Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1‐mediated calcium entry in heart failure

FIGURE 1. Pyridostigmine improves in vivo cardiac function following TAC surgery but does not prevent TAC‐induced structural remodelling of the heart. (A) Plot of acetylcholinesterase activity 28 days after osmotic pump implantation (*P < 0.05 vs. CTL, †P < 0.05 vs. TAC), 1‐way ANOVA + Tukey HSD. For acetylcholinesterase activity, n = 10 mice per group. (B) Plot of ventricular ejection fraction (EF, expressed as a %) TAC resulted in a significant reduction in EF vs. CTL mice (*P < 0.000001 vs. CTL). However, chronic pyridostigmine treatment significantly increased EF vs. TAC mice (†P < 0.0001 vs. TAC), 1‐way ANOVA + Tukey HSD. For echocardiographic measurements, analysis was conducted following acquisition using at least three non‐adjacent contractions. (C) Plot of heart weight (HW) to tibia length (TL) ratio. TAC mice exhibited a significant increase in HW/TL ratio vs. CTL mice (*P < 0.0001 vs. CTL) and chronic pyridostigmine treatment failed to reduce HW/TL following TAC (*P < 0.0001 vs. CTL), 1‐way ANOVA + Tukey HSD. (D) Plot of heart weight (HW) to bodyweight (BW) ratio. TAC hearts show a significant increase in HW/BW vs. CTL hearts (*P < 0.0001 vs. CTL), and pyridostigmine failed to…

FIGURE 2. Pyridostigmine reduces foetal gene expression in TAC mice. (A) Representative plot of MYH6 (myosin heavy chain 6) mRNA expression. TAC induced a significant decrease in adult MYH6 mRNA expression vs. CTL hearts (*P < 0.05 vs. CTL), while pyridostigmine did not attenuate MYH6 mRNA expression vs. CTL hearts (*P < 0.05 vs. CTL), 1‐way ANOVA + Tukey HSD. (B) Representative plot of MYH7 (myosin heavy chain 7) mRNA expression. TAC hearts exhibited a significant increase in MYH7 mRNA expression vs. CTL hearts (*P < 0.05 vs. CTL), while pyridostigmine significantly reduced MYH7 expression following TAC (*P < 0.05 vs. TAC), 1‐way ANOVA + Tukey HSD. (C) Representative plot of NPPA (atrial natriuretic peptide) mRNA expression. TAC hearts exhibit increased NPPA mRNA expression vs. CTL hearts (*P < 0.05 vs. CTL) and pyridostigmine treatment trends towards a decrease in NPPA expression (P = 0.08 vs. TAC), 1‐way ANOVA + Tukey HSD. (D) Representative plot of NPPB (brain natriuretic peptide) mRNA expression. TAC induced a significant increase in NPPB mRNA expression vs. CTL hearts (*P < 0.05 vs. CTL), while pyridostigmine trended towards a decrease in NPPB expression following TAC surgery (P =…

FIGURE 3. Pyridostigmine prevents cardiac arrhythmias in pressure overload HF. (A, B) Representative ECG traces obtained under baseline and 10‐min stress challenge with epinephrine (1.5 mg/kg) plus caffeine (120 mg/kg) in TAC (A) or pyridostigmine treated mice (B). Arrows indicate premature ventricular complex (PVC). Summary plots showing PVC frequency (C) and incidence of arrhythmias in TAC and TAC + PYR groups

FIGURE 4. Pyridostigmine reduces frequency of arrhythmogenic calcium waves in pressure overload–induced HF. (A) Representative confocal line‐scan images along with spatially averaged fluorescence profiles recorded in myocytes from CTL, TAC and TAC + PYR groups, respectively. Cells were stimulated at 1 Hz in a presence of 100 nM isoproterenol, a beta‐adrenergic receptor agonist. (B) Summary plot of time dependence of calcium wave probability. TAC myocytes showed significant increase in calcium wave incidence (P < 0.0001, CTL vs. TAC, log‐rank test). Pyridostigmine treatment resulted in a significant reduction of calcium waves in TAC myocytes (P < 0.02, TAC vs. TAC + PYR, log‐rank test). Data were obtained from CTL (h = 5, n = 49), TAC (h = 2, n = 18) and TAC + PYR (h = 4, n = 25) groups, where h is the number of cell isolations, and n is the number of myocytes studied

FIGURE 5. Pyridostigmine reduces TAC‐mediated increase in CAMKII activity. (A) Representative Western blot showing total and phosphorylated levels of CaMKII and RYR2. (B) and (C) Boxplots illustrating the effect of pyridostigmine treatment on TAC‐induced increase in RyR2 S2814 phosphorylation (*, P < 0.01 vs. CTL, †, P < 0.05 vs. TAC) and in CaMKII T287 phosphorylation (*P < 0.05 vs. CTL; †P < 0.05 vs. TAC). 1‐way ANOVA + Tukey HSD, n = 3‐5 mice per group, and minimum of three experiments per group

FIGURE 6. Pyridostigmine reduces STIM1‐dependent store‐operated Ca entry (SOCE) in HF myocytes. (A) Representative images of Fluo‐4 fluorescence illustrating SOCE response in cardiac myocytes from CTL, TAC and TAC + PYR groups. Myocyte sarcoplasmic reticulum (SR) Ca was depleted by 1 µM thapsigargin, a SR Ca ATPase inhibitor, and SOCE was induced by rapid increase in extracellular Ca from 0 to 2 mM. (B) Representative time‐courses of spatially averaged fluorescence before and following application of Ca were recorded in myocytes from CTL, TAC, and TAC + PYR groups. (C) Summary data on the amplitude of local Ca increase in CTL, TAC and TAC + PYR myocytes, 1‐way ANOVA + Tukey HSD. (D) Summary data for the fraction of cells exhibiting SOCE response in CTL, TAC and TAC + PYR groups, respectively. (E) Boxplot illustrating maximal changes in myocyte fluorescence in response to application of external Ca, 1‐way ANOVA + Tukey HSD. For SOCE experiments, n = 11‐34 cells per group, n = 3‐10 mice per group, minimum of three experiments

FIGURE 7. Pyridostigmine effects STIM1 levels in control, HF and HF + PYR myocytes. (A) Representative Western blot showing STIM1 levels detected in heart preparations from CTL, TAC and TAC + PYR groups, respectively. GAPDH was used as a loading control. (B) Boxplot illustrating the effect of pyridostigmine treatment on TAC‐induced increase in STIM1 levels (*P < 0.05 vs. CTL, †P < 0.05 vs. TAC, 1‐way ANOVA + Tukey HSD, n = 3‐4 mice per group, minimum of three experiments per group