The AMP-activated protein kinase (AMPK) couples cellular energy demand and ATP production and increases PGC-1α-dependent expression of mitochondrial proteins, an effect enhanced by the estrogen related receptor (ERR). Hypoxia also stimulates AMPK but decreases oxidative metabolism. Therefore, we tested the hypothesis that stimulation of AMPK with AICAR blunts impaired mitochondrial function in hypoxia to prevent functional impairment of cardiomyocytes. H-10 cardiomyocytes were treated with AICAR (1.5 mM, 24h) to stimulate AMPK in normoxia and hypoxia (1.5 % O₂; 24h).

AICAR, hypoxia, and their combination increased phosphorylated AMPK indicating stimulation. AICAR induced a 3-fold increase in mitochondrial oxygen consumption (JO₂) that depended on ERR, whereas hypoxia decreased JO₂ (-20%) and prevented the AICAR-induced increase. Hypoxia also prevented the AICAR-induced increase in mitochondrial proteins. The HIF-dependent genes GAPD (1.5-fold) and BNIP3 (4-fold) were increased in hypoxia, also in the presence of AICAR. Both, hypoxia and AICAR decreased the mitochondrial membrane potential measured with TMRE. Hypoxia decreased H₂O₂ production and increased lactate formation. Caspase-3 activity, an indicator of BNIP3-dependent apoptosis was not affected by the treatments.

These results indicate that hypoxia, a known stimulator of AMPK, prevents AMP- and AMPK-dependent stimulation of mitochondrial metabolism thus disproving our hypothesis. The inhibition by hypoxia seems to be caused by inhibition of the expression of mitochondrial proteins by blocking signaling downstream of AMPK, and by decreasing the substrate flux into mitochondria. Together this indicates that hypoxia decreases cardiomyocyte oxidative metabolism despite increased metabolic demand.