During and following
myocardial ischemia,
glucose oxidation rates are low and
fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on
fatty acid oxidation as a source of
ATP generation, a functional proteomics approach was utilized.
RESULTS: 2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow
ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in
protein abundance compared to aerobic controls. Of the five
proteins with the greatest change in abundance, two were increased (
long chain acyl-coenzyme A dehydrogenase (48 +/- 1 versus 39 +/- 3 arbitrary units, n = 3, P < 0.05) and alpha subunit of
ATP synthase (189 +/- 15 versus 113 +/- 23 arbitrary units, n = 3, P < 0.05)), while two were decreased (24 kDa subunit of
NADH-ubiquinone oxidoreductase (94 +/- 7 versus 127 +/- 9 arbitrary units, n = 3, P < 0.05) and D subunit of
ATP synthase (230 +/- 11 versus 368 +/- 47 arbitrary units, n = 3, P < 05)). Two forms of
pyruvate dehydrogenase betaE1 subunit, the rate-limiting
enzyme for
glucose oxidation, were also identified. The
protein level of the more acidic form of
pyruvate dehydrogenase was reduced during reperfusion (37 +/- 4 versus 56 +/- 7 arbitrary units, n = 3, P < 05), while the more basic form remained unchanged. The more acidic
isoform was found to be O-palmitoylated, while both
isoforms exhibited
ischemia/reperfusion-induced phosphorylation. In silico analysis identified the putative
kinases as the
insulin receptor kinase for the more basic form and
protein kinase Czeta or
protein kinase A for the more acidic form. These modifications of
pyruvate dehydrogenase are associated with a 35% decrease in
glucose oxidation during reperfusion.
CONCLUSIONS: Cardiac
ischemia/reperfusion induces significant changes to a number of metabolic
proteins of the mitochondrial
proteome. In particular,
ischemia/reperfusion induced the post-translational modification of
pyruvate dehydrogenase, the rate-limiting step of
glucose oxidation, which is associated with a 35% decrease in
glucose oxidation during reperfusion. Therefore these post-translational modifications may have important implications in the regulation of myocardial energy metabolism.