Abstract
AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that plays a pivotal role in regulating cellular metabolism for sustaining energy homeostasis under stress conditions. Activation of AMPK has been observed in the heart during acute and chronic stresses, but its functional role has not been completely understood because of the lack of effective activators and inhibitors of this kinase in the heart. We generated transgenic mice (TG) with cardiac-specific overexpression of a dominant negative mutant of the AMPK alpha2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia. In isolated perfused hearts subjected to a 10-min ischemia, AMPK alpha2 activity in wild type (WT) increased substantially (by 4.5-fold), whereas AMPK alpha2 activity in TG was similar to the level of WT at base line. Basal AMPK alpha1 activity was unchanged in TG and increased normally during ischemia. Ischemia stimulated a 2.5-fold increase in 2-deoxyglucose uptake over base line in WT, whereas the inactivation of AMPK alpha2 in TG significantly blunted this response. Using 31P NMR spectroscopy, we found that ATP depletion was accelerated in TG hearts during no-flow ischemia, and these hearts developed left ventricular dysfunction manifested by an early and more rapid increase in left ventricular end-diastolic pressure. The exacerbated ATP depletion could not be attributed to impaired glycolytic ATP synthesis because TG hearts consumed slightly more glycogen during this period of no-flow ischemia. Thus, AMPK alpha2 is necessary for maintaining myocardial energy homeostasis during ischemia. It is likely that the functional role of AMPK in myocardial energy metabolism resides both in energy supply and utilization.
Highlights
AMP-activated protein kinase (AMPK) is an energysensing enzyme that plays a pivotal role in regulating cellular metabolism for sustaining energy homeostasis under stress conditions
We generated transgenic mice (TG) with cardiac-specific overexpression of a dominant negative mutant of the AMPK ␣2 catalytic subunit to clarify the functional role of this kinase in myocardial ischemia
In isolated perfused hearts subjected to a 10-min ischemia, AMPK ␣2 activity in wild type (WT) increased substantially, whereas AMPK ␣2 activity in TG was similar to the level of WT at base line
Summary
Generation of Transgenic Mice—A full-length cDNA of rat AMPK ␣2 subunit was tagged at the 5Ј end with a HA epitope. One base-line 31P NMR spectrum was collected [16], and one-half of the hearts were subjected to a 10-min no-flow ischemia and the other half to a 10-min normal perfusion. Four consecutive 2-min 31P NMR spectra were collected to monitor the dynamic changes in high energy phosphate content. AMPK activity was measured after immunoprecipitating 200 g of protein using antibodies made against the amino acid sequences 339 –358 of rat AMPK ␣1, 352–366 of ␣2, and 2–16 of both ␣1 and ␣2 (pan-␣) [18]. HPLC Measurements and Glycogen Assay—Freeze-clamped tissues were used for determination of myocardial content of adenine nucleotides, nucleosides, and purine bases by a HPLC method as reported previously [20]. The mean value of [ATP] for WT or TG hearts was used to calibrate the ATP peak area of the base-line 31P NMR spectrum. All the statistical analyses were performed with Statview (Brainpower Inc.), and a value of p Ͻ 0.05 was considered significant
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