Abstract
The type 2 ryanodine receptor (RyR2) is the major Ca(2+) release channel required for Ca(2+)-induced Ca(2+) release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca(2+) signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca(2+) flux dictates local CICR that underlies Ca(2+) sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca(2+) sensor RCEPIA-1er was used to directly measure the ER [Ca(2+)] (Ca(2+)](ER)) in the T-Rex-293 the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca(2+)](ER) depletions in the form of spontaneous Ca(2+) waves due to propagating CICR. For each studied cell, the [Ca(2+)](ER) at which Ca(2+) waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca(2+)](ER) activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca(2+) lowered the [Ca(2+)](ER) at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca(2+) had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca(2+) load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.