Authors: | Kuster, D. W.; Govindan, S.; Springer, T. I.; Martin, J. L.; Finley, N. L.; Sadayappan, S |
Article Title: | A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction |
Abstract: | 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. |
Journal Title: | The Journal of biological chemistry |
Volume: | 290 |
Issue: | 9 |
ISSN: | 1083-351X; 0021-9258 |
Publisher: | The American Society for Biochemistry and Molecular Biology |
Date Published: | 2015 |
Start Page: | 5855 |
End Page: | 5867 |
Language: | ENG |
DOI/URL: |
jbc.M114.607911 |
Notes: | LR: 20150115; CI: Copyright (c) 2015; JID: 2985121R; OTO: NOTNLM; aheadofprint |