Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Abstract

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Fig. 1.

Calstabin-2 deficiency causes pacing-induced bidirectional VT and MAP alternans, which are prevented by JTV519. (A) Representative lead I and II ECG tracings and simultaneous left ventricular epicardial MAP recording from a placebo-treated calstabin-2^+/− mouse during overdrive pacing at CL 30 ms as documented by PES trace. Pacing triggered sustained bidirectional VT, as evidenced by simultaneous ECG and MAP recordings shown at higher resolution in B. Pacing stimuli (arrows) induced MAP alternans that continued after cessation of pacing in placebo-treated calstabin-2^+/− mice (CL 32 ms). (C) Bar graphs summarizing simultaneous occurrence of bidirectional MAP alternans (Left) and sustained VT (sVT) (Center) or nonsustained VT (nsVT) (Right) arrhythmias in placebo-treated (J−) or JTV519-treated (J+) calstabin-2^+/− (open bars) or calstabin-2^−/− (filled bars) mice during ISO stimulation (0.5 mg·kg⁻¹ body weight). Mouse numbers and dimensions are as indicated. sVT, >10 beats (Center); nsVT, 3–10 arrhythmogenic beats (Right). GT, genotype.

Fig. 2.

Spontaneous bidirectional VT and MAP alternans in a calstabin-2^+/− mouse (no pacing). (A) Simultaneous, representative ECG lead I tracing and left ventricular epicardial MAP recording from a placebo-treated calstabin-2^+/− mouse during early phase of sVT. CL, cycle length. Bidirectional ECG QRS alternans and MAP alternans are closely correlated. (B) Bar graphs summarizing alternans (Left), sVTs (Center), or nsVTs (Right) in placebo-treated (J−) or JTV519-treated (J+) calstabin-2^+/− (open bars) or calstabin-2^−/− (filled bars) mice during ISO stimulation. GT, genotype.

Fig. 3.

Example of discordant MAP alternans in a haploinsufficient calstabin-2^+/− mouse during pacing-induced sVT. Phase transition between epicardial MAP signal sites 1 and 2 is indicated by the letters “A” and “B” and by vertical lines.

Fig. 4.

Calstabin-2 deficiency causes aberrant diastolic Ca²⁺ release and after-contractions (AFCs) that are prevented by JTV519. (A) Alternating (indicated by “A”/“B”) intracellular Ca²⁺ transients and AFCs in calstabin-2^+/− cardiomyocyte paced at 0.5 Hz and aberrant Ca²⁺ release events after 6-Hz pacing. (B) JTV519 treatment prevented intracellular Ca²⁺ oscillations and AFCs in calstabin-2^+/− cardiomyocytes after rapid pacing. (C) Ca²⁺ oscillations (min⁻¹ cell⁻¹) are significantly increased at higher pacing rates in calstabin-2^+/− or calstabin-2^−/− cardiomyocytes compared with WT. (D) JTV519 treatment significantly decreased aberrant Ca²⁺ oscillations in heterozygous calstabin-2^+/−, but not calstabin-2^−/−, cardiomyocytes. (E) SR Ca²⁺ load measured by local caffeine application was significantly reduced in calstabin-2-deficient cardiomyocytes.

Fig. 5.

JTV519 increased calstabin-2 binding to RyR2 in cardiomyocytes during β-AR stimulation. (A) RyR2 immunoprecipitation from calstabin-2^+/− cardiomyocyte lysate demonstrating increased PKA phosphorylation of RyR2 at Ser-2808 and calstabin-2 depletion after a 30-min exposure to 100 nM isoproterenol (ISO). Pretreatment with 1 μM JTV519 prevented calstabin-2 release from RyR2 despite PKA Ser-2808 phosphorylation. (B) Bar graphs summarizing results from three myocyte isolations each assessed in triplicate. ∗, P < 0.05.

Fig. 6.

Inhibition of calcium-dependent transient inward current (I_TI) in haploinsufficient calstabin-2^+/− cardiomyocytes by JTV519. (A) Confocal Ca²⁺ line scan image of isolated WT cardiomyocyte current trace during 1 μM isoproterenol (ISO). After a depolarization–repolarization step, the I_Ca tail current rapidly activated a homogeneous intracellular [Ca²⁺]_i transient followed by a long electrically stable resting phase. (B) In contrast, in calstabin-2^+/− cardiomyocytes, intracellular Ca²⁺ sparks and Ca²⁺ waves (arrows) were frequent, and Ca²⁺ waves coincide with I_TI (arrows) under the same conditions. Scales are the same as in A. (C) Typical current traces were recorded at −75 mV after a preconditioning depolarization train under control conditions (CO) or after 4 min of 1 μM ISO in cells dialyzed with 11 or 1 mM EGTA (D) or 1 mM EGTA and 1 μM JTV519 pretreatment (E). Scales in C–E are as indicated. (F) Bar graph summarizes average number of I_TIs per cell under indicated experimental conditions. No I_TI was detected in cells dialyzed with 11 mM EGTA (n = 4, each CO and ISO). In calstabin-2^+/− cells dialyzed with 1 mm EGTA, ISO significantly increased the number of I_TIs per cell (CO, n = 6; ISO, n = 5; P < 0.05). The number of I_TIs in calstabin-2^+/− cells treated with 1 μM JTV519 and dialyzed with 1 mM EGTA was not significantly increased by ISO (CO, n = 7; ISO, n = 11).