Genetic Inhibition of Mitochondrial Permeability Transition Pore Exacerbates Ryanodine Receptor 2 Dysfunction in Arrhythmic Disease

Figure 1. Deletion of CypD in CASQ2−/− mice did not cause pathological cardiac remodeling. (A) Mean ± SEM of left ventricular EF in CypD−/− (n = 9), CASQ2−/− (n = 8), DKO (n = 8), and WT (n = 6) mice. (B) Representative images of M-mode echocardiography of left ventricle in the different groups. (C) Mean ± SEM of cross-sectional area of myocytes as measured with WGA staining in WT (n = 195 cells, N = 3 hearts), CypD−/− (n = 195 cells, N = 3 hearts), CASQ2−/− (n = 195 cells, N = 3 hearts), and DKO (n = 195 cells, N = 3 hearts). * p < 0.05 one-way ANOVA between groups. (D) Representative images of the WGA staining. (E) Mean ± SEM of heart weight/body weight (HW/BW) ratio in WT (n = 7), CypD−/− (n = 8), CASQ2−/− (n = 4) and DKO (n = 6) mice. (F) Representative images of H&E staining of the hearts from different groups.

Figure 2. Intracellular Ca2+ handling of myocytes isolated from WT, CypD−/−, CASQ2−/−, and DKO hearts. (A) Representative traces of time-dependent fluorescence profiles of Ca2+ transient and SCWs under baseline condition and in the presence of 100 nM ISO. (B) Mean ± SEM of average Fluo-3 amplitude (ΔF/F0) (n = 28–70 cells from N = 3–4 mice), 80% Ca2+ transient decay time (n = 28–70 cells from N = 3–4 mice), frequency of Ca2+ waves (n = 36–82 cells from N = 3–4 mice), and SR Ca2+ content (ΔF/F0) (n = 21–42 cells from N = 3–4 mice). * p < 0.05 one-way ANOVA between groups, # p < 0.05 vs. baseline.

Figure 3. Increased autonomous activation of CaMKII led to higher levels of Ser-2814 phosphorylation of RyR2 in DKO hearts. (A) Representative images of cellular ROS production in WT, CypD−/−, CASQ2−/−, and DKO. For each recording, 21 images were captured during a 5 min time window. The last image at the 5 min time point was shown as the representative. (B) Mean ± SEM of average ROS production rate in the three groups. N = 18–51 cells from N = 3–4 mice, * p < 0.05 one way-ANOVA between groups, # p < 0.05 vs. baseline. (C) Representative western blots detecting phosphorylation of CaMKII at Thr-286 and its expression in CypD−/−, CASQ2−/−, and DKO; GAPDH served as loading control. (D) Mean ± SEM, quantification of western blots; * p < 0.05 compared with DKO, analyzed by one-way ANOVA, N = 4 hearts for each group. (E) Representative western blots detecting phosphorylation of RyR2 at Ser-2814 and its expression in CypD−/−, CASQ2−/−, and DKO; GAPDH served as loading control. (F) Mean ± SEM, quantification of western blots; * p < 0.05 compared with DKO, analyzed by one-way ANOVA, N = 4 hearts for each group.

Figure 4. Transcriptome analysis identified altered gene expression patterns associated with electrical instability in DKO. (A) PCA plot showing the distinct gene expression between groups. (B) Number of differentially expressed genes when compared between the different genotypes. Number of significant upregulated genes (red) based on log2FC > 1&padj < 0.05, and number of significant down-regulated genes (blue) based on log2FC < −1&padj < 0.05. (C) Venn diagrams showing the intersection between significantly regulated genes (padj < 0.05 & log2FC > 1). (D) Volcano plot of all genes in different comparisons. Significant upregulated genes (red) based on log2FC > 1 and padj < 0.05, and significant downregulated genes (blue) based on log2FC < −1 and padj < 0.05. (E) GO term enrichment analysis of differentially expressed genes.

Figure 5. Genetic inhibition of mPTP exacerbates RyR2 dysfunction in CPVT by increasing the autonomous activation of CaMKII and subsequent hyperphosphorylation of RyR2.