MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

BACKGROUNDMYBPC3, 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 MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). 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 treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, 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 MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG