Cooling the ischemic heart by just a few degrees protects it from
infarction without affecting its mechanical function, but the mechanism of this protection is unknown. We investigated whether signal transduction pathways might be involved in the anti-
infarct effect of mild
hypothermia (35°C). Isolated rabbit hearts underwent 30 min of coronary artery occlusion/2 h of reperfusion. They were either maintained at 38.5°C or cooled to 35°C just before and only during
ischemia.
Infarct size was measured. The effects of the
protein kinase C inhibitor
chelerythrine, the
nitric oxide synthase inhibitor N (ω)-nitro-
L: -arginine methyl ester (
L: -NAME), the
phosphatidylinositol 3-kinase antagonist
wortmannin, or either of the
mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors
PD98059 or
U0126 on cooling's protection were examined. Myocardial
ATP assays were performed and the level of phosphorylation of
extracellular signal-regulated kinase (ERK) and
MEK was examined by western blotting. To investigate an effect of cooling on
protein phosphatase (PPase), a PPase inhibitor
cantharidin was tested in the
infarct model and the effect of mild
hypothermia on PP2A activity in vitro was measured.
Infarct size was 34.4 ± 2.2% of the ischemic zone in normothermic (38.5°C) hearts, but only 15.6 ± 8.7% in hearts cooled to 35°C during
ischemia. Mechanical function was unaffected. Neither
chelerythrine,
L: -NAME, nor
wortmannin had any effect, but both
PD98059 and
U0126 completely eliminated protection.
Ischemia rather than reperfusion was the critical time when ERK had to be active to realize protection. Phosphorylation of ERK and
MEK fell during normothermic
ischemia, but during hypothermic
ischemia phosphorylation of ERK remained high while that of
MEK was increased. Cooling only slightly delayed the rate at which
ATP fell during
ischemia, and ERK inhibition did not affect that attenuation suggesting
ATP preservation was unrelated to protection.
Cantharidin, like cooling, also protected during
ischemia but not at reperfusion, and its protection was dependent on ERK phosphorylation. However, mild
hypothermia had a negligible effect on PP2A activity in an in vitro assay. Hence, mild
hypothermia preserves ERK and
MEK activity during
ischemia which somehow protects the heart. While a PPase inhibitor mimicked cooling's protection, a direct effect of cooling on PP2A could not be demonstrated.