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Canadian Journal of Cardiology

NOVEL SYNTHETIC HSP90 INHIBITOR FORMULATION IMPROVES CARDIOPROTECTION IN RAT AND HUMAN IN VITRO ISCHEMIA/REPERFUSION CELL MODEL

      Background

      Ischemic heart disease continues to be one of the major causes of death in the developed world despite improvements in treatment. 30% of non-lethal myocardial infarctions will develop heart failure symptoms. Improved survival of the myocardium is a need in clinical practice. Our group has previously shown that Celastrol, along with a synthetic HSP90 inhibitor analog have the potential to reduce infarct size when given as a postconditioning agent at the moment of reperfusion. The objective is to evaluate the rapid cardioprotective mechanisms of a novel formulation of the HSP90 inhibitor compound on two cell lines: rat H9c2 cardiomyoblasts and Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

      Methods and Results

      H9c2 rat cardiomyoblasts and Human iPSC-CMs were cultured. Cell signaling was evaluated by western blot to evaluate pathway activations. Both cell lines were put in ischemic conditions (no glucose, 95% N2, 5% CO2) overnight, then reperfused (normal culture) with different concentrations of HSP90i after optimizing the human iPSC-CMs’ stress experiment. Cell viability and mitochondrial permeability transition pore (mPTP) opening were evaluated using assays, oxygen-free radical production by fluorescence assay and antioxidant gene messenger RNA expression via polymerase chain reaction (PCR). Results showed an increase in cytoprotective pathway activation when both cell lines were treated with 10-6M of the compound without any stress: HO-1 and HSP-70 in the first 30 minutes while AKT and ERK after 1 hour of treatment and 3 hours of recuperation. Interestingly, treatment with the compound at 10-6M at the moment of reperfusion showed decreased the viability of the cells while 10-7M improved it. Free radical production was also decreased at a concentration of 10-7M when compared to baseline, and as expected, the compound also decreased mPTP opening. These results were seen in both human and rat cell lines. Preliminary evaluation of the antioxidant gene expression in H9c2 cells only showed an increase in the expression of the cytoprotective HO-1 gene.

      Conclusion

      We have previously shown that Celastrol compounds reduce reperfusion damage in myocardial ischemia models, including myocardial infarction and donation after circulatory death. These experiments show that the effects of the novel HSP90i formulation include the expression of antioxidant genes and the launching of a series of cytoprotective pathways that stabilize the mitochondrial membrane, reduce free radical production, and improve cell survival. Further studies investigating the mechanisms further and the optimal dose are underway to fully understand the mode of action of the medication and move to animal trials.