Metabolic stress exacerbates Ca 2+ dysregulation and arrhythmias in catecholaminergic polymorphic ventricular tachycardia

FIGURE 1. Dysregulation of myocyte Ca2+ handling was exacerbated in Akita‐CASQ2−/−. (a) Representative traces of time‐dependent fluorescence profiles of Ca2+ transient under baseline conditions and in the presence of 100 nM ISO. (b, c) Mean ± SD of average Fluo‐3 amplitude (ΔF/F 0) (n = 52–91 cells from N = 4–5 mice) and 80% Ca2+ transient decay time (n = 52–91 cells from N = 4–5 mice). (d) Mean ± SD of frequency of Ca2+ waves (n = 99–133 cells from N = 4–5 mice). Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 2. Akita‐CASQ2−/− mice exhibited the most severe arrhythmic burden. (a) Representative surface ECG traces of WT, Akita, CASQ2−/−, and Akita‐CASQ2−/− after catecholamine challenge (2 mg/kg epinephrine and 120 mg/kg caffeine, i.p. injection). (b) Mean ± SD of arrhythmia score, N = 4–7 mice. Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 3. Akita‐CASQ2−/− mice exhibited systolic and diastolic dysfunction. (a) Representative images of M‐mode echocardiography of left ventricle in the different groups. (b) Mean ± SD of fractional shortening (WT, N = 7 mice; Akita, N = 9 mice; CASQ2−/−, N = 6 mice; and Akita‐CASQ2−/−, N = 6 mice). (c) Representative images of Pulse‐wave Doppler. (d) Mean ± SD of myocardial performance index (WT, N = 7 mice; Akita, N = 9 mice; CASQ2−/−, N = 6 mice; and Akita‐CASQ2−/−, N = 5 mice). Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 4. Akita‐CASQ2−/− mice developed cardiac hypertrophy. (a) Representative images of H&E staining of the hearts from WT, Akita, CASQ2−/−, and Akita‐CASQ2−/−. (b) Mean ± SD of heart weight normalized to body weight (WT, N = 5 mice; Akita, N = 9 mice; CASQ2−/−, N = 5 mice; and Akita‐CASQ2−/−, N = 7 mice). (c) Representative images of the WGA staining. (d) Mean ± SD of cross‐sectional area of myocytes as measured with WGA staining (n = 245–317 cells from N = 4–5 animals). Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 5. Akita‐CASQ2−/− mice showed limited cardiac fibrosis. (a) Representative images of picrosirius red staining in WT, Akita, CASQ2−/−, and Akita‐CASQ2−/−. (b) Mean ± SD of quantification of fibrosis normalized to area; N = 4–5 mice. Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 6. Akita‐CASQ2−/− mice showed few signs of apoptosis. (a) representative images of TUNEL staining in WT, Akita, CASQ2−/−, and Akita‐CASQ2−/−, and a positive control which is WT tissue treated by DNase. (b) Mean ± SD of quantification of TUNEL positive signal normalized to area; N = 4–5 mice. Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 7. Assessment of mitochondrial function by high‐resolution respirometry (HRR). (a) Basal fatty acid oxidation activity (FAO) of mitochondria in permeabilized myocardium. (b) Fatty acid oxidation activity of mitochondria in permeabilized myocardium in the presence of added ADP (oxidative phosphorylation, OxPhos). (c) The OxPhos activity mediated by FAO, complex I (CI), and complex II (CII). (d) Maximal respiration activity in the presence of FCCP. (e) Spare respiratory capacity of mitochondria in permeabilized myocardium. N = 4–6 mice. Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.

FIGURE 8. Akita‐CASQ2−/− myocytes had a higher rate of mitochondrial ROS production. (a) representative images of mitochondrial ROS production in WT, Akita, CASQ2−/−, and Akita‐CASQ2−/−. (b) Mean ± SD of mitochondrial ROS production rate in the four groups; n = 29–87 cells from N = 3–5 animals. Data were analysed using two‐way ANOVA followed by Tukey's post hoc test.