Abstract 14422: Sarcomere Gene and Protein Expression Studies Suggest Mutation-Specific Disease Mechanisms in Human Hypertrophic Cardiomyopathy
Heterozygous mutations in sarcomere genes cause hypertrophic cardiomyopathy (HCM), although the mechanisms by which these mutant proteins exert their effects in human disease are largely unknown. We hypothesize that heterozygous expression of sarcomere gene mutations in HCM alters sarcomere stoichiometry in a gene and mutation-specific manner. Sarcomere transcript and protein expression was analyzed in septal myectomy specimens from HCM patients with no sarcomere mutations (n=10), missense and nonsense mutations in cardiac myosin binding protein C (MyBPC, n=15) and missense mutations in myosin (n=4), troponin T (n=1), tropomyosin (n=1), and myosin light chain 2 (n=1). By qrtPCR, total MyBPC mRNA expression was significantly reduced in samples with nonsense (truncating) MyBPC mutations compared to control hearts (0.78 ± 0.06 vs 1.12 ± 0.02 respectively, P<0.05). Next, we used either qrtPCR or single base extension with the Sequenom MassARRAY platform to examine the relationship between wild-type and mutant sarcomere transcripts within each sample. With one notable exception (Asp1076fs), nonsense mutant transcripts in MyBPC consistently accounted for < 20% of total MyBPC mRNA, implying eradication by nonsense mediated RNA decay. In contrast, missense mutant transcripts in all genes were present in a ~1:1 ratio with wild-type transcripts. By western blotting, total MyBPC protein content was reduced in samples with MyBPC mutations (0.89 ± 0.8%) compared to HCM samples without sarcomere mutations (1.49 ± 0.21%, P<0.05) but not when compared to control samples (0.98 ± 0.05%). A linear regression model showed a high degree of correlation between MyBPC protein content and total and mutant fraction of MyBPC RNA (P=0.002). Next, we used absolute quantification of abundance (AQUA) with LC/MRM to examine relative expression of mutant and wild-type proteins. Stoichiometric ratios of full length mutant proteins relative to wild-type were strikingly variable, with fraction of the mutant protein ranging from 30-84%. These results challenge the exclusive distinction of haploinsufficiency or dominant negative effects of sarcomere gene mutations, and indicate mutation-specific mechanisms with important implications for gene-targeted therapies.
- © 2011 by American Heart Association, Inc.