Abstract 1291: Mitochondrial Binding and Glucose Phosphorylation are Both Needed for the Protective Effects of Hexokinase I and II
Alterations in glucose metabolism have been demonstrated in heart failure and cardiac hypertrophy. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. Overexpression of HKI and HKII in tissue culture protects against oxidant-induced cell death. The protective effects of these enzymes are thought to be due to either an increase in glucose phosphorylation or closure of the mitochondrial permeability transition pore (mPTP) as a result of HK binding to the voltage dependent anion channel (VDAC) on the mitochondria. VDAC is believed to form part of mPTP, opening of which causes cellular injury. The relative contribution of HK binding to the mitochondria and the increase in glucose phosphorylation to the overall protective effects of HKs is not clear. Furthermore, there is no clear evidence supporting the hypothesis that HK binding to mitochondria inhibits mPTP. In order to better understand the mechanism(s) behind the protective effects of HKs, we made adenoviruses for full length HKI and HKII (FL-HKI and FL-HKII, respectively) and their truncated proteins lacking the N-terminal hydrophobic domains (Tr-HKI and Tr-HKII, respectively) and transfected them into neonatal rat cardiomyocytes. The truncated constructs cannot bind to the mitochondria since they lack the hydrophobic N-terminal sequence. Overexpression of FL-HKI, FL-HKII resulted in complete protection against H2O2-induced loss of mitochondrial membrane potential and cell death (survival percentage of 96±9 and 95±5 for FL-HKI and FL-HKII, respectively). Although overexpression of Tr-HKI and Tr-HKII reduced cell death, the degree of protection was about 40–50% less than that of the full-length proteins. Similar results were obtained when these proteins were expressed in HEK293 cells. Furthermore, FL-HKI and FL-HKII inhibited mitochondrial permeability transition (MPT) in the presence of H2O2, while the truncated forms only caused partial inhibition. These results suggest that both glucose phosphorylation and inhibition of mPTP contribute to the protective effects of HKI and HKII. These findings bear implications of HK overexpression and binding to the mitochondria as a potential clinical treatment strategy for various forms of heart disease.