Novel Functional Role of Heat Shock Protein 90 in Mitochondrial Connexin 43-Mediated Hypoxic Postconditioning

Abstract

Background/Aims: Previous studies have shown that heat shock protein 90 (HSP90)-mediated mitochondrial import of connexin 43 (Cx43) is critical in preconditioning cardioprotection. The present study was designed to test whether postconditioning has the same effect as preconditioning in promoting Cx43 translocation to mitochondria and whether mitochondrial HSP90 modulates this effect. Methods: Cellular models of hypoxic postconditioning (HPC) from rat heart-derived H9c2 cells and neonatal rat cardiomyocytes were employed. The effects of HPC on cardiomyocytes apoptosis were examined by flow cytometry and Hoechst 33342 fluorescent staining. Reactive oxidative species (ROS) production was assessed with the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescin in diacetate (DCFH-DA). The anti- and pro-apoptotic markers Bcl-2 and Bax, HSP90 and Cx43 protein levels were studied by Western blot analysis in total cell homogenate and sarcolemmal and mitochondrial fractions. The effects on HPC of the HSP90 inhibitor geldanamycin (GA), ROS scavengers superoxide dismutase (SOD) and catalase (CAT), and small interfering RNA (siRNA) targeting Cx43 and HSP90 were also investigated. Results: HPC significantly reduced hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. These beneficial effects were accompanied by an increase in Bcl-2 levels and a decrease in Bax levels in both sarcolemmal and mitochondrial fractions. HPC with siRNA targeting Cx43 or the ROS scavengers SOD plus CAT significantly prevented ROS generation and HPC cardioprotection, but HPC with either SOD or CAT did not. These data strongly supported the involvement of Cx43 in HPC cardioprotection, likely via modulation of the ROS balance which plays a central role in HPC protection. Furthermore, HPC increased total and mitochondrial levels of HSP90 and the mitochondria-to-sarcolemma ratio of Cx43; blocking the function of HSP90 with the HSP90 inhibitor geldanamycin (GA) or siRNA targeting HSP90 prevented the protection of HPC and the HPC-induced association of Cx43, indicating that mitochondrial HSP90 was important for mitochondrial translocation of Cx43 during HPC. Conclusion: Mitochondrial HSP90 played a central role in HPC cardioprotection, and its activity was linked to the mitochondrial targeting of Cx43, the activation of which triggered ROS signaling and the subsequent reduction of redox stress. Consequently, its target gene, Bcl-2, was upregulated, and proapoptotic Bax was inhibited in the sarcolemma and mitochondria, ultimately attenuating H/R-induced cardiomyocyte apoptosis. These data reveal a novel mechanism of HPC protection.

Ischemic Postconditioning Reduces Reperfusion Arrhythmias by Adenosine Receptors and Protein Kinase C Activation but Is Independent of KATP Channels or Connexin 43

“…Finally, our results also confirm previous works demonstrating that, as occurred with the antiarrhythmic effect of IPoC during reperfusion, Cx43 is not involved in protection against infarction induced by the maneuver [49]. However, others have suggested that mitochondrial Cx43 might play a role in hypoxic postconditioning against infarction in H9c2 cells and neonatal rat cardiomyocytes [50]. These discrepancies deserve further research in the future.…”

Acquired Resilience: An Evolved System of Tissue Protection in Mammals

“…Reinforcing the idea that HSPs are important in tissue resilience generally, they are reported to be effective in slowing muscular dystrophy in a rat model, 368,369 in slowing the development of cortical pathology in a rodent model of dementia, 370,371 in mitigating the tissue pathologies of diabetes, 372 in protecting the failing heart, 373 in reducing neuroinflammatory markers in astrocytes, and in conditioning of cardiomyocytes 374 and fibroblasts 375 in vitro. In short, HSPs are upregulated by a range of stresses and are protective to a range of tissues, ranges comparable those of the inducers of resilience and of tissues protected, considered in previous sections.…”

Doxorubicin‑induced oxidative and nitrosative stress: Mitochondrial connexin 43 is at the crossroads

“…For this purpose, H9c2 cells were treated with 1 µ M Doxo for 3 and 6 h, in both the absence and presence of radicicol, an Hsp90 inhibitor. Indeed, although it is well known that Hsp90 is involved in a number of other cellular processes ( 22 ), radicicol is widely used as a pharmacological tool to block the Doxo-induced translocation of Cx43 to the mitochondria ( 4 , 9 ), just as another Hsp90 inhibitor has been used to study the role of mitochondrial Cx43 in cardioprotection ( 35 ).…”