Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes

Abstract

Protection achieved by ischemic preconditioning is dependent on A_2B adenosine receptors (A_2BAR) in rabbit and mouse hearts and, predictably, an A_2BAR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A_2BAR. The present study tested whether A_2BAR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A_2BAR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A_2BAR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A_2BAR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A_2BAR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A_2BAR on the cardiomyocyte’s sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A_2BAR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A_2BAR band. Thus, the present study demonstrates that activation of A_2BAR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A_2BAR are expressed in individual cardiomyocytes. However, surprisingly, A_2BAR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A_2BAR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.