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Published on: September 17, 2015
MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids
Insights
The MYBPC3 D389V variant causes hypertrophic cardiomyopathy (HCM) by increasing cardiac cell contraction. This hypercontractility, linked to reduced protein binding, can be improved with myosin inhibitors like mavacamten.
Area of Science:
- Cardiovascular Biology
- Genetics
- Stem Cell Biology
Background:
- MYBPC3 mutations are a leading cause of hypertrophic cardiomyopathy (HCM).
- The MYBPC3 D389V variant, prevalent in South Asians, is linked to hyperdynamic heart function.
- The precise molecular mechanisms of D389V-associated HCM remain unclear.
Purpose of the Study:
- Investigate the molecular mechanisms of HCM pathogenesis linked to the MYBPC3 D389V variant.
- Utilize human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs) to model the variant's effects.
- Define the cellular and functional changes caused by the MYBPC3 D389V variant.
Main Methods:
- Generated hiPSC-derived cardiomyocytes and cardiac organoids from carriers and non-carriers of the MYBPC3 D389V variant.
- Performed confocal and electron microscopy, functional assays (contraction, calcium cycling), and biochemical analyses (protein phosphorylation, oxidative stress).
- Utilized recombinant proteins for in vitro binding and motility assays and treated organoids with myosin inhibitor mavacamten.
Main Results:
- MYBPC3 D389V cardiac organoids exhibited hypercontractility, faster calcium cycling, and increased contraction velocity.
- Increased cMyBP-C phosphorylation, higher oxidative stress, and lower mitochondrial membrane potential were observed in D389V organoids.
- Reduced binding affinity between D389V cMyBP-C and myosin S2 was identified as a potential cause of hypercontraction, which was reversed by mavacamten.
Conclusions:
- Human cardiac organoids are a feasible model for studying HCM mechanisms, demonstrating the MYBPC3 D389V hypercontractile phenotype.
- Faster sarcomere kinetics due to reduced protein binding affinity underlie the hypercontractility, leading to secondary mitochondrial defects.
- Mavacamten effectively rescued the hypercontractile phenotype, suggesting potential therapeutic benefits for MYBPC3 D389V carriers.
Background:
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 human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs).
Methods:
The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V 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 .
Results:
Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V 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 MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 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 treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction.
Conclusions:
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 MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V 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. 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. The MYBPC3 D389V -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 by in vitro studies, 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 of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.
