SEARCHING FOR NEW PLAYERS IN CARDIOPROTECTION

Abstract

Exercise training induces adaptations that improve pump performance and protect the heart from disease-related challenges. To understand the molecular signaling mechanisms of cardioprotection, it will be necessary to identify the proteins involved, as well as the transcriptional, translational, and posttranslational changes they undergo in response to exercise. PURPOSE Our long-term goal was to identify novel cardiac proteins that regulate exercise effects. The short-term goals were to evaluate the potential of a proteomic approach, and to optimize quantitation of protein abundance and peptide mass fingerprinting (PMF). METHODS Proteins were prepared from left ventricles of 8 sedentary and 8 exercise-trained rats. Two-dimensional electrophoresis (2-DE) consisted of isoelectric focusing with immobilized pH gradient strips (pH 5–8) and vertical polyacrylamide slab gels (8–12% gradient). A modified silver-staining (SS) technique was compared with ruthenium-based (RU) fluorescent dyes for quantitative linearity and for compatibility with PMF. Automated PMF (APMF) was compared to a manual procedure (MPMF) for identification of proteins extracted from the 2-DE gels. RESULTS The average coefficient of variation (CV) between gels was 49% for SS, and 32% for RU. Log transformation reduced the CV of spot ratios for SS gels from 49% to 14%, and for RU from 32% to 3%. Using MPMF, 6 of 34 reference spots (18%) were identified. APMF identified 36 of 45 reference spots (80%). Nineteen of 22 (86%) spots from SS gels were identified, and 26 of 36 (72%) spots from RU gels were identified. Among candidate spots that differed in abundance (p < 0.05) between exercise-trained and sedentary proteomes were 3α-hydroxysteroid dehydrogenase and a 20 kDa heat shock protein. CONCLUSION Reliability in protein abundance measurements was greatest with ruthenium-based dyes and was markedly improved by log transformation of the data. APMF is superior to MPMF and is equally compatible with SS and RU. Candidate proteins for the cardioprotective effects of exercise have been identified using a proteomic approach and require further study. Supported by the American Heart Association-Midwest Affiliate