Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Abstract

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in "leaky" RyR2 channels and a diastolic SR Ca(2+) leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca(2+) leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca(2+) leak prevents fatal sudden cardiac arrhythmias in DMD.

Conflict of interest statement

Conflict of interest statement: A.R.M. is on the scientific advisory board and owns shares in ARMGO Pharma, Inc., a startup company developing RyR-targeted drugs for clinical use in the treatment of heart failure and sudden death.

Figures

Fig. 1.

RyR2 is S-nitrosylated and depleted of calstabin2 in mdx mice hearts. RyR2 was immunoprecipitated from heart homogenate of mdx and WT littermates at 21, 35, and 180 days after birth, as described in SI Materials and Methods. In addition, a group of 5-week-old mdx mice were treated for 2 weeks with S107. (A) Immunoblots prepared for RyR2, S-nitrosylation of cysteine residues on RyR2 (Cys-NO), and calstabin2 bound to RyR2. The blots are representative of three independent experiments. (B) Bar graph depicting the relative amount of RyR2 S-nitrosylation for each group, determined by dividing the Cys-NO signals by the total amount of RyR2 immunoprecipitated. (C) Bar graph depicting the relative amount of calstabin2 associated with the channel complex for each group, determined by dividing the calstabin2 signals by the total amount of RyR2 immunoprecipitated. (D) Immunoblots of heart lysates (100 μg) separated by 15% PAGE, prepared using an anti-calstabin antibody. (E) Immunoblots for nNOS, eNOS, and iNOS in 35- and 180-day-old mdx and WT littermate heart muscle lysate. (F) Coimmunoprecipitation of NOS enzymes with RyR2. RyR2 was immunoprecipitated from 500 μg of WT or mdx heart lysate and probed for RyR2 and the NOS enzymes. (G) Coimmunoprecipitation of RyR2 with NOS enzymes. NOS enzymes were immunoprecipitated separately from 500 μg of mdx heart lysate (35 days for nNOS; 180 days for eNOS and iNOS) and probed for RyR2 and the NOS enzymes. In F and G, positive controls for immunoblotting were 100 μg of 180-day-old WT heart lysate for RyR2, nNOS, and eNOS and 100 μg of 180-day-old mdx heart lysate for iNOS.

Fig. 2.

SR Ca2+ leak assessed by Ca2+ sparks analysis in mdx mice. Spontaneous SR Ca2+ release events were recorded in fluo-4 AM–loaded intact cardiomyocytes by laser scanning confocal microscopy, as described in SI Materials and Methods. (A) Representative ΔF/F line scan images (1.54 ms/line) were recorded in WT (Top) and mdx mice without (Middle) and with (Bottom) S107 treatment. Diastolic SR Ca2+ leak is estimated by the average sparks frequency (D). Average spatiotemporal properties of Ca2+ sparks, such as amplitude (B), full duration at half maximum (C), and spatial spread (full width at half maximum) (E). Data are expressed as mean ± SEM. *P < 0.05 WT vs. mdx; #P < 0.05 mdx vs. S107-mdx. n = 195 sparks in 20 cells in WT, 1,272 sparks in 58 cells in mdx, and 889 sparks in 60 cells in S107-treated mdx mice.

Fig. 3.

Elevated diastolic Ca2+ concentration in mdx mice. Isolated cardiomyocytes, loaded with indo-1 AM as described in SI Materials and Methods, were paced at 1 Hz. After 1 min, cells were maintained quiescent for 30 s before a second train of stimulation. (A) Global Ca2+ was recorded simultaneously, as illustrated by two typical recordings in WT and in mdx cardiomyocytes. (B) During the stimulation, diastolic Ca2+ was elevated in mdx cardiomyocyte. This was prevented by NAC or S107 treatment. (C) During the 30-s rest period, ∼50% of mdx cardiomyocytes exhibited Ca2+ waves that were not observed in WT or in mdx after NAC or S107 treatment. (D) The peak amplitude of the Ca2+ transients did not differ significantly in all conditions. Data are expressed as mean ± SEM. WT, n = 24; mdx, n = 29; mdx-S107, n = 15; mdx-NAC, n = 28. *P < 0.05.

Fig. 4.

ECG recordings in young mdx mice. ECGs were recorded as described in SI Materials and Methods. (A) Typical ECGs acquired by telemetry in 35-day-old vigil mice over 24 h and analyzed specifically during the 12-h overnight period in WT (Top; n = 5), mdx (Middle; n = 5), and S107-mdx treated (Bottom; n = 5) mice in normal condition (Left) and after i.p. injection of isoproterenol (2.5 mg · kg-1) (Right). (B–F) ECG analysis provides various functional parameters, including cardiac frequency given by the RR interval (B), heart rate variability expressed as SDNN (C), PVCs (D), QRS duration (E), and QT interval (F). (G) The QT interval also was measured after isoproterenol challenge. (H) Typical spontaneous sustained VT recorded in mdx mice after isoproterenol challenge. Note that VT was never triggered by isoproterenol treatment in either WT or S107-treated mice. *P < 0.05 control vs. mdx; #P < 0.05 mdx vs. S107-treated mdx.

Fig. 5.

ECG recordings in older mdx mice. (A) Typical ECGs acquired by telemetry in 180-day-old vigil mice over 24 h and analyzed specifically during the 12-h overnight period in WT (Top; n = 5), mdx (Middle; n = 5), and S107-treated mdx (Bottom; n = 5) mice in normal condition. (B and C) As in young mice, QT intervals in baseline condition (B) and after injection of isoproterenol (C) were enlarged in mdx mice (black bars) compared with WT mice (open bars). (D) PVCs also were increased in mdx mice. These electrophysiological abnormalities were fully abolished after S107 treatment (dashed bars). (E) Typical spontaneous sustained VT recorded in mdx mice after isoproterenol challenge. This mouse died from SCD during the acquisition. Note that VT was never triggered under the same treatment in ether WT or S107-treated mdx mice. *P < 0.05 control vs. mdx; #P < 0.05 mdx vs. S107-treated mdx.