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 (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~ 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 ~ 20 min, basal [Ca2 +]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca2 +]i rise occurred at ~ 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 μM diltiazem or nifedipine) prevented [Ca2 +]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca2 + store by thapsigargin did not have any significant effect. Importantly, β-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 β-adrenergic agonist was present. Taken together, we conclude that Ca2 + entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca2 +]i overload and the ensuing loss of mitochondrial function in intact heart.