No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning

Totzeck A, Boengler K, van de Sand A, Konietzka I, Gres P, Garcia-Dorado D, Heusch G, Schulz R. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning. Am J Physiol Heart Circ Physiol 295: H2106-H2112, 2008. First published October 3, 2008; doi: 10.1152/ajpheart.00456.2008.-Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1 alpha, PP2A alpha, and PP2B alpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in % area at risk) in left ventricular anterior myocardium of Gottinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [ n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2A alpha and PP1 alpha, but not PP2B alpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2A alpha but not with PP1 alpha. Although the total PP2A alpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2A alpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2A alpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2A alpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.