Heat shock protects cardiomyocytes from hypoxia-mediated apoptosis by attenuation of nitric oxide.
BACKGROUND
Considerable data exists on the beneficial effect of Heat Shock (HS) on myocardial necrosis. However, the effect of HS on apoptosis or programmed cell death, the other mode of cell death in the heart, is unknown. Hypoxia leads to apoptosis and increased nitric oxide (NO) production in myocardium. NO has also been shown to cause apoptosis. We sought to investigate if HS could protect cardiomyocytes (CM) from hypoxia-induced apoptosis and if this protection was NO-mediated.


METHODS
CM were isolated from 1-2 day old rats and incubated at 37 degrees C or exposed to HS at 42 degrees C for 90 minutes before overnight incubation. CM were then subjected to hypoxic incubation in an argon/CO2 chamber (O2 concentration between 0.8-1.3 parts per million) for 90 minutes, followed by reoxygenation for 24 hours. Apoptosis was measured at 48 hours by TUNEL assay for in-situ DNA fragmentation and by immunohistochemical detection of c-myc protein, a proto-oncogene upregulated with apoptosis (grade 0 = no stain; +++ = most dense stain). NO was detected using the Griess reaction (nM +/- SEM). N > 5 for each group, performed in triplicate.


RESULTS
CM viability demonstrated by spontaneously beating cells in culture and trypan blue staining was > 85%. (*, # = p < 0.01, ANOVA).
Bhora
F Y
FY
Division of Cardiothoracic Surgery, George Washington University Medical Center, Washington, DC 20037, USA. FYBhora@aol.com
Wise
R M
RM
Foster
A H
AH
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D053938:DNA Fragmentation; D019084:Fluorescent Antibody Technique, Indirect; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D018869:Heat-Shock Response / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D009569:Nitric Oxide / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D015854:Up-Regulation
D016271:Proto-Oncogene Proteins c-myc; D009569:Nitric Oxide