Beta-Adrenergic Stimulation Does Not Affect Calcium Sparks Refractoriness in Ventricular Myocytes

Abstract

Cardiac Ca2+ sparks are intracellular Ca2+ release events from clusters of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum (jSR). L-type Ca2+ channels (LCC) are located in the nearby apposing sarcolemma (SL) mainly at the transverse tubules. Cellular depolarization permits local Ca2+ influx through LCC that activates RyR2 clusters by Ca2+-induced Ca2+-release (CICR). Ca2+ sparks also occur during diastole due to the finite opening rate of the RyR2s that are sensitive to both cytosolic Ca2+ ([Ca2+]i) and to SR Ca2+ ([Ca2+]SR). There is a significant (at least 50%) depletion of jSR Ca2+ during each Ca2+ spark and this depletion (measured as Ca2+ blinks, Brochet et al. 2005) suggests that refractoriness of Ca2+ sparks is due to the reduction of [Ca2+]SR (Sobie et al. 2006). Additional factors (beyond [Ca2+]i and [Ca2+]SR) have been reported to affect the opening and closing rates of RyR2. Here we examine RyR2 modulation by protein kinase A (PKA) during Beta-adrenergic stimulation. Studying RyR2 refractoriness is complicated because it overlaps with ICa restitution and with the slower SR Ca2+ uptake by SERCA. We assessed RyR2 refractoriness in permeabilized ventricular myocytes from phospholamban-KO mice by studying repeated spontaneous Ca2+ sparks at the same Ca2+ release location in the absence and presence of cAMP (10 μM). We observed under control conditions that Ca2+ spark amplitude restoration (time constant ∼70 ms) was ∼2 fold slower than the reported jSR Ca2+ refilling. RyR2 phosphorylation did not affect Ca2+ sparks amplitude restoration, and the Ca2+ spark frequency distribution peak was slightly diminished, with small increases at longer delays. We conclude that under conditions when neither LCC nor [Ca2+]SR can change to influence Ca2+ sparks rate or Ca2+ sparks refractoriness, RyR2 phosphorylation by PKA activation does not alter RyR2 refractoriness.