Abstract
BackgroundDuring 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.Results2-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 α 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 βE1 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 Cζ 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.ConclusionsCardiac 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.
Highlights
During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism
2-D Electrophoresis and identification of proteins The mitochondrial isolation resulted in a similar yield of mitochondria (97 ± 8 and 102 ± 8 mg mitochondria/g wet heart weight) with enriched expression of Mitochondrial creatine kinase (mtCK) and free of contamination by cytosolic Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) (Figure 2A). 2-D PAGE identified 32 I/R-induced changes in protein levels out of approximately 260 protein spots observed in all of the gels and we chose to identify the five spots with the greatest change in abundance (Figure 2B, C and Table 1)
Both aerobic and I/R groups expressed two molecular forms of PDHβE1
Summary
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. During reperfusion fatty acid oxidation quickly recovers, at the expense of glucose oxidation, and predominates as the main source of mitochondrial oxidative metabolism [1012]. If the pyruvate from glycolysis is aerobically metabolized (i.e. glucose oxidation), lactate and protons are not produced, leading to improved functional recovery during reperfusion [21]
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