Abstract 1488: Inhibition of Endosomal Trafficking Alters PC-mediated Phosphorylation and Expression of ATP synthase, Complex I, PDH, and 3-ketoacyl-CoA-thiolase
The mechanisms by which preconditioning (PC) decreases injury during a subsequent sustained ischemia are not well understood. It has been proposed that PC initiates signaling cascades which converge on mitochondrial end-effectors and result in cardioprotection. We previously observed that inhibiting endosomal recycling by bafilomycin (Baf) blocks PC. We propose that endosomal signaling may facilitate signaling to the mitochondria. To test this hypothesis, we utilized 2D electrophoresis and mass spectrometry to detect differences in protein levels and post-translational modifications of mitochondrial fractions from Langendorff perfused mouse hearts preconditioned (+/− Baf) with 4 cycles of 5 min ischemia and 5 min reflow and time matched controls. Using comparative gel analysis, we focused on proteins altered in PC but not in control or PC + Baf hearts. Levels of ATP synthase beta and complex I were increased in PC relative to control and PC + Baf. In contrast, 3-ketoacyl-CoA-thiolase expression was decreased in PC compared to control and PC + Baf. PC + Baf hearts also exhibited reduced pyruvate dehydrogenase (PDH) levels. These differences appear to be due to post-translational modifications as evident by protein isoelectric point shifts. Consistent with the shifts, Pro-Q Diamond phosphoprotein staining revealed PC hearts relative to control and PC + Baf had reduced PDH phosphorylation and enhanced phosphorylation of ATP synthase beta, complex I, and 3-ketoacyl-CoA-thiolase; these proteins have been implicated in cardioprotection. Because the ATP synthase (F1F0 ATPase) has been shown to run in reverse and consume ATP during ischemia, decreased F1F0 ATPase activity during ischemia would reduce ischemic ATP breakdown and promote cardioprotection. Modulation of complex I activity has also been associated with protection. PDH activation and 3-ketoacyl-CoA-thiolase inhibition have been shown to be beneficial, reportedly by stimulating glucose oxidation. Our data suggests that PC is associated with alterations in phosphorylation and expression levels of ATP synthase beta, complex I, PDH, and 3-ketoacyl-CoA-thiolase, which can be influenced by endosomal trafficking.