Abstract 18174: Loss of Ischemic Preconditioning-Induced Cardioprotection and Protein S-Nitrosylation in Mouse Hearts by Caveolae Disruption via Methyl-β-Cyclodextrin Treatment
Recent studies suggest that caveolae, lipid and signaling molecules enriched microdomains of the plasma membrane, play an important role in cardioprotection. In this study, we found that treatment of Langendorff perfused mouse hearts with the cholesterol sequestering agent methyl-β-cyclodextrin (MβCD) to disrupt caveolae structure abolished cardioprotection induced by ischemic preconditioning (IPC). We hypothesize that cardioprotection by IPC involve changes in the structure and composition of caveolae and caveolin-3-associated signaling. We are particularly interested in nitric oxide (NO) signaling and protein S-nitrosylation (SNO), which have been found to play crucial role in IPC-induced cardioprotection. The caveolae-enriched fractions were isolated from perfusion control and IPC mouse hearts by a non-detergent sucrose ultracentrifugation and analyzed by two dimensional (2D) CyDye fluorescence difference gel electrophoresis (DIGE) proteomic method to study protein expression levels. The threshold for differences was set as greater than 1.5 fold change of labeled CyDye-fluorescence (Cy3 for perfusion control or Cy5 for IPC) vs the internal standard labeled with Cy2 consisting of equal amount of perfusion control and IPC samples. There were no differences in labeled CyDye-fluorescence between perfusion control and IPC hearts, indicating comparable protein expression between these two samples. Surprisingly, most of identified proteins by 2D CyDye DIGE in caveolae-enriched fraction were mitochondrial proteins, suggesting that caveolae might interact with mitochondria. To study SNO levels we used a modified DyLight switch and 2D DyLight DIGE. With 2D DyLight DIGE, a significant increase in protein SNO was found in caveolae-enriched fraction isolated from IPC hearts compared to perfusion control. Interestingly, disruption of caveolae structure by MβCD treatment not only blocked IPC-mediated cardioprotection, but also abolished the IPC-induced increase in SNO, suggesting an important role of caveolae and caveolin-3-associated NO/SNO signaling involved in IPC-mediated cardioprotection.
- © 2010 by American Heart Association, Inc.