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...

Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.

Quantitative fluorescent PCR (QF-PCR

The presence of a PCO morphology in women with a PP history was found to relate to prenatal growth. It would be of interest to verify whether a similar relationship exists in anovulatory and/or hyperandrogenic women without PP history.