MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

<i>MYBPC3</i>, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in <i>MYBPC3</i> by using isogenic human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, functional, and energetic changes caused by the <i>MYBPC3</i>^D389V variant, which is associated with increased fractional shortening and highly prevalent in South Asian descendants. Recombinant C0-C2, N' region of cMyBP-C (wild-type and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the <i>MYBPC3</i>^D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility, faster calcium cycling, and faster contractile kinetics in hCOs expressing <i>MYBPC3</i>^D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in <i>MYBPC3</i>^D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after the treatment of the myosin inhibitor mavacamten (CAMZYOS^®) in <i>MYBPC3</i>^D389V hCOs. Lastly, various vitro binding assays revealed a significant loss of affinity in the presence of <i>MYBPC3</i>^D389V with myosin S2 region as a likely mechanism for hypercontraction. Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the <i>MYBPC3</i>^D389V hypercontractile phenotype, which was rescued by the administration of a myosin inhibitor.