Β-adrenergic-stimulated L-type channel Ca²+ entry mediates hypoxic Ca²+ overload in intact heart

Ca(2+) mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca(2+) ([Ca(2+)]i) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca(2+)]i overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca(2+) indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca(2+)]i, mitochondrial membrane potential (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~20 mmHg in glucose-free perfusate), [Ca(2+)]i transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12 min. At ~20 min, basal [Ca(2+)]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca(2+)]i rise occurred at ~2h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca(2+) or blockade of the l-type Ca(2+) channel (LTCC) (10 μM diltiazem or nifedipine) prevented [Ca(2+)]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca(2+) store by thapsigargin did not have any significant effect. Importantly, β-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca(2+) and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca(2+)]i overload was reproduced in isolated cardiomyocytes when β-adrenergic agonist was present. Taken together, we conclude that Ca(2+) entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca(2+)]i overload and the ensuing loss of mitochondrial function in intact heart.