Expression level of cardiac ryanodine receptors dictates properties of Ca2+-induced Ca2+ release

Abstract

The type 2 ryanodine receptor (RyR2) is the major Ca²⁺ release channel required for Ca²⁺-induced Ca²⁺ 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 Ca²⁺ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca²⁺ flux dictates local CICR that underlies Ca²⁺ 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 Ca²⁺ sensor RCEPIA-1er was used to directly measure the ER [Ca²⁺] (Ca²⁺]_ER) in the T-Rex-293 the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca²⁺]_ER depletions in the form of spontaneous Ca²⁺ waves due to propagating CICR. For each studied cell, the [Ca²⁺]_ER at which Ca²⁺ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca²⁺]_ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca²⁺ lowered the [Ca²⁺]_ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca²⁺ 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 Ca²⁺ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.