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Updated: Apr 23, 2026
Functional Characterization of Endogenously Expressed Human RYR1 Variants
Published on: June 9, 2021
Hyperphosphorylation of RyRs underlies triggered activity in transgenic rabbit model of LQT2 syndrome
Dmitry Terentyev1, Colin M Rees2, Weiyan Li2
1From the Department of Medicine, Division of Cardiology, Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University, Providence (D.T., W.L., L.L.C., H.K.J., Y.L., R.T., J.D., K.B., B.-R.C., G.K.); Physics Department, Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, MA (C.M.R., A.K.); Department of Comparative Medicine, Pennsylvania State University College of Medicine, Hershey (X.P.); and Department of Cardiology and Angiology I, Heart Center Freiburg University, Freiburg, Germany (K.E.O.). dmitry_terentyev@brown.edu gideon_koren@brown.edu.
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 Ca(2+) handling remained unexplored.
Objective:
We investigated the remodeling of Ca(2+) 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 Ca(2+) imaging revealed decrease in amplitude of Ca(2+) transients and sarcoplasmic reticulum Ca(2+) content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca(2+) indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca(2+) 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 Ca(2+) releases and EADs, which were abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca(2+) releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na(+)/Ca(2+) 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 Ca(2+) releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na(+)/Ca(2+) exchanger type 1, thereby slowing repolarization and enabling L-type Ca(2+) current reactivation.
Insights
Loss-of-function mutations in human ether go-go (HERG) channels cause Long QT syndrome type 2 (LQT2), leading to fatal arrhythmias. This study reveals that altered intracellular calcium handling, specifically hyperactive ryanodine receptors, drives early afterdepolarizations in LQT2.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Ion Channelopathies
Background:
- Long QT syndrome type 2 (LQT2) is linked to fatal ventricular arrhythmias due to HERG channel mutations.
- Previous research focused on plasma membrane ion channels, neglecting intracellular calcium (Ca2+) handling in LQT2 arrhythmogenesis.
Purpose of the Study:
- Investigate Ca2+ homeostasis remodeling in LQT2 ventricular cardiomyocytes.
- Determine if Ca2+ handling changes contribute to early afterdepolarizations (EADs) and triggered activity in LQT2.
Main Methods:
- Confocal Ca2+ imaging in cardiomyocytes from a transgenic rabbit LQT2 model.
- Ryanodine receptor (RyR) Ca2+ leak assessment using specific indicators.
- Western blot and coimmunoprecipitation for RyR phosphorylation and phosphatase association.
- Pharmacological stimulation (isoproterenol) and inhibition (CaMKII inhibitor) studies.
- Computational modeling of Ca2+ release and action potential dynamics.
Main Results:
- LQT2 myocytes showed reduced Ca2+ transient amplitude and sarcoplasmic reticulum (SR) Ca2+ content.
- Enhanced RyR-mediated SR Ca2+ leak and increased RyR phosphorylation were observed in LQT2 cells.
- Reduced association of protein phosphatases with RyR complex in LQT2.
- Isoproterenol induced EADs in LQT2 cells, linked to aberrant Ca2+ release and abolished by CaMKII inhibition.
- Simulations confirmed RyR hyperactivity promotes EADs via increased Na+/Ca2+ exchanger (NCX1) forward mode.
Conclusions:
- Hyperphosphorylated and hyperactive RyRs, due to decreased phosphatase activity, enhance triggered activity in LQT2.
- Aberrant RyR-mediated Ca2+ releases, despite reduced SR Ca2+ content, promote EADs in LQT2.
- These Ca2+ releases increase NCX1 forward mode, prolonging repolarization and enabling L-type Ca2+ current reactivation, contributing to LQT2 arrhythmias.
