Hyperphosphorylation of RyRs underlies triggered activity in transgenic rabbit model of LQT2 syndrome

Rationale: Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane-related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca²⁺ handling remained unexplored. Objective: We investigated the remodeling of Ca²⁺ homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). Methods and Results: Confocal Ca²⁺ imaging revealed decrease in amplitude of Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca²⁺ indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca²⁺ leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with β-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca²⁺ releases and EADs, which were abolished by inhibition of Ca²⁺/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca²⁺ releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na⁺/Ca²⁺ exchanger type 1. Conclusions: Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca²⁺ releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na⁺/Ca²⁺ exchanger type 1, thereby slowing repolarization and enabling L-type Ca²⁺ current reactivation. (Circ Res. 2014;115:919-928.)