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

Abstract

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.

Keywords: arrhythmias, cardiac; calcium release; long QT syndrome; protein phosphatase; ryanodine receptor.

Figure 1. Decreased [Ca²⁺] Transient Amplitude and SR [Ca²⁺] Content in Intact LQT2 Myocytes

A, Representative Ca²⁺ transients recorded in intact LMC (black) and LQT2 (red) ventricular myocytes undergoing repetitive stimulation at 0.25 Hz at baseline conditions and in the presence of 50 nmol/L ISO. B, Bar Graphs present pooled data for Ca²⁺ transient amplitudes at different stimulation frequencies. Grey bars: LMC; black bars: LQT2.C, Representative traces of Ca²⁺ transients induced by application of 10 mmol/L caffeine. D, Bar Graphs depict averaged amplitudes and decay time constants for caffeine transients. *p<0.05, unpaired Student’s t-test, n=6–33.

Figure 2. Increased SR [Ca²⁺] Content and Increased Frequency and Amplitude of [Ca²⁺] Sparks in Permeabilized LQT2 Myocytes

A, Representative line scan images of Ca²⁺ sparks recorded in permeabilized myocytes using intracellular solution with 100 nmol/L [Ca²⁺]free. BC, Bar Graphs present averaged frequency and amplitude of Ca²⁺ sparks. D, Representative traces of Ca²⁺ release induced by 10 mmol/L caffeine application; and E, pooled data for Caffeine-induced Ca²⁺ transients. *p<0.05; **p<0.01. and ***p<0.001 vs. LMC, unpaired Student’s t test.

Figure 3. Accelerated SR [Ca²⁺] Uptake and Leak in Permeabilized LQT2 Myocytes

SR Ca²⁺ uptake and leak was measured in myocytes with SR-entrapped low affinity Ca²⁺ indicator Fluo-5N. A, To measure SERCA-mediated SR Ca²⁺ uptake saponin-permeabilized myocytes were exposed to 10 mmol/L caffeine to exhaust Ca²⁺ stores, then RyRs were blocked with 40 μmol/L RuRed in Ca²⁺-free solution. Time constant of increase in Fluo-5N fluorescence upon re-introduction of 250 nmol/L [Ca²⁺] into cytosol was used as a measure of SERCA activity (B). C, RyR-mediated SR Ca²⁺ leak was unmasked by application of specific inhibitor of SERCA thapsigargin (Tg, 10 μmol/L ). D, Pooled data for SR-Ca²⁺ leak. *p<0.05 vs. LMC, unpaired Student’s t test.

Figure 4. Changes in RyR and PLB Phosphorylation in LQT2 Myocytes

A, Representative Western blots for RyR and PLB phosphorylation at PKA and CaMKII sites. RyR2 phosphorylation at sites Ser-2815 (CaMKII) and Ser-2809 (PKA) and PLB phosphorylation at Ser-16 (PKA) and Thr-17 (CaMKII) was measured with phospho-specific antibodies. Total RyR/PLB protein content was measured in the same samples on a different blot and used as a control for loading. Ventricular cells isolated from 1 heart were split into 3 samples; one sample was kept at baseline conditions, second sample was exposed to 50 nmol/L ISO for 3 min., and third sample was treated with 1 μmol/L Calyculin A and 1 μmol/L ISO to achieve maximum phosphorylation and used for normalization of the signals. B, Data pooled from 6 experiments in LQT2 and 4 experiments in LMC cells. *p<0.05 vs. LMC, unpaired Student’s t test.

Figure 5. Reduced Levels of Phosphatases in RyR Macromolecular Complex in LQT2 Myocytes

A, Representative western blots of PP1 and PP2A catalytic subunits in samples from LV myocytes from LMC and LQT2 hearts. B, Pooled Data for PP1 and PP2A phosphatase activity of in samples from ventricular myocytes (n=3); and averaged normalized optical density (OD) for PP1 and PP2A catalytic subunits from cell lysates (n=7). C, Representative western blots of PP1c and PP2Ac immuno-precipitated with anti-RyR abs. E, Bar Graph demonstrates data for normalized OD for PP1c and PP2Ac subunit pooled down with RyRs, *p<0.05 vs. LMC, paired Student’s t test, n=5.

Figure 6. Inhibition of CaMKII Abolishes EADs in LQT2 Myocytes Exposed to ISO

A, Membrane potential traces (black) and corresponding averaged time dependent profiles (red) and confocal line scan images of Ca²⁺ transients recorded in current clamped LMC and LQT2 myocytes undergoing repetitive stimulation at 0.25 Hz in the presence of 50 nmol/L ISO. Application of RyR sensitizer caffeine (250 μmol/L) prolongs APD and Ca²⁺ transient, and promotes EADs in LMC cells, while preincubation of LQT2 myocytes with CaMKII inhibitor attenuates pro-arrhythmic Ca²⁺ mishandling. BCDEFG, Bar Graphs present pooled data for APD (B), incidence of EADs (C), Duration of AP plateau from +10 to −50 mV (D), amplitude (E), time to peak (F) and time of decay of Ca²⁺ transients to 25% of peak amplitude (G). *, **p<0.05 vs. LMC and LQT2 respectively, unpaired Student’s t test, n=4–18.

Figure 7. Causal Link between RyR Hyperactivity and EAD Formation in LQT2 Myocytes under β–Adrenergic Stimulation

Comparison of computer modeling results of LQT2 myocytes paced at 0.25 Hz under ISO stimulation with hyperactive (blue) and stabilized (red) RyRs highlighting the causal relationship of RyR hyperactivity to AP prolongation and EAD formation. Aberrant late Ca²⁺ releases in the hyperactive case increase the forward mode depolarizing NCX1 current thereby slowing repolarization and allowing more time for reactivation of LTCCs when the transmembrane voltage V_m traverses the window (i.e. the V_m range where the steady-state I_Ca,L current is appreciable). A, V_m traces for 4 consecutive beats in steady-state demonstrating elimination of EADs by RyR stabilization. B-I shows a detailed comparison for the third beat in A of the cytosolic Ca²⁺ concentration (B) the SR Ca²⁺ concentration (C), confocal line scan equivalents for stabilized (D) and hyperactive (E) RyRs, V_m traces (F), and three key sarcolemmal currents including I_Ca,L (G), I_NCX (H), and I_K,s (I). The origin of time in B to I corresponds to the start of the third beat in A.