Research ArticleCardiology

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression

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Science Translational Medicine  12 Sep 2018:
Vol. 10, Issue 458, eaan0724
DOI: 10.1126/scitranslmed.aan0724
  • Fig. 1 Rycal S36 reduces SR Ca2+ leak in murine CMs.

    (A) Line-scan images of Ca2+ spark in the presence of isoproterenol (ISO; 10 nM) with or without rycal S36 (1 μM). (B and C) Mean CaSpF (B) and relative SR Ca2+ leak normalized to placebo (C). (D) Representative traces of intracellular Ca2+ recordings showing measurement of SR Ca2+ leak in placebo- and rycal S36–treated CMs. (E to G) Mean total SR Ca2+ leak (E), SR Ca2+ leak–to–SR Ca2+ load ratio (F), and SR Ca2+ load (G). Data are means ± SEM, n = 5 mice per group in (A) to (C); 7 mice per group in (D) to (G). *P < 0.05 versus placebo, two-tailed unpaired Student’s t test. Numbers within columns indicate CMs/mice. AU, arbitary units.

  • Fig. 2 Improved survival in TAC–rycal S36 mice.

    (A to C) Mean total SR Ca2+ leak (A), SR Ca2+ leak per SR Ca2+ load ratio (B), and SR Ca2+ load (C) at 3 weeks after TAC measured by the tetracaine protocol in isolated murine CMs. (D) Kaplan-Meier survival curves, TAC-placebo versus TAC–rycal S36; §P < 0.05, log-rank test. (E) Echocardiographic M-mode representative images at 9 weeks after TAC. (F to I) Mean radial diastolic peak velocity (F), septum thickness (G), LV end-diastolic diameter (LVEDD) (H), and ejection fraction (EF) (I). Data are means ± SEM, n = 5 mice per group in (A) to (C); 5 mice per sham and 8 to 15 mice per TAC in (F) to (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus corresponding sham, §§§P < 0.001 versus corresponding placebo, one-way analysis of variance (ANOVA) with Bonferroni posttest. Numbers within parentheses or columns indicate CMs/mice or mice.

  • Fig. 3 Similar pathological remodeling in placebo- and rycal S36–treated animals at 3 and 9 weeks after TAC.

    (A) Cardiac cross sections of the hematoxylin and eosin (H&E; top), Masson’s Trichome (MT; middle), and wheat germ agglutinin (WGA; bottom). (B and C) Quantification of cardiac fibrosis (B) and myocyte CSA (C). (D and E) HW/TL (D) and LungW/TL ratios (E). (F) Quantitative reverse transcription polymerase chain reaction (PCR) expression analyses of Nppa, Nppb, Serca2a, and Rcan1.4 at 9 weeks after TAC. Data are means ± SEM, n = 3 to 6 mice per group in (A) to (C); 5 to 8 mice per group in (D) and (E); 3 to 5 mice per group in (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus corresponding sham, one-way ANOVA with Bonferroni posttest. Numbers within columns indicate CM/mice or mice.

  • Fig. 4 Rycal S36 mitigates TAC-induced arrhythmias.

    (A) Mean APD (90%). (B) Average DAD incidence. Analyzed CMs/mice were 18/3 in TAC-placebo and 22/3 in TAC–rycal S36. (C) Telemetry ECGs recording at 9 weeks after TAC. PVCs (arrowheads) and polymorphic VT (arrow) are shown. (D to F) Incidence of PVCs (D), VT (E), and mean arrhythmia score (F) after TAC. (G) Representative traces of monophasic APs. (H) Ventricular arrhythmia inducibility. Data are means ± SEM, n = 2 to 3 mice per group in (A) and (B); 3 to 8 mice per group in (C) to (H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus corresponding sham, §P < 0.05, §§P < 0.01, §§§P < 0.001 versus corresponding placebo, two-tailed unpaired Student’s t test (B), and one-way ANOVA with Bonferroni posttest (A and D to H). Numbers within columns indicate CMs/mice or mice.

  • Fig. 5 Reduced mortality in MI–rycal S36 mice.

    (A) Kaplan-Meier survival curves, MI-placebo versus MI–rycal S36; §P < 0.05, log-rank test. (B) Representative echocardiographic M-mode images at 6 weeks after MI. (C) Average values of LVEDD and (D) EF. Data are means ± SEM, n = 5 mice per sham and 13 to 17 mice per MI in (B) to (D). **P < 0.01, ***P < 0.001 versus corresponding sham, one-way ANOVA with Bonferroni posttest. Numbers within parentheses or columns indicate mice.

  • Fig. 6 Rycal S36 abates MI-induced arrhythmias but has no beneficial effect on myocardial remodeling.

    (A) Cardiac cross sections of H&E (top) and WGA (bottom). (B to D) Mean myocyte CSA (B), HW/TL and LungW/TL ratios (C), and Nppa and Nppb mRNA expressions (D) at 6 weeks after MI. (E and F) Average PVCs and arrhythmia score. Data are means ± SEM, n = 3 to 4 mice per group in (A) and (B); 7 mice per sham and 14 to 18 mice per MI in (C); 4 to 5 mice per sham and 7 to 8 mice per MI in (D); 4 to 5 mice per group in (E) and (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus corresponding sham, §P < 0.05, §§§§P < 0.0001 versus corresponding placebo, one-way ANOVA with Bonferroni posttest. Numbers within columns indicate cells/mice.

  • Fig. 7 Rycal S36 averts proarrhythmic phenotype in human failing ventricular CMs.

    (A) Original line-scan images of Ca2+ spark, and (B) mean CaSpF, amplitude, width [full width at half maximum (FWHM)], and duration [full duration at half maximum (FDHM)]. (C) Representative traces of intracellular CaT and AP simultaneously recorded in ventricular CMs. (D to H) Mean DADs/min (D), APD (E), RMP (F), CaT amplitude (G), and fluorescence decay time (τ) (H). Data are means ± SEM, n = 4 patients per group in (A) and (B); 4 CMs per group in (C) to (H). *P < 0.05 versus placebo, two-tailed unpaired Student’s t test. Numbers within columns indicate cells/patients.

  • Fig. 8 Rycal S36 aborts proarrhythmic events in human nonfailing atrial CMs.

    (A) Original line-scan images of Ca2+ spark and (B) mean CaSpF. (C and D) Representative traces of recorded intracellular Ca2+ and membrane voltage in atrial CMs. (E to G) Representative traces showing AP (E), mean APD (F), and RMP (G). Data are means ± SEM, n = 4 to 5 patients per group in (A); 6 to 9 CMs per group in (F) and (G). *P < 0.05, one-way ANOVA with Bonferroni posttest (A), two-tailed unpaired Student’s t test (G). Numbers within columns indicate CMs/patients.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/458/eaan0724/DC1

    Materials and Methods

    Fig. S1. Global intracellular CaT and sarcomere shortening in placebo- and rycal S36–treated CMs.

    Fig. S2. Trans-stenotic systolic pressure gradient measurement after TAC.

    Fig. S3. SR Ca2+ release events in ventricular CMs at 3 weeks after TAC.

    Fig. S4. Representative traces of intracellular Ca2+ recordings illustrating measurement of total SR Ca2+ leak in CMs isolated from placebo- and rycal S36–treated mice at 3 weeks after TAC.

    Fig. S5. Effect of threefold higher dose of rycal S36 (≈4 μM) on SR Ca2+ leak and myocardial remodeling at 3 weeks after TAC.

    Fig. S6. Comparable perivascular fibrosis in placebo- and rycal S36–TAC mice.

    Fig. S7. Unaltered intracellular Ca2+ homeostasis in placebo- and rycal S36–treated mice at 3 weeks after TAC.

    Fig. S8. Western blot analyses of Ca2+ regulatory proteins at 9 weeks after TAC.

    Fig. S9. Patch-clamp and telemetric analyses of the TAC-operated mice.

    Fig. S10. Rycal S36 rescues SR Ca2+ leak in CPVT-E4076K CMs.

    Fig. S11. Electrophysiological analyses of the WT and Ryr2RS/WT mice at 3 weeks after shunt.

    Fig. S12. Increased SR Ca2+ leak in Ryr2RS/WT mice at 3 weeks after shunt.

    Fig. S13. Increased SR Ca2+ leak but comparable survival and functional deficit in Ryr2RS/WT versus WT mice after shunt.

    Fig. S14. Pathological myocardial remodeling is not different in WT and Ryr2RS/WT animals at 9 weeks after shunt.

    Fig. S15. Western blot analyses of Ca2+ regulatory proteins in WT/WT and Ryr2RS/WT (RS/WT) mice at 9 weeks after shunt.

    Table S1. Echocardiographic parameters of placebo- and rycal S36–treated mice at 3 and 9 weeks after TAC.

    Table S2. Echocardiographic parameters at 3 weeks after surgery of mice treated with a threefold higher dose of rycal S36.

    Table S3. Echocardiographic parameters of placebo- and dantrolene-treated mice at 7 weeks after TAC.

    Table S4. Raw APD time values at 9 weeks after surgery.

    Table S5. ECG parameters at 9 weeks after surgery.

    Table S6. Echocardiographic parameters of placebo- and rycal S36–treated mice at 3 and 6 weeks after MI.

    Table S7. Clinical data of explanted human hearts.

    Table S8. Echocardiographic parameters of WT and Ryr2RS/WT mice at 20 weeks after shunt.

    Table S9. Primary data (Excel file).

    References (5357)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Global intracellular CaT and sarcomere shortening in placebo- and rycal S36–treated CMs.
    • Fig. S2. Trans-stenotic systolic pressure gradient measurement after TAC.
    • Fig. S3. SR Ca2+ release events in ventricular CMs at 3 weeks after TAC.
    • Fig. S4. Representative traces of intracellular Ca2+ recordings illustrating measurement of total SR Ca2+ leak in CMs isolated from placebo- and rycal S36–treated mice at 3 weeks after TAC.
    • Fig. S5. Effect of threefold higher dose of rycal S36 (≈4 μM) on SR Ca2+ leak and myocardial remodeling at 3 weeks after TAC.
    • Fig. S6. Comparable perivascular fibrosis in placebo- and rycal S36–TAC mice.
    • Fig. S7. Unaltered intracellular Ca2+ homeostasis in placebo- and rycal S36–treated mice at 3 weeks after TAC.
    • Fig. S8. Western blot analyses of Ca2+ regulatory proteins at 9 weeks after TAC.
    • Fig. S9. Patch-clamp and telemetric analyses of the TAC-operated mice.
    • Fig. S10. Rycal S36 rescues SR Ca2+ leak in CPVT-E4076K CMs.
    • Fig. S11. Electrophysiological analyses of the WT and Ryr2RS/WT mice at 3 weeks after shunt.
    • Fig. S12. Increased SR Ca2+ leak in Ryr2RS/WT mice at 3 weeks after shunt.
    • Fig. S13. Increased SR Ca2+ leak but comparable survival and functional deficit in Ryr2RS/WT versus WT mice after shunt.
    • Fig. S14. Pathological myocardial remodeling is not different in WT and Ryr2RS/WT animals at 9 weeks after shunt.
    • Fig. S15. Western blot analyses of Ca2+ regulatory proteins in WT/WT and Ryr2RS/WT (RS/WT) mice at 9 weeks after shunt.
    • Table S1. Echocardiographic parameters of placebo- and rycal S36–treated mice at 3 and 9 weeks after TAC.
    • Table S2. Echocardiographic parameters at 3 weeks after surgery of mice treated with a threefold higher dose of rycal S36.
    • Table S3. Echocardiographic parameters of placebo- and dantrolene-treated mice at 7 weeks after TAC.
    • Table S4. Raw APD time values at 9 weeks after surgery.
    • Table S5. ECG parameters at 9 weeks after surgery.
    • Table S6. Echocardiographic parameters of placebo- and rycal S36–treated mice at 3 and 6 weeks after MI.
    • Table S7. Clinical data of explanted human hearts.
    • Table S8. Echocardiographic parameters of WT and Ryr2RS/WT mice at 20 weeks after shunt.
    • References (5357)

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    Other Supplementary Material for this manuscript includes the following:

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