Protein densitometry profiling in mouse hearts carrying an HCM cardiac troponin T mutation.

Abstract

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases caused by mutations in over a dozen genes encoding sarcomere-associated proteins. The cardiac troponin T (TnT) I79N mutation has been linked to familial HCM in humans and in transgenic mice expressing the mutant human TnT. Many studies from molecular to physiological assays have shown altered cardiac myofilament performance, increased myofilament Ca2+ sensitivity, enhanced cardiac contractility, and impaired relaxation in mice carrying the cTnT-I79N mutation. However, the overall protein expression profile by which a gene mutation causes the disease pathogenesis is still not well understood. In this study we aim to apply protein densitometry profiling of myocardial tissue from non-transgenic (NTg) mice and transgenic mice bearing cTnT-I79N to explore global alterations in cellular and myofibrillar protein expression levels. We used the traditional 1D SDS-PAGE in conjunction with protein identification by mass spectrometry. Whole cell protein extract was obtained from seven hearts of NTg mice and seven HcTnT-I79N mice of 5 months of age. After electrophoresis, gels were stained with Coomassie brilliant blue and imaged for densitometry analysis. ImageJ was used to determine the relative peak areas of the different gel bands. From cell extract, we observed a wide range of protein abundances including the presence of myofilament proteins. Thus our next goal is to extract myofibril proteins to acquire more distinct peaks, and to determine the myofibrillar protein ratios in NTg and I79N hearts. Secondary confirmation of each myofibrillar protein expression levels will be done with western blot and mass spectrometry. This work on the analysis of the major proteins in the HCM heart can provide new insights into cellular mechanisms involved in cardiac dysfunction.