Dynamic calcium-calmodulin-dependent stabilization of leaky RyR2 in live cardiac myocytes.

Abstract

Calmodulin (CaM) is a major regulator of cardiac ryanodine receptors (RyR2). CaM binding shifts RyR2 channel conformation to a state that suppresses diastolic SR Ca leak that can become pro arrhythmic as commonly observed in cardiac diseases such as heart failure (HF) or diabetic cardiomyopathy. In these diseases, CaM affinity for RyR2 is reduced, promoting a leaky pathological RyR2 structural state that is arrhythmogenic. Conversely, increased CaM binding can suppress RyR2-mediated Ca leak and promote a conformational shift to the stable physiological non-leaky state. Still, there are unknowns regarding the dynamic interplay between CaM-RyR2 during each heartbeat and in pathogenic RyR states in live cardiac myocytes. We hypothesize that high [Ca] elevations reached during Ca transients in a healthy beating myocyte would increase CaM-RyR binding throughout the cardiac cycle, promoting RyR stability. Further, the lower CaM-RyR2 affinity in disease, exacerbated by reduced Ca transient amplitude, resulting in lower CaM-RyR2 binding, destabilized RyR2s and enhance arrhythmogenic diastolic SR Ca leak. We used permeabilized cardiac myocytes from WT and HF mouse models as a controlled approach to measuring both the [Ca]i and [CaM] dependence of CaM-RyR2 binding via FRET between donor-labeled FKBP12.6 and acceptor-labeled CaM. Moreover, we simultaneously recorded [Ca]i transients within myocytes, enabling measurement of dynamic CaM-RyR2 saturation during periodic Ca waves that emulate paced Ca transients. We find that CaM-RyR2 FRET is reduced in the leaky pathological state and associated with alterations in Ca leak event frequencies. We infer that the healthy higher CaM-RyR2 affinity favors more complete saturation of RyR2 with CaM throughout the cardiac cycle, versus pathological states. These data suggest that a modest fraction of CaM-free RyR2s with pathological diastolic conformation might suffice to elicit proarrhythmic SR Ca leak in cardiac disease.