardial I/R injury. F, G Heat map, gene ontology analysis and KEGG analysis of –/– differentially expressed proteins in WT and SLP-2 mice were obtained by mitochondrial proteomics after myocardial I/R injury. (n = 3 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. WT Saline group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. WT Ang II group, 1-way ANOVA, Tukey test). Cell Death Discovery (2023) 9:63 Y. Hu et al. myocardial I/R injury. To explore how SLP-2 is involved in pathological factors [32, 33]. The main factors that promote the myocardial I/R injury, we extracted cardiomyocyte mitochondria development of cardiac remodeling include myocardial ﬁbrosis, –/– for proteomics from WT and SLP-2 mice with ischemia for excessive deposition of extracellular matrix, oxidative stress and 45 min followed by reperfusion for 6 h. Through the functional apoptosis [34, 35]. Up to now, the progress of improving and analysis of 53 differentially expressed proteins obtained from alleviating cardiac remodeling is still the focus and difﬁculty in the proteomics (Fig. 6F), we found that ubiquitination was involved in treatment of cardiovascular disease . Our study focused on the biological process, cellular component and molecular function of role of SLP-2 in different stimulus-induced cardiac remodeling or gene ontology analysis and KEGG enrichment. ubiquitination is H9C2 cell injury under Dox stimulation, aiming to explore the closely related to apoptosis, autophagy and mitochondrial quality mechanism of the development of cardiac remodeling in order to control system [29–31]. We speculate that SLP-2 deﬁciency may seek to improve and alleviate the progression of cardiac affect the level of ubiquitination of intramitochondrial proteins, remodeling. then promote apoptosis and autophagy, damage the mitochon- Excessive deposition of extracellular matrix and myocardial drial quality control system, and ﬁnally aggravate myocardial I/R ﬁbrosis are involved in cardiac remodeling [32, 35, 36]. Under the injury (Fig. 6G). stimulation of pathological stress, cardiac ﬁbroblasts are trans- formed into myoﬁbroblasts, which can secrete extracellular matrix Low SLP-2 expression leads to increased apoptosis and components such as collagen I, collagen III and ﬁbronectin. mitochondrial dysfunction in H9C2 cells under Dox Excessive deposition of extracellular matrix and ﬁbrosis lead to stimulation myocardial tissue disorder, cardiac systolic and diastolic dysfunc- Western blot analysis showed that the expression of SLP-2 was tion, and eventually develop into cardiac remodeling . In our signiﬁcantly decreased in H9C2 cells transfected with siRNA1, study, the degree of myocardial ﬁbrosis and the expression of α- –/– siRNA2 and siRNA3, and siRNA2 SLP-2 most effectively reduced SMA in cardiac ﬁbroblasts in SLP-2 group were higher than WT SLP-2 expression (Fig. S5A, B). We selected siRNA2 SLP-2 for cell group after Dox treatment, which indicated that SLP-2 deﬁciency transfection in the follow-up experiment. promoted the transformation of cardiac ﬁbroblasts into myoﬁ- We then evaluated the effects of low SLP-2 expression on broblasts. At the same time, the expression of collagen I, collagen –/– apoptosis and mitochondrial function of H9C2 cells. The results III and ﬁbronectin in SLP-2 Dox group was signiﬁcantly higher showed that low SLP-2 expression signiﬁcantly increased the level than WT Dox group. HE staining and echocardiography showed of apoptosis (TUNEL, Bax/Bcl-2, C-caspase-3), decreased ATP that compared with WT Dox group, the ventricular cavity of –/– content and mitochondrial membrane potential, increased mtROS SLP-2 Dox group was further dilated, the ventricular wall and production under Dox stimulation (Fig. 7A–E, S5C, D). These results IVS were thinner, and the cardiac function was signiﬁcantly indicate that low SLP-2 expression leads to increased apoptosis decreased. These results indicate that SLP-2 deﬁciency aggravates and mitochondrial dysfunction in H9C2 cells under Dox extracellular matrix deposition and myocardial ﬁbrosis, and stimulation. promotes the progression of adverse cardiac remodeling. Excessive accumulation of ROS can lead to oxidative stress Low SLP-2 expression aggravates H9C2 cells injury under Dox damage and damage to lipids, proteins and DNA, resulting in cell stimulation by regulating PI3K-Akt-mTOR signaling pathway death . Mammalian cardiomyocytes almost completely lose to promote autophagy their ability to proliferate in adulthood, and will be replaced by We further examined the effect of low SLP-2 expression on scar tissue after cardiomyocyte death, resulting in myocardial autophagy of H9C2 cells. The results showed that the expression structural and electrophysiological dysfunction, eventually of autophagy-associated proteins (LC3B II, Beclin1 and ATG5) and develop into cardiac remodeling [38, 39]. Previous studies have mitophagy-associated proteins (PINK1 and Parkin) in si-SLP-2 Dox shown that Dox can mediate mitochondrial dysfunction and group was signiﬁcantly higher than si-NC Dox group, and there aggravate oxidative stress injury and cardiomyocyte apoptosis was no signiﬁcant difference between si-NC group and si-SLP-2 . SLP-2, as a mitochondrial inner membrane protein, is closely group (Fig. 7F, H), which was consistent with previous in vivo related to oxidative stress . A study showed that over- results. expression of SLP-2 signiﬁcantly increased the cell viability and 3-MA is a commonly used autophagy inhibitor. After pretreat- decreased apoptosis and ROS production in neurons injured by ment of H9C2 cells with 3-MA, the expression of autophagy- oxygen-glucose deprivation /reoxygenation, while inhibition of associated proteins (LC3B II, Beclin1 and ATG5) decreased SLP-2 showed the opposite effect . Consistent with this, our signiﬁcantly in si-SLP-2 Dox group (Fig. S6A, B), while the study shows that SLP-2 deﬁciency further aggravates the damage mitochondrial membrane potential increased and mtROS produc- effect of Dox, the levels of ROS, MDA and SOD in myocardium are tion decreased (Fig. S6C, D), suggesting that H9C2 cells injury signiﬁcantly increased, and the decrease of Bcl-2/Bax ratio and the caused by low SLP-2 expression under Dox stimulation was increase of Cleaved-caspase-3 level indicate that myocardial alleviated after inhibition of autophagy. apoptosis is further increased. Therefore, SLP-2 deﬁciency may We further explored the expression of PI3K-Akt-mTOR signaling aggravate the progression of cardiac remodeling by increasing pathway. Western blot analysis showed that the ratios of p-PI3K/ myocardial oxidative stress injury and apoptosis. PI3K, p-Akt/Akt and p-mTOR/mTOR in H9C2 cells decreased A great deal of evidence shows that autophagy is closely related signiﬁcantly after Dox stimulation, and low SLP-2 expression to the progression of a variety of heart diseases, such as coronary further reduced the above ratios. These results suggest that low artery disease, DCM, hypertrophic cardiomyopathy and myocar- SLP-2 expression suppresses the PI3K-Akt-mTOR signaling path- dial I/R injury [42–45]. Studies suggest that excessive autophagy way of H9C2 cells under Dox stimulation, activates autophagy and leads to DCM, while inhibition of autophagy can reduce leads to H9C2 cells injury. myocardial infarction area, inhibit cardiac remodeling and improve cardiac function [43, 46, 47]. Studies have shown that excessive mitochondrial ﬁssion can lead to excessive mitophagy, DISCUSSION which exacerbates cardiac remodeling, and overexpression of SLP- Cardiovascular disease has become the primary disease that 2 can signiﬁcantly increase the content of mitochondrial fusion threatens human life [1, 2]. Most cardiovascular diseases develop protein Mfn2, thereby reducing mitochondrial ﬁssion and into irreversible cardiac remodeling and heart failure at the end oxidative stress injury [48–51]. Silencing SLP-2 can inhibit gastric stage. The mechanism of cardiac remodeling is very complex, cancer cell proliferation and induce apoptosis and autophagy via which is the result of the comprehensive action of a variety of ANXA2/ β-catenin signal pathway . In our study, the expression Cell Death Discovery (2023) 9:63 Y. Hu et al. Fig. 7 Low SLP-2 expression aggravates H9C2 cells injury under Dox stimulation by promoting mitochondrial dysfunction and autophagy. A, B Representative images of expression of Bcl-2, Bax and C-caspase-3, and quantitative analysis in different groups of H9C2 cells. C ATP levels in different groups of H9C2 cells. D Representative images of mitochondrial peroxide staining in different groups of H9C2 cells. E Representative ﬂuorescence images of JC-1 staining in different groups of H9C2 cells. F, H Representative images of expression of LC3B, Beclin1, ATG5, PINK1 and Parkin, and quantitative analysis in different groups of H9C2 cells. G, I Representative images of expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR, and quantitative analysis in different groups of H9C2 cells. (n = 3–4 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. si-NC group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. si-NC Dox group, 1-way ANOVA, Tukey test). Cell Death Discovery (2023) 9:63 Y. Hu et al. –/– levels of LC3B II, Beclin1 and ATG5 in SLP-2 Dox group were mitochondrial dysfunction of H9C2 cells, and promotes autophagy signiﬁcantly higher than WT Dox group, suggesting that SLP-2 via PI3K-Akt-mTOR signaling pathway under Dox stimulation, deﬁciency aggravated cardiomyocyte autophagy induced by Dox. which ultimately leads to H9C2 cells injury. After using autophagy Then we further explored the effect of SLP-2 deﬁciency on inhibitor 3-MA to inhibit autophagy, the damaged mitochondrial –/– mitophagy in DCM. In SLP-2 Dox group, the expression of membrane potential was restored and the production of mitophagy-related proteins PINK1 and Parkin and mitochondrial mitochondrial ROS was decreased, which conﬁrmed that excessive ﬁssion protein Drp1 were signiﬁcantly increased, while the autophagy induced by low SLP-2 expression played an important mitochondrial fusion protein Mfn2 was signiﬁcantly decreased. role in the injury of H9C2 cells. These evidences suggest that SLP-2 deﬁciency promotes excessive In summary, our research revealed for the ﬁrst time that SLP-2 is autophagy of cardiomyocytes, causing abnormal mitochondrial closely related to the development of adverse cardiac remodeling. ﬁssion and excessive mitophagy, and ultimately exacerbating the Pathological cardiac remodeling eventually develops into cardiac progression of Dox-induced adverse cardiac remodeling. ejection dysfunction and heart failure, the latter is the main cause PI3K-Akt-mTOR signaling pathway is closely related to a variety of death in the population. Therefore, it is necessary to explore of physiological activities and is a well-known pathway involved in how to improve and reverse adverse cardiac remodeling. Our autophagy regulation in mammals . Phosphoinositide 3-kinase research shows that SLP-2 deﬁciency exacerbates the progression (PI3K) regulates cell signal transduction, energy metabolism and of cardiac remodeling, so how to promote the expression of SLP-2 cell cycle [53, 54]. Akt known also as protein kinase B is regulated may be a vital direction of cardiac remodeling therapy in the by PI3K and is mainly involved in apoptosis, autophagy and cell future. cycle regulation, while Akt can activate its downstream mamma- lian target of rapamycin (mTOR) . mTOR is relatively conservative in evolution. It can integrate a variety of extracellular MATERIALS AND METHODS signals such as nutrition, energy and growth factors, participate in Animals and experimental models –/– SLP-2 and wild-type (C57BL/6, WT) male mice (6–24 weeks old) were a variety of biological processes of transcription, translation and purchased from GemPharmatech, Inc. (Nanjing, China) and underwent ribosome synthesis, and play an important role in cell growth and cultivation in the Model Animal Research Center of Nanjing University metabolism, apoptosis and autophagy [56–58]. One study showed (Nanjing, China). All mice were housed in pathogen-free cages under a 12- that the activation of PI3K-Akt-mTOR signaling pathway can hour light/12-hour dark cycle, controlled room temperature and given promote mitochondrial fusion and suppress mitochondrial ﬁssion, freely available diet and water. All animal experiments were approved by thus attenuating ischemia-reperfusion injury in diabetic cardio- the Ethics Committee of Experimental Animal of Nanjing First Hospital, myopathy . As far as we know, the relationship between PI3K- Nanjing Medical University. –/– Akt-mTOR signaling pathway, cardiomyocyte autophagy, and All mice (22–25 g) were randomly divided into WT control, SLP-2 cardiac remodeling is unclear. In our study, we found that SLP-2 control and corresponding experimental groups. We construct Dox- induced and Ang II-induced cardiac remodeling, and myocardial I/R injury deﬁciency inhibits PI3K-Akt-mTOR signaling pathway in mouse animal models (All mice began to undergo experimental surgery when heart under Dox treatment, promotes oxidative stress injury, they were 8 weeks old, each experimental group included 8 mice.). Dox- apoptosis and mitochondrial dysfunction, leads to excessive induced DCM group mice were injected with a cumulative dose of 30 mg/ activation of autophagy, and eventually develops into adverse kg doxorubicin (Sigma-Aldrich; Merck KGaA) within 30 days after 6 times of cardiac remodeling. intraperitoneal injection (5 mg/kg i.p.). The sham group received the same Ang II can increase cardiac preload and postload, cause changes amount of sterile isotonic saline. Echocardiography was performed 2 weeks in myocardial morphology and function, increase extracellular after the last injection. To establish Ang II-induced cardiac remodeling matrix deposition and cardiac ﬁbroblasts activation, and promote model, 5% chloral hydrate (400 mg/kg) was used to anesthetize mice and myocardial oxidative stress injury [60–62]. In our study, SLP-2 osmotic pumps (Model 2004, Alzet Scientiﬁc Products, USA) supplemented with Ang II (1000 ng/kg/min) or sterile isotonic saline were implanted deﬁciency induced cardiomyocyte hypertrophy, promoted myo- subcutaneously. Echocardiography was performed 28 days later. To cardial ﬁbrosis, and signiﬁcantly increased ROS production and establish myocardial I/R injury model, the mice were anesthetized with apoptosis levels under Ang II stimulation. Similarly, SLP-2 the above method, and the left fourth intercostal small incision was made deﬁciency further inhibits PI3K-Akt-mTOR signaling pathway and to expose heart. In total, 3 mm from the starting point, the left anterior leads to increased autophagy of cardiomyocytes under Ang II descending (LAD) coronary artery was ligated and the incision was sutured. stimulation, which is consistent with the results in Dox-induced The slipknot was released for reperfusion after 45 min. The sham group cardiac remodeling, suggesting that SLP-2 deﬁciency leads to received the same operation but the LAD were not strangled. Echocardio- excessive activation of autophagy, which induces cardiomyocyte graphy was performed 28 days after reperfusion. After echocardiography, injury in mice and ultimately exacerbates adverse cardiac the mice were sacriﬁced immediately after anesthesia, then heart tissues and serum were harvested. remodeling. Myocardial I/R injury can cause acute cardiac injury in a short time. Complications of myocardial I/R injury can lead to adverse Cell culture and transfection cardiac remodeling, including inﬂammation, activation of apop- H9C2 cardiomyocytes were purchased from the Cell Bank of the Chinese tosis, loss of cardiomyocytes and so on, and then develop into Academy of Sciences (Shanghai, China). H9C2 cells were cultured in DMEM (Gibco, Thermo Fisher Scientiﬁc, USA) containing 10% FBS (Gibco, Thermo heart failure [63, 64]. Similarly, in our study, SLP-2 deﬁciency Fisher Scientiﬁc, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher aggravated myocardial I/R injury and myocardial ﬁbrosis, further Scientiﬁc, USA) and placed in incubator at 37 °C with 5% CO . decreased cardiac function, and eventually led to adverse cardiac Small interfering RNA (siRNA) and siRNA negative control (NC) for SLP-2 remodeling. We further extracted the mitochondrial proteins of (siRNA1 SLP-2, sense: 5ʹ-GGUAUGUGCAGAGUCUCAATT-3ʹ, anti-sense: 5ʹ- mouse cardiomyocytes for proteomics and analyzed the function UUGAGACUCUGCACAUACCTT-3ʹ; siRNA2 SLP-2, sense: 5ʹ-GCAUUAUG- of the differentially expressed proteins. The results showed that GAUCCUUACAATT-3ʹ, anti-sense: 5ʹ-UUGUAAGGAUCCAUAAUGCTT-3ʹ; SLP-2 deﬁciency affected the ubiquitination of intramitochondrial siRNA3 SLP-2, sense: 5ʹ-CCAGCGAUGUGACAAGUAUTT-3ʹ, anti-sense: 5ʹ- proteins. ubiquitination is widely involved in the processes of AUACUUGUCACAUCGCUGGTT-3ʹ; siRNA NC, sense: 5ʹ-UUCUCCGAACGU- apoptosis and autophagy. We speculate that SLP-2 deﬁciency GUCACGUTT-3ʹ, anti-sense: 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ) were pur- chased from GenePharma biotech Co., Ltd (Suzhou, China) and transfected regulates autophagy by affecting the level of ubiquitination, which into H9C2 cells using Lipofectamine3000 (Invitrogen, Thermo Fisher aggravates cardiac remodeling after I/R injury. The speciﬁc Scientiﬁc, USA). In short, 5 μL siRNA or 5 μL Lipofectamine 3000 were mechanism remains to be veriﬁed by further research. diluted in 62.5 µL Opti-MEM (Gibco, Thermo Fisher Scientiﬁc, USA) and Finally, by silencing SLP-2 expression in H9C2 cells, we veriﬁed incubated at room temperature for 5 min before being mixed and that low SLP-2 expression leads to increased apoptosis level and Cell Death Discovery (2023) 9:63 Y. Hu et al. incubated for another 15 min. H9C2 cells were then cultured with the proteintech, USA), Bcl-2 (26593-1-AP, proteintech, USA), Bax (2772 S, cell mixture at 37 °C for 4 h and then replaced with fresh preheated DMEM signaling technology, USA), Cleaved-caspase-3 (9661 S, cell signaling medium for 48 h. The cell proteins were harvested for western blot analysis technology, USA), LC3B (ab51520, abcam, USA), Beclin1 (66665-1-Ig, to select the most effective sequences for subsequent experiments. proteintech, USA), ATG5 (12994 S, cell signaling technology, USA), PINK1 To inhibit autophagy, successfully transfected H9C2 cells were (23274-1-AP, proteintech, USA), Parkin (66674-1-Ig, proteintech, USA), pretreated with 10 mM 3-methyladenine (3-MA, MCE, USA) for 4 h, then Mfn2 (12186-1-AP, proteintech, USA), Drp1 (12957-1-AP, proteintech, cultured in DMEM medium containing 1 μM Dox for 24 h, followed by USA), p-PI3K (4228 S, cell signaling technology, USA), PI3K (4257 S, cell follow-up experiments. signaling technology, USA), p-Akt (4060 S, cell signaling technology, USA), Akt (9272 S, cell signaling technology, USA), p-mTOR (2971 S, cell signaling technology, USA) and mTOR (2983 S, cell signaling technology, USA). The Echocardiographic evaluation membranes were washed 3 times with Tris Buffered Saline with Tween 20 Mice were anesthetized with 1.5–2.0% isoﬂurane inhalation, and the (TBST), and incubated with the secondary antibody at room temperature cardiac function indexes were measured at the papillary muscle level by for 1 h. The protein bands emerged by Immobilon western chemilumi- using the Vevo2100 instrument (VisualSonics, Canada) with M-mode nescence HRP substrates (WBKLS0500, Millipore, USA) were photographed echocardiography. Finally, left ventricular ejection fraction (EF), left using ChemiScope (Clinx Science Instruments, China). The gray value of ventricular fractional shortening (FS), left ventricular mass (LV mass), each band was measured by Chemi analysis software (Clinx Science interventricular septum (IVS) thickness, and left ventricular posterior wall Instruments, China). (LVPW) thickness were automatically calculated. ROS staining Histological analysis Levels of intracellular ROS and mitochondrial ROS (mtROS) generation Mice hearts were immersed in 4% neutral formaldehyde tissue ﬁxative for were detected using 2’,7’- dichlorodihydroﬂuorescein diacetate (DCFH-DA) 24 hours, then dehydrated with ethanole of gradient concentration and and MitoSOX (Yeasen, China). The prepared tissue sections were treated xylen, embedded in parafﬁn, and ﬁnally cut into 5μm thick slice. The with 10 M DCFH-DA for 20 min at 37 °C in the dark, humid chamber. The sections were stained with hematoxylin-eosin (HE), Masson’s trichrome and cell slides were treated with 2 mL MitoSOX red mitochondrial superoxide Picro-Sirius Red (PSR), then imaged under the light microscope to evaluate indicator for 10 min at 37 °C in the dark, humid chamber. The ROS positive the myocardial morphology and ﬁbrosis. All histological analysis were counts from four randomly chosen ﬁelds observed under a ﬂuorescence performed blind. microscope were measured. Immunoﬂuorescence (IF) staining Mice heart tissues were ﬁxed in the frozen section embedding agent TUNEL staining (O.C.T., Sakura, USA) and cut into 5μm sections. The sections were dried at The tissue sections/cell slides were incubated at room temperature with room temperature for 30 min and then ﬁxed with 4% neutral formalde- 0.2% TritonX-100 for 15 minutes, washed with 1×PBS for 3 times and hyde tissue ﬁxative for 20 min. After 1×PBS ﬂushing, the tissues were incubated with 1×Equilibration buffer at room temperature for 30 min. treated with 0.1% TritonX-100 for 5 min and 3% goat serum (ZLI-9022, Then the sections were incubated with TDT incubation buffer at room Beijing Zhongshan Biotechnology) for 1 h, then separately incubated with temperature for 1 hour. After washing with 1×PBS for 3 times, the nuclei antibodies against Vimentin (ab8978, abcam, USA), LC3B (ab51520, were stained with Hoechst33342. The reagents used are from Vazyme abcam, USA), PINK1 (23274-1-AP, proteintech, USA) and Parkin (66674-1- biotech Co., Ltd (Nanjing, China). The images were observed under a Ig, proteintech, USA) at 4 °C overnight, followed by incubation with ﬂuorescence microscope. secondary antibody for 1 h at room temperature. Finally, the nuclei were stained with Hoechst33342. The tissue sections were incubated with Determination of ATP content and serum biomarkers wheat germ agglutinin (WGA) working solution (W7024, Invitrogen, USA) The ATP content and serum biomarkers were measured strictly based on at room temperature for 10 min to detect the cardiomyocyte cross- the respective kit instructions (ATP, A095-1-1; AST, C010-2-1; ALT, C009-2-1; sectional area. The images were observed under a ﬂuorescence TBil, C019-1-1; BUN, C013-2-1; Cre, C011-2-1; MDA, A003-1-1; SOD, A001-3- microscope. 2, Nanjing Jiancheng Bioengineering Institute, China). Immunohistochemistry (IHC) staining Evans Blue/TTC Staining After dewaxing and rehydration, tissue sections were inﬁltrated with 3% The LAD was re-ligation after I/R surgery, and 2 mL 2% Evans Blue (Sigma- hydrogen peroxide for 15 min to remove endogenous peroxidase, then the Aldrich, USA) was injected into inferior vena cava. Isolated hearts were antigen was repaired by using citric acid solution, and incubated with goat frozen at −20 °C for 10 min and cut into ﬁve pieces (approximately 1 mm serum (ZLI-9022, Beijing Zhongshan Biotechnology) for 1 h to prevent non- thick) and incubated with 1% 2,3,5-Triphenyltetrazolium chloride (TTC) speciﬁc binding of antibodies. The sections were separately incubated with solution at 37 °C for 20 min. Finally, the heart sections were ﬁxed with 4% primary antibodies against Collagen I (14695-1-AP, proteintech, USA), α- neutral formaldehyde for 2 h, and the stained areas were analyzed by SMA (A5228, sigma-aldrich, USA) and Collagen III (22734-1-AP, proteintech, ImageJ software. USA) at 4 °C overnight, followed by goat anti-rabbit or anti-mouse IgG (KIT- 5004 and KIT-5001, MXB, China) for 1 hour at room temperature. Finally, immunohistochemical reactions were analyzed using a DAB kit. The results Bioinformatics analysis of IHC staining were evaluated using ImageJ software. The negative result The proteomic and bioinformatics analyses were performed by Applied score of staining intensity was 0, while the low positive, positive or high Protein Technology Co., Ltd (Shanghai, China). positive result scores were 1, 2 and 3, respectively. The score of positive staining area <5% was 0, when the area of positive staining area is 5–25%, Detection of mitochondrial membrane potential 26–50%, 51–75% and >75%, scores were 1, 2, 3 and 4, respectively. The H9C2 cells were incubated with JC-1 working solution (C2006, Beyotime, ﬁnal total score is the product of the intensity score and the area score. China) at 37 °C for 20 minutes, then washed with JC-1 buffer for 3 times and observed under ﬂuorescence microscope. Western blot analysis The mice left ventricular tissues were lysed with RIPA buffer on ice, and Statistical analysis then the protein concentration was measured using a BCA protein assay Results are expressed as mean ± standard error of the mean. The kit (KGP902, KeyGEN BioTECH, China). Equal amounts of proteins (30 μg) Shapiro–Wilk test was used to evaluate the normality of data distribution. were separated by 8%–12% SDS-PAGE and transferred to PVDF Student’st test was used for comparison between the two groups. membrane. After sealed with 5% skimmed milk for 1 h at room Multiple group comparisons were performed using 1-way ANOVA, temperature, membranes were incubated with following primary anti- bodies overnight at 4 °C: α-SMA (A5228, sigma-aldrich, USA), Collagen I followed by the Tukey test. All statistical analyses were performed (14695-1-AP, proteintech, USA), Collagen III (22734-1-AP, proteintech, with GraphPad Prism 8.0 software (GraphPad Software Inc.). We USA), Fibronectin (15613-1-AP, proteintech, USA), GAPDH (HRP-60004, considered P < 0.05 as statistically signiﬁcant. Cell Death Discovery (2023) 9:63 Y. Hu et al. DATA AVAILABILITY 30. Sulkshane P, Ram J, Glickman MH. 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Cardiac ﬁbroblast-speciﬁc knockout of ADDITIONAL INFORMATION PGC-1α accelerates angii-induced cardiac remodeling. Front Cardiovasc Med. Supplementary information The online version contains supplementary material 2021;8:664626. available at https://doi.org/10.1038/s41420-023-01350-z. 61. Rosin NL, Sopel M, Falkenham A, Myers TL, Légaré JF. Myocardial migration by ﬁbroblast progenitor cells is blood pressure dependent in a model of angII Correspondence and requests for materials should be addressed to Rui Wang or myocardial ﬁbrosis. Hypertens Res. 2012;35:449–56. Zhibing Qiu. 62. Deng L, Liu W, Xu Q, Guo R, Zhang D, Ni J, et al. Tianma Gouteng Decoction regulates oxidative stress and inﬂammation in AngII-induced hypertensive mice Reprints and permission information is available at http://www.nature.com/ via transcription factor EB to exert anti-hypertension effect. Biomed Pharmac- reprints other. 2022;145:112383. 63. Calderón-Sánchez EM, Falcón D, Martín-Bórnez M, Ordoñez A, Smani T. Urocortin Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims role in ischemia cardioprotection and the adverse cardiac remodeling. Int J Mol in published maps and institutional afﬁliations. Sci. 2021;22:12115. 64. Bai Y, Wu J, Yang Z, Wang X, Zhang D, Ma J, Mitochondrial quality control in cardiac ischemia/reperfusion injury: new insights into mechanisms and implica- tions. Cell Biol Toxicol. 2022.[Epub ahead of print]. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give ACKNOWLEDGEMENTS appropriate credit to the original author(s) and the source, provide a link to the Creative Thanks to all the staff in the lab for their help. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory AUTHOR CONTRIBUTIONS regulation or exceeds the permitted use, you will need to obtain permission directly YH and HJ designed and performed the experiments, and wrote the original from the copyright holder. To view a copy of this license, visit http:// manuscript. YX, GC and RF designed and constructed animal disease models. YZ, YL, creativecommons.org/licenses/by/4.0/. YY and RL performed experiments and analyzed data. WC, KZ and XC provided experimental guidance and suggestions. RW and ZQ reviewed data, gave writing guidance and provided important suggestions. © The Author(s) 2023 Cell Death Discovery (2023) 9:63
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