Kcne4 deletion sex-specifically predisposes to cardiac arrhythmia via testosterone-dependent impairment of RISK/SAFE pathway induction in aged mice

Figure 1. Kcne4 deletion sex-independently delays ventricular repolarization in aging mice. (A) Exemplar surface ECGs from male and female Kcne4+/+ and Kcne4−/− mice showing QT prolongation (arrow) in the latter. (B) Quantification of parameters from electrocardiograms as in A showing QT and QTc prolongation in Kcne4−/− mice of either sex (n = 12–18). *P < 0.05, **P < 0.01 between genotypes. All other group comparisons P > 0.05. RR, RR interval; HR, heart rate; PR, PR interval; QRS, QRS interval. (C) Exemplar surface ECG traces belonging to the only male Kcne4−/− mouse exhibiting frequent ventricular premature beats (VPBs).

Figure 2. Male sex exacerbates effects of Kcne4 deletion on predisposition to post-IR arrhythmias. Exemplar surface ECGs of Kcne4+/+ and Kcne4−/− mice of either sex during the cardiac reperfusion period (n = 10–15). PVT indicates polymorphic VT; VT, ventricular tachycardia; AVB, AV block.

Figure 3. Male Kcne4−/− mice are especially predisposed to longer and more severe post-IR ventricular tachyarrhythmias. (A) Quantification of cardiac arrhythmia incidence during post-ischemia reperfusion in male Kcne4+/+ (n = 10) and Kcne4−/− (n = 17) and female Kcne4+/+ (n = 15) and Kcne4−/− (n = 20) mice. Numbers of mice exhibiting each arrhythmia category are indicated in parentheses above columns. AVB, AV block; SCD, sudden cardiac death; VT, ventricular tachycardia; PVT, polymorphic VT; SVT, sustained ventricular tachycardia over 20 seconds. *P < 0.05; **P < 0.01 between genotypes or sexes. (B) Mean VT durations for male and female Kcne4+/+ and Kcne4−/− mice; n as in panel A. Mice without VT were indicated as 0 s duration. *P < 0.05, **P < 0.01, vs. male Kcne4−/− mice (by one-way ANOVA). (C) Latency to first run of VT after the onset of reperfusion in male and female Kcne4+/+ and Kcne4−/− mice; n as in panel A. NS, P > 0.05 among groups (by one-way ANOVA).

Figure 4. Kcne4 deletion activates RISK and SAFE pathways at baseline in female mice. (A) Schematic of the RISK and SAFE cardioprotective pathways. mPTP, mitochondrial permeability transition pore. (B–E) Left, representative western blots of phospho-(p) ERK1/2 and total (t) ERK1/2 (B), phospho-(p) AKT and total (t) AKT (C), phospho-(p) GSK3β and total (t) GSK3β (D), phospho-(p) STAT-3 and total (t) STAT-3 (E) from baseline male and female Kcne4+/+ and Kcne4−/− ventricles; one mouse per lane. Right, mean ratio of pERK/tERK (B, n = 5–7), pAKT/tAKT (C, n = 5–7), pGSK3β/tGSK3β (D, n = 7–10), pSTAT-3/tSTAT-3 (E, n = 5–9) band densities from blots as in left. *P < 0.05, **P < 0.01, between genotypes.

Figure 5. Kcne4 deletion impairs post-IR ventricular RISK/SAFE pathway induction in male mice. (A) Left, representative western blots of phosphorylated (p) ERK1/2 and total (t) ERK1/2 isolated from baseline and post-IRI male Kcne4+/+ and Kcne4−/− ventricles. IR: hearts subjected to 10 min left coronary ligation followed by 20 min of reperfusion; one mouse per lane. Right, mean ratio of pERK/tERK band density from blots as in left; n = 8–12. ***P < 0.001, compared with baseline male Kcne4+/+ mice; ###P < 0.001, compared with baseline male Kcne4−/− mice; †††P < 0.001, compared with male Kcne4+/+ mice post-IR (by One-way ANOVA). (B) Left, representative western blots of phospho- (p) AKT and total (t) AKT isolated from baseline and post-IR male Kcne4+/+ and Kcne4−/− ventricles; one mouse per lane. Right, mean ratio of pAKT/tAKT band density from blots as in left. n = 8–10; **P < 0.01, compared with baseline male Kcne4+/+ mice; ##P < 0.01, compared with baseline male Kcne4−/− mice; ††P < 0.01, compared with male Kcne4+/+ mice post-IR (by One-way ANOVA). (C) Left, representative western blots of phospho- (p) GSK3β and total (t) GSK3β isolated from baseline and post-IR male Kcne4+/+ and Kcne4−/−…

Figure 6. Kcne4 deletion does not impair post-IR ventricular RISK/SAFE pathway induction in female mice. (A) Left, representative western blots of phosphorylated (p) ERK1/2 and total (t) ERK1/2 isolated from baseline and post-IRI female Kcne4+/+ and Kcne4−/− ventricles. IR: hearts subjected to 10 min left coronary ligation followed by 20 min of reperfusion; one mouse per lane. Right, mean ratio of pERK/tERK band density from blots as in left; n = 5–8. *P < 0.05, **P < 0.01, compared with baseline female Kcne4+/+ mice; #P < 0.05, compared with baseline female Kcne4−/− mice (by One-way ANOVA). (B) Left, representative western blots of phospho- (p) AKT and total (t) AKT isolated from baseline and post-IR female Kcne4+/+ and Kcne4−/− ventricles; one mouse per lane. Right, mean ratio of pAKT/tAKT band density from blots as in left; n = 7–8. *P < 0.05, ***P < 0.001 compared with baseline female Kcne4+/+ mice; #P < 0.05,###P < 0.001 compared with baseline female Kcne4−/− mice (by One-way ANOVA). (C) Left, representative western blots of phospho- (p) GSK3β and total (t) GSK3β isolated from baseline and post-IR female Kcne4+/+ and Kcne4−/− ventricles; one mouse per lane. Right, mean ratio of…

Figure 7. Inhibition of ERK1/2, AKT, or STAT-3 phosphorylation increases arrhythmia predisposition in female mice post-IR. (A) Left, representative western blots of phosphorylated (p) ERK1/2 and total (t) ERK1/2 isolated from baseline and post-IRI female Kcne4+/+ and Kcne4−/− ventricles in the absence or presence of U0126. IR: hearts subjected to 10 min left coronary ligation followed by 20 min of reperfusion; one mouse per lane. Right, mean ratio of pERK/tERK band density from blots as in left; n = 5–6. *P < 0.05, ***P < 0.001, compared with baseline female Kcne4+/+ mice; #P < 0.05, ###P < 0.001, compared with baseline female Kcne4−/− mice (by One-way ANOVA). (B) Left, representative western blots of phosphorylated (p) AKT and total (t) AKT isolated from baseline and post-IRI female Kcne4+/+ and Kcne4−/− ventricles in the absence or presence of wortmannin (wort). IR: hearts subjected to 10 min left coronary ligation followed by 20 min of reperfusion; one mouse per lane. Right, mean ratio of pAKT/tAKT band density from blots as in left; n = 5–6. *P < 0.05, ***P < 0.001, compared with baseline female Kcne4+/+ mice; #P < 0.05, ###P < 0.001, compared with baseline female Kcne4−/− mice (by…

Figure 8. Castration decreases predisposition to severe IR ventricular arrhythmias in male Kcne4−/− mice. (A–D) Upper, representative western blots of phosphorylated (p) ERK1/2 and total (t) ERK1/2 (A), phospho- (p) AKT and total (t) AKT (B), phospho- (p) GSK3β and total (t) GSK3β (C), phospho- (p) STAT-3 and total (t) STAT-3 (D), isolated from castrated and non-castrated male Kcne4+/+ and Kcne4−/− left ventricles. Lower, mean ratio of pERK/tERK, pAKT/AKT, pGSK3β/GSK3β, pSTAT-3/STAT-3 band densities from blots as in upper; n = 5. *P < 0.05, **P < 0.01, compared with castrated male Kcne4−/− mice (by one-way ANOVA). (E) Bar graph showing serum concentrations of total testosterone in male (castrated and non-castrated) and female Kcne4+/+ and Kcne4−/− mice at baseline; n = 6–7 each group. ***P < 0.01, compared with non-castrated male Kcne4+/+ mice, ###P < 0.001, compared with non-castrated male Kcne4−/− mice (by One-way ANOVA). (F–H) Quantification of incidence of cardiac arrhythmia (F), ventricular tachycardia (VT) (G) and polymorphic VT (PVT) (H) during post-ischemia reperfusion in castrated and non-castrated male Kcne4+/+ and Kcne4−/− mice (n = 8–17). Numbers of mice per category are…