Abstract Body (Do not enter title and authors here): Introduction: Hypertrophic cardiomyopathy (HCM) is a hereditary disorder of cardiac muscle and is typically caused by mutations in genes encoding sarcomere proteins. Despite calsarcin-1 being identified as an important modulator of HCM, its role in HCM patients has not been well studied.
Hypothesis: We hypothesize that the deficiency of calsarcin-1 leads to destabilization of the muscle LIM protein/Z-disc complex, which subsequently contributes to the development of HCM.
Methods: Calsarcin-1 expression level in the HCM proband heart, cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs), was measured. Isogenic control iPSCs were generated by gene editing. In combination with viral infection and various other techniques, we explored the potential role of calsarcin-1 in HCM.
Results: Immunofluorescence staining revealed largely disrupted striated patterns of myofilaments in the MLP-W4R;MYH7-R723C proband heart, in contrast to the well-defined striated patterns and organized myofilaments observed in the healthy control (CTL) heart. In MLP-W4R;MYH7-R723C iPSC-CMs, mRNA levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) were elevated, confirming that the hypertrophic phenotype was maintained in proband iPSC-CMs. Importantly, calsarcin-1 protein expression in the proband iPSC-CMs was significantly decreased compared to that in the CTL iPSC-CMs. Moreover, ectopic expression of calsarcin-1 was able to significantly rescue the HCM phenotype, including the enlarged cardiomyocyte size and the elevated ANF and BNP mRNA levels in MLP-W4R;MYH7-R723C iPSC-CMs. Calsarcin-1 significantly enhanced MLP expression in the proband iPSC-CMs. Importantly, the effects of MLP on decreasing cell size and BNP mRNA level in the MLP-W4R;MYH7-R723C iPSC-CMs were markedly blunted when calsarcin-1 was knocked down using the shRNA lentivirus targeting calsarcin-1. Two additional iPSC lines generated from HCM patients carrying pathogenic variants in MYH7-R663H (myosin heavy chain) and MYBPC3-V321M (myosin-binding protein C) were investigated. Notably, calsarcin-1 was able to significantly reduce the hypertrophic phenotype of cardiomyocytes derived from these two iPSC lines.
Conclusions: Calsarcin-1 is reduced in MLP-W4R;MYH7-R723C iPSC-CMs. Elevating the expression level of calsarcin-1 may have a broader role in mitigating hypertrophic cardiomyopathic defects and could be an attractive therapeutic target for treating HCM.