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Published on: September 17, 2015
MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids
Darshini Desai1, Taejeong Song1, Rohit R Singh1
1Center for Cardiovascular Research, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
Insights
The MYBPC3 D389V variant causes hypertrophic cardiomyopathy (HCM) by inducing hypercontractility in cardiac organoids. This phenotype, linked to altered protein binding and mitochondrial dysfunction, was reversed by a myosin inhibitor.
Area of Science:
- Cardiovascular Biology
- Genetics
- Stem Cell Biology
Background:
- Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in the MYBPC3 gene.
- The molecular mechanisms underlying MYBPC3-associated HCM require further elucidation.
- A specific MYBPC3 variant (D389V) is prevalent in South Asian populations and linked to increased cardiac function.
Purpose of the Study:
- To investigate the molecular and cellular mechanisms of HCM caused by the MYBPC3 D389V variant.
- To utilize isogenic human-induced pluripotent stem cell-derived cardiac organoids (hCOs) for studying disease pathogenesis.
- To define the functional and energetic consequences of the MYBPC3 D389V variant.
Main Methods:
- Generation of isogenic human cardiac organoids (hCOs) from MYBPC3 D389V variant carriers and non-carriers.
- Confocal and electron microscopy for sarcomere organization analysis.
- Functional assays measuring contractility, calcium cycling, and cellular energetics.
- In vitro binding assays using recombinant proteins.
- Treatment with myosin inhibitor mavacamten.
Main Results:
- MYBPC3 D389V hCOs exhibited hypercontractility, faster calcium cycling, and accelerated contractile kinetics.
- Increased MYBPC3 phosphorylation, oxidative stress, and reduced mitochondrial membrane potential were observed in MYBPC3 D389V hCOs.
- In vitro binding assays showed reduced affinity between MYBPC3 D389V and myosin S2.
- Mavacamten treatment rescued the hypercontractile phenotype in MYBPC3 D389V hCOs.
Conclusions:
- The MYBPC3 D389V variant induces a hypercontractile phenotype in cardiac organoids.
- Altered protein binding affinity and mitochondrial dysfunction contribute to HCM pathogenesis.
- Human cardiac organoids are a viable model for studying HCM mechanisms.
- Myosin inhibition represents a potential therapeutic strategy for MYBPC3-associated HCM.
Abstract:
MYBPC3, 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 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 MYBPC3D389V 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 MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility, 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 (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 MYBPC3D389V hCOs. Lastly, various vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V 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 MYBPC3D389V hypercontractile phenotype, which was rescued by the administration of a myosin inhibitor.
