| Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotetive effect via the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (GA, 1μg/ml). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis.Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c.Adult mouse cardiomyocytes were subjected to isoproterenol (ISO, 0.1μmol/ml) in the absence or presence of the HSP90 inhibitor GA. GA neither affected the myocyte viability nor enhanced the ISO induced myocyte death.In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3K/Akt pathway. The cardioprotective effect of HSP90 is not mediated via attenuating the activation of adrenergic signaling pathways.Cardiac diseases persistently increase the contractility demands of cardiac myocytes which met by activating the sympathetic nervous system and subsequently increasing myocyte Ca2+ transients. Persistent exposure to sympathetic and/or Ca2+ stress is associated with myocyte death. This study examined the respective roles of persistentβ-adrenergic receptor (β-AR) agonist exposure and high [Ca2+]i in myocyte death. Methods: Ventricular myocytes (VMs) were isolated from transgenic (TG) mice with cardiac specific and inducible expression of theβ2a subunit of the L-type Ca2+ channel (LTCC). VMs were cultured and the rate of myocyte death was measured in the presence of isoproterenol (ISO), other modulators of Ca2+ handling and theβ-adrenergic system, and inhibitors of caspases and reactive oxygen species (ROS) generation.Results and Conclusions: The rate of myocyte death was greater in TG versus WT myocytes and accelerated by ISO in both groups although ISO did not increase LTCC current (ICa-L) in TG-VMs. Nifedipine, an LTCC antagonist, only partially prevented myocyte death. These results suggest both LTCC-dependent and independent mechanisms in ISO induced myocyte death. ISO increased the contractility of WT and TG-VMs by enhancing SR function and inhibiting SERCA, Na+/Ca2+ exchanger, and CaMK II partially protected myocyte from death induced by both Ca2+ and ISO. Caspase and ROS inhibitors did not butβ2-AR activation did reduce myocyte death induced by enhanced ICa-L and ISO stimulation. Our results suggest that catecholamines induce myocyte necrosis primarily throughβ1-AR mediated increases in ICa-L but other mechanisms are also involved in rodents. |
