MYBPC3D389V Variant Induces Hypercontractility in Cardiac Organoids

Abstract

AbstractBACKGROUNDMYBPC3, 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, additionalin vitrostudies 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) inMYBPC3by 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 theMYBPC3D389Vvariant. 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 assaysin vitro.RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of theMYBPC3D389Vvariant 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 expressingMYBPC3D389Vthan NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation inMYBPC3D389VhCOs 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 treatment with the myosin inhibitor mavacamten (CAMZYOS®) inMYBPC3D389VhCOs. Lastly, variousin vitrobinding assays revealed a significant loss of affinity in the presence ofMYBPC3D389Vwith 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 theMYBPC3D389Vhypercontractile phenotype, which was rescued by administration of a myosin inhibitor.Novelty and SignificanceWhat Is Known?MYBPC3mutations have been implicated in hypertrophic cardiomyopathy.D389V is a polymorphic variant ofMYBPC3predicted 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.The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute?The 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.TheMYBPC3D389V-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.Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced byin vitrostudies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor.In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes.Early treatment ofMYBPC3D389Vcarriers with mavacamten may prevent or reduce early HCM-related pathology.GRAPHICAL ABSTRACTA graphical abstract is available for this article.