Alterations to Cardiac Muscle Function and Sarcomeric Proteins Following Myocardial Infarction

Post-translational modifications of the proteins that make up the cardiac sarcomere have been suggested as a source of pathological muscle dysfunction. Reactive oxygen species (ROS) can induce post-translational modifications to proteins, and an increase in ROS levels is recognized as a feature of heart failure following myocardial infarction (MI). The experiments described here test the hypothesis that MI exerts a functional effect through alteration of myofibrillar proteins, which can be detected within days after the infarction. Experimental MI was induced by ligation of the left anterior descending coronary artery in 6-month old female CD1 mice. Samples were collected 3-4 days after ligation or sham surgery (n = 10). We performed functional analysis through force-calcium measurements of detergent-extracted fiber bundles (“skinned fibers”) dissected from non-infarcted papillary muscle. Our findings included an increase in Ca++ sensitivity in fibers from MI hearts compared to those from sham-operated animals and a decreased cooperativity of activation (p < 0.05). Biochemical data derived from electrophoresis of isolated myofibrillar proteins from these hearts revealed both oxidation and modified phosphorylation. We used ProQ Diamond phosphoprotein gel stain to analyze myofilament protein phosphorylation, and nonreducing-reducing “diagonal” SDS-PAGE to detect the formation of disulfide products. Total troponin I phosphorylation levels were decreased after MI (p < 0.01). We observed evidence of increased tropomyosin oxidation by reversible modification of sulfhydryls, and confirmed the oxidized protein's identity using mass spectrometry. These data characterize the relatively unexplored structural and functional modifications to sarcomeres in the early aftermath of MI, and may provide insight into the initial changes that trigger remodeling and heart failure, as well as the contribution of ROS to this process.