Translocation of Connexin 43 to the Inner Mitochondrial Membrane of Cardiomyocytes Through the Heat Shock Protein 90–Dependent TOM Pathway and Its Importance for Cardioprotection

Abstract

Abstract-We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia-and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning. Key Words: mitochondria Ⅲ heat shock protein Ⅲ geldanamycin Ⅲ connexin 43 Ⅲ TOM (Translocase of the Outer Membrane) complex C ardiomyocyte death during acute coronary syndromes determines survival and quality of life of patients with coronary artery disease. 1 In the majority of these patients, cardiomyocyte death is the consequence of transient, prolonged ischemia, and there is strong evidence that a substantial part of cell death occurs at the time of reperfusion. 2,3 Preconditioning, a state of increased resistance against cell death induced by ischemia-reperfusion, is elicited by brief ischemia/reperfusion episodes or by certain pharmacological stimuli and has received particular attention. 4 A wealth of information has been collected on the molecular mechanisms involved in preconditioning, but many aspects of the signaling pathways and of the end effectors of the protection remain unknown. 4,5 An intriguing and unresolved aspect is the involvement of connexin 43 (Cx43), the protein forming gap junctions connecting adjacent ventricular cardiomyocytes, 6,7 in the genesis of preconditioning. 8,9 The protection of preconditioning is abolished in Cx43-deficient mice 10 but also in isolated cardiomyocytes from Cx43-deficient hearts, 11 indicating that it cannot be explained by effects of preconditioning on gap junction-mediated cell-to-cell c...

Identification of rab20 as a Potential Regulator of Connexin43 Trafficking

“…The cell response to 4-PBA is complex and includes changes in gene expression and inhibition of histone acetylation (Chen et al 1997;Wright et al 2004), which can also affect Cx43 expression Asklund et al 2004;Hattori et al 2007;Khan et al 2007). Interestingly, geldanamycin, an agent also shown to alter Cx43 regulation (Carystinos et al 2003;Lidington et al 2000Lidington et al , 2002Rodriguez-Sinovas et al 2006) had no effect on 3(3) cells.…”

Protein kinase Cε mediates salutary effects on electrical coupling induced by ischemic preconditioning

“…43 PKCε overexpression also modulates expression of an inducible form of the chaperone protein, heat shock protein 70 (Hsp70), in rat neonatal cardiomyocytes 44 raising the possibility that PKCε regulates the formation of a Cx43-Hsp90-Hsp70 complex for translocation to the inner mitochondrial membrane. 43 It is important to note that the significance of Cx43 phosphorylation at Ser368 in the heart remains controversial as several groups have reported conflicting findings. 26,45,46 Our finding that Cx43 phosphorylation at Ser368 increases after 30 min of global ischemia is consistent with a recent study, also in the mouse, from Ek-Vitorin et al 26 However, Matsushita et al and Axelsen et al report decreased Cx43 phosphorylation at Ser368 in the rat heart subjected to hypoxic perfusion 45 or global ischemia.…”

Recurrent and founder mutations in the Netherlands

“…This was also true for HSP90, a protein that interacts with Cx43 and is known to be essential for the survival of cardiomyocytes. 29,30 Finally, the expression of p.Arg79X led to the inability of the mutated PKP2 protein to interact with both DSP and Cx43. Together, these results suggest that the p.Arg79X mutation leads to a loss of myocardial integrity characteristic of ARVC/D.…”