L7	Connexin 43 and myocardial ischemia/ reperfusion injury.
1K.Boengler, 1G.Dodoni, 2A.Rodriguez-Sinovas, 2A.Cabestrero, 2M.Ruiz-Meana, 1P.Gres, 1I.Konietzka, 2D.Garcia-Dorado, 3F.Di Lisa, 1G.Heusch, 1R.Schulz
1Institut für Pathophysiologie, Universität Essen, Essen, DE; 2Servicio de Cardiologia, Hospital Vall d'Hebron, Barcelona, ES; 3Dipartimento di Chimica Biologica, Universita di Padova, Padova, IT.

Connexin 43 (Cx43) is known to be involved in the infarct size reduction by ischemic preconditioning (IP). The protein is not exclusively localized at the sarcolemma, but we have recently detected Cx43 at cardiomyocyte mitochondria, where its content is increased by IP. The aim of the present study was to identify the exact submitochondrial localization of Cx43 and the pathways by which Cx43 is potentially imported into the mitochondria. Confocal laser scan microscopy demonstrated Cx43 in pig left ventricular mitochondria devoid of the outer membrane. Western blot analysis on fractionated mitochondria for Cx43 and marker proteins of different mitochondrial compartments indicated that Cx43 is located at the inner mitochondrial membrane. An interaction of Cx43 with the translocase of the outer membrane (TOM) was shown by co-immunoprecipitation of Cx43 with TOM20, which is – beside TOM5, 6, 7, 22, 40 and 70 - part of the TOM protein complex and thereby of the mitochondrial import machinery. We then investigated 1) whether ischemia-induced changes in TOM20 occur and 2) whether such changes are affected by IP. In pigs undergoing 90 min low-flow ischemia without (n=8) or with (n=7) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion, mitochondria were isolated from the anterior ischemic and the posterior control wall and subjected to Western blot analysis. The mitochondrial protein level of TOM20 was decreased by ischemia as shown by the TOM20 ratio between the anterior and the control posterior wall of 0.56±0.15 arbitrary units. However, in mitochondria of preconditioned myocardium the TOM20 protein level was preserved (TOM20 ratio between anterior and posterior wall: 1±0.13 a.u., p<0.05). Taken together, our data show that Cx43 is located at the inner membrane of cardiomyocyte mitochondria. The preserved TOM20 protein level may reflect overall preservation of the TOM complex during ischemia following IP. The preservation of the TOM complex may enhance the translocation of Cx43 to cardiomyocyte mitochondria and may thereby contribute to the increased mitochondrial Cx43 protein level following IP.

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