Post-ischemic interventions that activate
phosphatidylinositol-3-OH kinase (PI3K)-Akt or ERK1/2 pro-survival
kinases (the so-called "
reperfusion injury signalling
kinase (RISK) pathway") during the first few minutes of reperfusion protect against lethal reperfusion-induced injury. We have previously shown that
insulin protects against reperfusion-induced injury via activation of the PI3K-Akt pathway. In addition, opening of the
mitochondrial permeability transition pore (mPTP) at the time of reperfusion is a major determinant of lethal reperfusion-induced injury, and pharmacologically inhibiting it is cardioprotective. In this study, we examined the relationship between the pro-survival
kinase pathways and
mPTP opening. Specifically, we tested the hypothesis that activation of the pro-survival
kinase pathway by
insulin protects cardiomyocytes by reducing the probability of
mPTP opening upon reperfusion.
Laser illumination of the fluorophore, tetramethyl
rhodamine methyl
ester (TMRM), was used to induce oxidative stress in the preparation of adult rat ventricular cardiomyocytes. Maintained illumination ultimately induces
mPTP opening, detected as a global mitochondrial depolarization, followed by
ATP depletion and rigor
contracture.
Insulin significantly delayed
mPTP opening by
a factor of approximately 1.7-fold (P<0.001). The effect of
insulin was prevented by
Wortmannin and by
LY-294002, inhibitors of the PI3K pathway, by SH-6, a selective inhibitor of Akt, and by
L-NAME, an inhibitor of
nitric oxide production. The expression of a dominant negative construct of Akt eliminated the effect of
insulin in delaying
mPTP opening in a cardiac cell line. Furthermore, the overexpression of constitutively active Akt was sufficient to maximally delay
mPTP opening. These results indicate that activation of the PI3K-Akt pro-survival
kinase pathway inhibits opening of the
mPTP, and demonstrate an important link between the survival
kinases and the
mPTP.