β-Adrenergic-stimulated L-type channel Ca2+ entry mediates hypoxic Ca2+ overload in intact heart

Ca2+ mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca2+ ([Ca2+](i)) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca2+](i) overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca2+ indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca2+]i, mitochondrial membrane potential (Delta Psi(m), by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO(2) similar to 20 mm Hg in glucose-free perfusate), [Ca2+](i) transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12 min. At similar to 20 min, basal [Ca2+](i) rose to its first peak at a supraphysiological level, coincident with loss of Delta Psi(m), and onset of rigor. A greater [Ca2+](i) rise occurred at similar to 2 h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca2+ or blockade of the L-type Ca2+ channel (LTCC) (10 mu M diltiazem or nifedipine) prevented [Ca2+](i) overload and markedly delayed the loss of Delta Psi(m); by contrast, depletion of the sarcoplasmic reticulum Ca2+ store by thapsigargin did not have any significant effect. Importantly, beta-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca2+ and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca2+](i) overload was reproduced in isolated cardiomyocytes when beta-adrenergic agonist was present. Taken together, we conclude that Ca2+ entry through beta-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca2+](i) overload and the ensuing loss of mitochondrial function in intact heart. (C) 2013 Elsevier Ltd. All rights reserved.