A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction [Protein Synthesis and Degradation]

January 12th, 2015 by Kuster, D. W. D., Govindan, S., Springer, T. I., Martin, J. L., Finley, N. L., Sadayappan, S.

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-basepair deletion in MYBPC3 is inherited in millions worldwide. While 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 to 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, while 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, as Ca2+-transients were unaffected. Co-sedimentation and protein crosslinking 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.
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