AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury

Abstract

AMP-activated protein kinase (AMPK) is an important regulator of diverse cellular pathways in the setting of energetic stress. Whether AMPK plays a critical role in the metabolic and functional responses to myocardial ischemia and reperfusion remains uncertain. We examined the cardiac consequences of long-term inhibition of AMPK activity in transgenic mice expressing a kinase dead (KD) form of the enzyme. The KD mice had normal fractional shortening and no heart failure, cardiac hypertrophy, or fibrosis, although the in vivo left ventricular (LV) dP/dt was lower than that in WT hearts. During low-flow ischemia and postischemic reperfusion in vitro, KD hearts failed to augment glucose uptake and glycolysis, although glucose transporter content and insulin-stimulated glucose uptake were normal. KD hearts also failed to increase fatty acid oxidation during reperfusion. Furthermore, KD hearts demonstrated significantly impaired recovery of LV contractile function during postischemic reperfusion that was associated with a lower ATP content and increased injury compared with WT hearts. Caspase-3 activity and TUNEL-staining were increased in KD hearts after ischemia and reperfusion. Thus, AMPK is responsible for activation of glucose uptake and glycolysis during low-flow ischemia and plays an important protective role in limiting damage and apoptotic activity associated with ischemia and reperfusion in the heart.

Figure 1

Protein expression of the native and mutant catalytic α subunits of AMPK in WT and AMPK KD hearts. Immunoblots showing the expression of native and mutant α isoforms of the catalytic subunit of AMPK in hearts from KD (K45R α2 mutation) and WT mice (A). Immunoblot confirming the expression of the c-myc–tagged transgene in KD hearts (B). Note that the KD transgenic α2 isoform containing the K45R mutation and c-myc tag migrates more slowly than the WT α2 isoform. TG, transgene project.

Figure 2

Light microscopy of trichrome-stained paraffin sections (upper panels: original magnification, ×40) and electron micrographs (lower panels: original magnification:, ×1,200) of hearts from WT (left panels) and AMPK KD (right panels) hearts. There is no evidence of increased fibrosis or ultrastructural changes in the KD hearts.

Figure 3

α2- and α1-specific AMPK activity from hearts from KD and WT mice after baseline perfusion period (n = 3 for both), after ischemia (n = 4 for both), and following 30 minutes of ischemia and 30 minutes of reperfusion (n = 7 for WT and n = 6 for KD). *P < 0.01 versus baseline; ^† P < 0.01 versus WT.

Figure 4

Glucose uptake, glycolysis, and GLUT expression in WT and KD hearts. Glucose uptake in hearts from KD transgenic (n = 6) and WT (n = 7) mice under baseline conditions, low-flow ischemia, and reperfusion (A). Lactate release from the same hearts (B). Stimulation of glucose uptake by insulin (1,000 μU/ml) in WT and KD hearts (n = 3 for both) (C) and immunoblot analysis of GLUT1 and GLUT4 expression in WT and KD hearts following baseline perfusion or ischemia/reperfusion (Isc/Rep) (D). *P < 0.05 versus baseline; ^† P < 0.05 versus WT.

Figure 5

Rates of oleate oxidation (A) and glucose oxidation (B) in hearts from WT (n = 7) and KD transgenic (n = 6) mice under baseline conditions, low-flow ischemia, and reperfusion. *P < 0.05 versus baseline.

Figure 6

LVDP, heart rate (HR), +dP/dt, and –dP/dt for WT (n = 7) and KD (n = 6) transgenic hearts before, during, and after low-flow ischemia. Horizontal lines identify time points with values that are significantly different (P < 0.05) from t = 0. *P < 0.05 versus WT.

Figure 7

Myocardial injury and apoptotic activity following low-flow ischemia. Release of myocardial creatine kinase (CK) and lactate dehydrogenase (LDH) from WT (n = 7) and KD (n = 6) transgenic hearts during ischemia and reperfusion (A and B). Caspase-3 activity following baseline perfusion (n = 3 for both groups) and following ischemia/reperfusion (n = 7 for WT and n = 6 for KD) (C). Quantification of apoptotic nuclei from WT and KD hearts (n = 3 for both) following ischemia/reperfusion (D). Representative composite confocal photomicrographs of sections from WT and KD hearts following reperfusion (E and F). Hearts underwent TUNEL staining with fluorescein-labeled dUTP and counterstaining with propidium iodide. TUNEL-positive nuclei are stained yellow (arrows). *P < 0.05 versus WT; P < 0.05 versus ischemia; ^†P < 0.05 versus baseline.