Inhibition of HSC70 alleviates hypertrophic cardiomyopathy pathology in human induced pluripotent stem cell‐derived cardiomyocytes with a MYBPC3 mutation

We performed a comprehensive study to assess the pathogenicity of a cardiac myosin binding protein C3 (MYBPC3) variant (L460fs) and to pinpoint underlying molecular mechanisms by utilizing human-induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model. L460fs iPSC-CMs exhibited a variety of deleterious phenotypes in response to angiotensin II (Ang II), including reduced MYBPC3 expression, hypertrophy, arrhythmia and elevated diastolic intracellular Ca2+ [Ca2+]i. Mechanistically, heat shock protein family A (HSP70) member 8 (HSC70) accelerated MYBPC3 degradation via lysosomal pathway under Ang II stress. The reduced MYBPC3-binding ryanodine receptor 2 (RYR2) caused by insufficiency of MYBPC3 protein may give rise to excessive free destabilized RYR2, which in turn promoted RYR2-mediated Ca2+ leak. The resultant elevated Ca2+ loading may trigger the development of both hypertrophy and arrhythmogenesis, particularly under stress conditions.1 Hypertrophic cardiomyopathy (HCM), featured by asymmetric ventricular hypertrophy, arrhythmias and sudden cardiac death (SCD), is the most common form of inherited cardiac disease.2, 3 To note, HCM has been repeatedly regarded as a major cause of SCD in young people.4 MYBPC3, which encodes cardiac myosin-binding protein C, is the most frequent HCM-associated gene, accounting for more than 50% of the HCM patients.5 However, the exact mechanism of MYBPC3-related HCM remains to be resolved.6, 7 In this study, a MYBPC3 variant (c.1377delC; p.L460fs) was identified in four-unrelated individuals who developed HCM in middle age (Figure 1A,B). The echocardiography and cardiac magnetic resonance imaging exhibited normal ventricular function, severe interventricular septum hypertrophy but without left ventricular outflow tract obstruction (Figure 1C, Figure S1A-D and Table S1). With treatment of β blockers or Ca2+ channel blockers, the symptoms were significantly relieved. Family history of SCD was presented in family 1 and 4 (Figure 1A). For proband 1, implantable cardioverter defibrillator (ICD) was implanted to prevent SCD, and radiofrequency ablation was applied to eliminate atrial fibrillation. Proband 4 suffered a lethal ventricular fibrillation before ICD implantation with much more diffused late gadolinium enhancement and frequent non-sustained ventricular tarchycadia (VT) compared to others (Figure 1C,D). Trans-epicardial ablation was applied to relief the burden of VT. As for proband 2 and 3, only mild symptom was presented and medical therapy was sufficient (Figure S1E and Table S1). To characterize the MYBPC3 variant in vitro, we created human WT and L460fs MYBPC3 constructs, which were overexpressed in healthy iPSC-CMs (Figure 1E-J). A known mutation (G853fs), previously identified as pathogenic in HCM patients, was utilized as a positive control.8 Both mutations are nonsense, resulting in truncated proteins that can be observed when overexpressed in human embryonic kidney 293T cells (Figure 1I). Total mRNA level of MYBPC3 was significantly decreased in both mutant iPSC-CMs as compared to WT (Figure 1K and Table S2). Baseline characteristics of WT and mutant iPSC-CMs were comparable (Figure 2 and Figures S2-S3). However, Ang II-treated mutant iPSC-CMs showed clear hypertrophy phenotype, including enlarged cell size and significant up-regulation of hypertrophic marker expression (Figure 2A-F and Figures S2-S3). Along with the hypertrophy phenotype, MYBPC3 protein expression was significantly decreased in Ang II-treated mutant iPSC-CMs (Figure 2G-H). Moreover, fura-2 ratiometric Ca2+ imaging demonstrated a predisposition of arrhythmia-like Ca2+ transients and higher level of diastolic [Ca2+]i in Ang II-treated mutant iPSC-CMs (Figure 2I-K, Figure S4A-D and Table S3). It has been reported that MYBPC3 may bind to RYR2 to form a complex for stabilizing RYR2-dependent Ca2+ release.9 Notably, we found that largely enhanced RYR2-mediated Ca2+ leak contributed to Ca2+ handling abnormalities in Ang II-treated mutant iPSC-CMs, whereas sarcoplasmic reticulum (SR) Ca2+ load was unchanged (Figure 2L-N, Figure S4E and Table S4). We next performed genome-wide RNA sequencing by comparing Ang II-treated WT and L460fs iPSC-CMs (Figure 3A and Figure S5). A significant enrichment of pathways involved in cardiac hypertrophy and cardiac muscle contraction was detected, suggesting a common gene signature of HCM in Ang II-treated L460fs iPSC-CMs (Figure 3B-C). Interestingly, heat shock cognate 70 kDa (HSC70) expression was significantly up-regulated in Ang II-treated L460fs iPSC-CMs at both mRNA and protein levels (Figure 3D-G and Figure S6A-C). To investigate if changes of HSC70 expression may give rise to functional consequences, either HSC70 activator (YM-1) or inhibitor (VER-155008) was applied to WT and mutant iPSC-CMs. Addition to YM-1 in mutant iPSC-CMs caused hypertrophic response whereas VER-155008 treatment or HSC70 knockdown in Ang II-treated mutant iPSC-CMs effectively rescued the hypertrophy phenotype (Figure 3H-K, Figure S6D-G and Figure S7). Nuclear translocation of nuclear factor of activated T cells triggered by Ang II was significantly inhibited by VER-155008 treatment (Figure S8). Moreover, elevated diastolic [Ca2+]i and enhanced RYR2-mediated Ca2+ leak in Ang II-treated mutant iPSC-CMs were markedly restored by VER-155008 treatment (Figure 3L-O and Table S4). It has been previously shown that HSC70 interacted with MYBPC3 and participated in its degradation.10 We thus reasoned that up-regulated HSC70 may accelerate MYBPC3 degradation. When applied to YM-1, mutant iPSC-CMs showed significantly reduced MYBPC3 protein expression in comparison with WT (Figure 4A,B). However, MYBPC3 protein expression in Ang II-treated mutant iPSC-CMs was markedly restored by VER-155008 treatment (Figure 4C,D). We further assessed the effect of HSC70 on MYBPC3 degradation in healthy iPSC-CMs. After cycloheximide (CHX) treatment, protein expression level of MYBPC3 in healthy iPSC-CMs was dramatically reduced at baseline, which can be partially rescued by HSC70 inhibition (Figure 4E and Figure S9A). Notably, treatment of chloroquine (CQ) but not MG-132 greatly rescued the CHX-induced MYBPC3 degradation in healthy iPSC-CMs (Figure 4F and Figure S9B). Collectively, these results suggest that increased HSC70 accelerates MYBPC3 degradation via lysosomal pathway in response to Ang II, while HSC70 inhibition restores MYBPC3 expression, thus alleviating hypertrophy phenotype and Ca2+ mishandling in mutant iPSC-CMs (Figure 4G). In conclusion, we present accelerated HSC70-mediated MYBPC3 protein degradation as a novel mechanism of enhanced diastolic Ca2+ leak from RYR2 in HCM, leading to hypertrophy and aberrant Ca2+ handling. Our findings will be helpful for elucidating the molecular mechanisms underlying MYBPC3-related HCM and for identifying novel therapeutic drugs for the disease. We would like to thank the core facility of Zhejiang University Institute of Translational Medicine for assistance with flow cytometry and confocal microscopy experiments. Ping Liang would like to thank Natalie Liang and Michael Liang for their encouragement and consistent support. National Key R&D Program of China, Grant Number: 2017YFA0103700; National Natural Science Foundation of China, Grant Numbers: 81922006, 81870175 and 81970269; Natural Science Foundation of Zhejiang Province, Grant Number: LD21H020001; National Natural Science Foundation of China, Grant Number: 81970269; Key Research and Development Program of Zhejiang Province, Grant Number: 2019C03022. The authors declare that they have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.