Determinants of Ca(2+) release restitution: Insights from genetically altered animals and mathematical modeling

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca(2+) release restitution (CRR), and its alterations are potential triggers of Ca(2+) arrhythmias. Although the control of CRR has been associated with SR Ca(2+) load and RYR2 Ca(2+) sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca(2+) content versus the velocity of SR Ca(2+) refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca(2+) (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S(1)/S(2)) protocol. CRR and ICaL restitution increased as a function of the (S(2)-S(1)) coupling interval, following an exponential curve. When SR Ca(2+) load was increased by increasing extracellular [Ca(2+)] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca(2+)]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca(2+) load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca(2+)] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca(2+) load.