| Contraction of cardiac muscle is achieved through the coordinated and tightly regulated interactions of the contractile proteins, including actin, myosin, tropomyosin, and troponin. The three subunits of troponin, troponin I (TnI), troponin T, and troponin C, collectively serve as the calcium sensor of the contractile apparatus, which 'turns on' contraction with elevated cytosolic calcium. Diseases of the myocardium which result in alterations to contractile function may therefore result from modifications to these proteins. In particular, acute episodes of blood flow occlusion to the myocardium (ischemia), followed by subsequent restoration of perfusion (reperfusion) result in depressed cardiac function, the severity of which depends upon the degree and duration of the ischemic insult.; Here we have found that TnI undergoes progressive and selective posttranslational modification with increasing severity of ischemia in the isolated perfused rat heart, in particular a C-terminal proteolytic cleavage that occurs with mild ischemia/reperfusion (known as stunning). The clinical relevance of this phenomenon was then explored by analysis of myocardial biopsies from coronary artery bypass patients. Similar post-translational modifications to TnI, including C-terminal proteolytic cleavage, were present both before and after the surgical procedure.; Since many other proteins are likely to be modified under ischemia/reperfusion, a proteomic approach to the analysis of myocardial biopsy samples (from bypass patients) was developed. Previously limited by the small amount of protein in the biopsy sample, we have applied a sequential Coomassie-Silver staining procedure coupled with a technique for removal of silver stain that will permit concurrent mass spectrometry and western blotting of a single 2-dimensionally separated protein spot. In addition, we have optimized the separation and detection of troponin T, which along with TnI has presented particular challenges to proteomic analysis.; These studies lay the foundation for the application of proteomics to analysis of small biopsy samples, and contractile proteins in particular. Degradation of TnI is already in the process of becoming a new marker for cardiac disease, and the identification of more disease-induced protein modifications may provide additional diagnostic tools and therapeutic targets for myocardial ischemic conditions. |
