A Hypertrophic Cardiomyopathy-associated MYBPC3 Mutation Common in Populations of South Asian Descent Causes Contractile Dysfunction

• Published in: The Journal of biological chemistry 2015-02, Vol.290 (9), p.5855-5867
• Main Authors: Kuster, Diederik W.D., Govindan, Suresh, Springer, Tzvia I., Martin, Jody L., Finley, Natosha L., Sadayappan, Sakthivel
• Format: Article
• Language: English
• Subjects: Animals; Asia; Asian Continental Ancestry Group - genetics; Cardiomyopathy; Cardiomyopathy, Hypertrophic - ethnology; Cardiomyopathy, Hypertrophic - genetics; Cardiomyopathy, Hypertrophic - physiopathology; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Size; Cells, Cultured; Contractile Protein; Genetic Polymorphism; Genetic Predisposition to Disease - ethnology; Genetic Predisposition to Disease - genetics; Heart Failure; Humans; Immunoblotting; Male; Microscopy, Fluorescence; Models, Molecular; Molecular Bases of Disease; Muscle Contraction - genetics; Muscle Physiology; Mutation; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Myosin Subfragments - metabolism; Protein Binding - genetics; Protein Structure, Tertiary; Rats, Sprague-Dawley; Sarcomeres - metabolism; Subcellular Fractions - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.607911

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-CC10mut), 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-CC10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca2+ transients, compared with wild-type cMyBP-C expression (cMyBP-CWT) and controls. Forty-eight hours after infection, 44% of cMyBP-CWT and 36% of cMyBP-CC10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-CC10mut to the C-zone, whereas cMyBP-CWT mostly showed C-zone staining, suggesting that cMyBP-CC10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-CC10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-CC10mut 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 C10mut 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. A 25-base pair deletion in the MYBPC3 gene is the most prevalent hypertrophic cardiomyopathy-associated mutation among South Asian populations. Expression of mutant protein (cMyBP-CC10mut) in cultured adult rat cardiomyocytes led to improper localization and contractile dysfunction. cMyBP-CC10mut expression is sufficient to cause contractile dysfunction. The study sheds light on the etiology of this ethnic-specific hypertrophic cardiomyopathy mutation.