Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase.
Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.
Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210, USA. ilangovan-1@medctr.osu.edu
Govindasamy
G
Ilangovan
Sola
S
Osinbowale
Anna
A
Bratasz
Mary
M
Bonar
Arturo J
AJ
Cardounel
Jay L
JL
Zweier
Periannan
P
Kuppusamy
D000818:Animals; D015153:Blotting, Western; D019069:Cell Respiration / Q000502:physiology*; D002478:Cells, Cultured; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis*; D006979:Hyperthermia, Induced; D018613:Microscopy, Confocal; D008928:Mitochondria; D032383:Myocytes, Cardiac / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D015854:Up-Regulation
D018841:HSP90 Heat-Shock Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase