Proteomics characterization of the cardiac sarcolemma

As the plasma membrane of the cardiac muscle cell (sarcolemma) plays a major role in the cardiac physiology and homeostasis, the aim of this study is to identify using mass spectrometry (MS) and data base mining the integral and peripheral proteins that are making its composition and to uncover putative novel proteins and to ultimately reveal the differences in protein composition between the sarcolemma of control rat hearts and hearts submitted to ischemia and ischemia-reperfusion to find disease-specific markers. Toward this goal we have developed a new method to purify cardiac sarcolemma using an approach that avoids denaturing conditions and combines subcellular fractionation and immunoadsorption: An initial cardiac sarcolemma preparation obtained by sucrose gradient centrifugation was enriched 27-fold in 5-nucleotidase activity and 5-12-fold in sarcolemmal specific markers. Subsequently, this enriched preparation was incubated with antibodies directed against intercalated disc proteins, N-cadherin and Connexin43, and then immunoadsorbed to superparamagnetic beads (Dynabeads Pan Mouse IgG). Samples from the enriched sarcolemma and immunoabsorbed fractions were separated by 1-D SDS-PAGE and in-gel digested with trypsin. The tryptic fragments were identified, analyzed and assigned to proteins using tandem MS-MS spectrometry in combination with Mascot search software. Out of the 518 identified proteins, 40% were attributed to the sarcolemma and associated cytoskeleton, of which 65% were peripheral proteins and the remainders were either membrane spanning or lipid anchored proteins. Further functional classification of the sarcolemma proteins indicates that the majority (65%) are involved in signaling, trafficking and cell adhesion. The MS analysis also reveals the presence of 71 novel proteins, which are currently being evaluated as relevant candidates for further study. Comparing the sarcolemma and immunoselected intercalated disc preparations; we noticed that the distribution of protein abundance in all of the localization and functional categories follows almost the same trend. This led us to conclude that in order to have an easy, fast and a reliable technique to compare different heart states in terms of sarcolemma protein composition the sarcolemma preparation would serve just as well as an immunoselected preparation with less time, effort and materials. In order to find out differences in protein composition between normal sarcolemma and sarcolemma submitted to global ischemia/ischemia-reperfusion; sarcolemma fractions (SF) obtained from Langendorff rat heart models were analyzed by mass spectrometry. The results of this study revealed that global ischemia induced an increase in calcium binding proteins (i.e. annexins) concomitantly with a decrease in transport proteins (i.e. Na/K ATPase). After ischemia, the signaling proteins category showed a decrease followed by a return to the control level after reperfusion. The amount of G alpha inhibiting 2 protein, caveolin, flotilin and myosin varied significantly during the ischemia/reperfusion protocol