A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction

Hypertrophic cardiomyopathy(HCM)results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C^C10mut), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C^C10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca²⁺ transients, compared with wildtype cMyBP-C expression (cMyBP-C^WT) and controls. Fortyeight hours after infection, 44% of cMyBP-C^WT and 36% of cMyBP-C^C10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C^C10mut to the C-zone, whereas cMyBP-C^WT mostly showed C-zone staining, suggesting that cMyBP-C^C10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C^C10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C^C10mut displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca²⁺ transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10^mut causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-CC10mut, providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-CC10mut protein is sufficient to cause contractile dysfunction in vitro.