Mechanisms of Spontaneous Calcium Wave Generation During Beta-Adrenergic Stimulation in Rabbit Ventricular Myocytes

The beta-adrenergic signaling pathway represents the principal positive inotropic mechanism of the heart. While the effects of beta-adrenergic stimulation on L-type Ca channel Ca influx and SERCA-mediated sarcoplasmic reticulum (SR) Ca uptake are well established, the effects on SR Ca release through ryanodine receptor (RyR) release clusters remains highly controversial. Here, we examine SR Ca release in rabbit ventricular myocytes in the form of spontaneous Ca waves during beta-adrenergic stimulation with isoproterenol under controlled cytosolic and SR [Ca]. Cytosolic Ca was monitored using high-affinity Ca indicators indo-1 or rhod-2, while SR Ca was measured directly using the low-affinity Ca indicator fluo-5N or indirectly using the amplitude of the cytosolic Ca transient in response to 10 mM caffeine. Under control conditions, Ca waves were not observed following rest from 0.75 Hz pacing. In the presence of isoproterenol (500 nM), SR Ca content increased by 34% and spontaneous Ca waves were observed in 67% of cells during rest after pacing. However, when post-rest cytosolic Ca and SR Ca content were experimentally matched to control conditions using low extracellular Ca (100 uM versus 2 mM) and SERCA inhibition (7.5 uM cyclopiazonic acid), spontaneous Ca waves were never observed in the presence of isoproterenol. In contrast, pharmacological sensitization of the RyR with 250 uM caffeine induced Ca waves under control conditions (8/12 cells) and in the presence of isoproterenol at matched cytosolic Ca and SR Ca content (7/12). Together, these data suggest that spontaneous Ca release during beta-adrenergic stimulation is a result of increased RyR sensitivity in response to increased SR Ca content, and is not due to direct alterations in RyR function by the beta-adrenergic signaling cascade.