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

Dear Editor, 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 reducedMYBPC3 expression, hypertrophy, arrhythmia and elevated diastolic intracellular Ca2+ [Ca]i. Mechanistically, heat shock protein family A (HSP70) member 8 (HSC70) accelerated MYBPC3 degradation via lysosomal pathway under Ang II stress. The reduced MYBPC3binding ryanodine receptor 2 (RYR2) caused by insufficiency of MYBPC3 protein may give rise to excessive free destabilized RYR2, which in turn promoted RYR2mediated 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 (  F I G U R E 1 Identification of a myosin binding protein C3 (MYBPC3) mutation in four hypertrophic cardiomyopathy (HCM) probands and overexpression of the MYBPC3 truncation mutations in healthy induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). (A) Pedigree of four HCM-affected families carrying a MYBPC3 mutation (c.1377del; p.L460fs). Individuals who were dead are marked with a fork. The probands are indicated by red arrow. Since June 2019, 374 HCM patients who presented with significant left ventricular (LV) hypertrophy and typical symptoms such as palpitation, angina and syncope have been enrolled in our study. In family 1, the brother of index patient died suddenly. The index patient developed a paroxysmal atrial fibrillation (AF), and radiofrequency ablation was applied to restore sinus rhythm. Implantable cardioverter defibrillator (ICD) was also implanted for sudden cardiac death (SCD) prevention. After procedure, amiodaron and β-blocker were administrated, and no AF-like event has happened. The son of index patient carried the same MYBPC3 mutation but present no symptom. The fourth probands experienced a lethal ventricular fibrillation at age of 56. Typical characterization of HCM was presented in echocardiography and cardiac magnetic resonance imaging. Much more diffused late gadolinium enhancement and more frequent non-sustained VT was observed compared to other probands. Besides ICD implantation, a trans-epicardial VT ablation was applied. The radiofrequency energy was delivered to isolate the scar. Since then, the patient has recovered well without ICD discharge. The Whole-exome sequencing (WES) of his brother and daughter revealed no mutation of MYBPC3. For family 2 and 3, symptom was slight and only presented in index patients. None of the family members reported an unexpected sudden death. In these four HCM families, there is no hint for an early manifestation of HCM but seems to have a higher risk of SCD. (B) Representative Sanger sequencing results of the MYBPC3 Ca 2+ release. 9 Notably, we found that largely enhanced RYR2-mediated Ca 2+ leak contributed to Ca 2+ handling abnormalities in Ang II-treated mutant iPSC-CMs, whereas sarcoplasmic reticulum (SR) Ca 2+ 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 [Ca 2+ ] i and enhanced RYR2mediated Ca 2+ 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 Ca 2+ mishandling in mutant iPSC-CMs ( Figure 4G).
In conclusion, we present accelerated HSC70-mediated MYBPC3 protein degradation as a novel mechanism of enhanced diastolic Ca 2+ leak from RYR2 in HCM, leading to hypertrophy and aberrant Ca 2+ handling. Our findings will be helpful for elucidating the molecular mechanisms underlying MYBPC3-related HCM and for identifying novel therapeutic drugs for the disease.  Figure S9A. n = 3-5 culture replicates. *** p < .001 and **** p < .0001. (F) Bar graph to compare the MYBPC3 expression between different groups in Figure S9B. n = 4-5 culture replicates. ** p < .01, *** p < .001 and **** p < .0001. (G) Proposed work model. Ang II stress in iPSC-CMs overexpressing MYBPC3 truncation mutations (L460fs and G853fs) causes a variety of deleterious phenotypes, including reduced MYBPC3 expression, hypertrophy, arrhythmia and elevated diastolic [Ca 2+ ] i . HSC70 plays a pivotal role in the turnover of MYBPC3 via lysosomal pathway, which can be inhibited by chloroquine (CQ). The manipulation of HSC70 activity by YM-1 (HSC70 activator) or VER-155008 (HSC70 inhibitor) can regulate the content of MYBPC3 protein. In Ang II-treated mutant iPSC-CMs, the up-regulated HSC70 accelerates the MYBPC3 degradation and results in the deficiency of MYBPC3 protein, which can be partially rescued by VER-155008. 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 causes larger RYR2-mediated Ca 2+ leak. The resultant elevated Ca 2+ loading may trigger the development of both hypertrophy and arrhythmogenesis, particularly under stress conditions

A C K N O W L E D G E M E N T S
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.