Involvement of mitochondrial permeability transition pore (mPTP) in cardiac arrhythmias: Evidence from cyclophilin D knockout mice

Abstract

In the present study, we have used a genetic mouse model that lacks cyclophilin D (CypD KO) to assess the cardioprotective effect of mitochondrial permeability transition pore (mPTP) inhibition on Ca²⁺ waves and Ca²⁺ alternans at the single cell level, and cardiac arrhythmias in whole-heart preparations. The protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) caused mitochondrial membrane potential depolarization to the same extent in cardiomyocytes from both WT and CypD KO mice, however, cardiomyocytes from CypD KO mice exhibited significantly less mPTP opening than cardiomyocytes from WT mice (p<0.05). Consistent with these results, FCCP caused significant increases in CaW rate in WT cardiomyocytes (p<0.05) but not in CypD KO cardiomyocytes. Furthermore, the incidence of Ca²⁺ alternans after treatment with FCCP and programmed stimulation was significantly higher in WT cardiomyocytes (11 of 13), than in WT cardiomyocytes treated with CsA (2 of 8; p<0.05) or CypD KO cardiomyocytes (2 of 10; p<0.01). (Pseudo-)Lead II ECGs were recorded from ex vivo hearts. We observed ST-T-wave alternans (a precursor of lethal arrhythmias) in 5 of 7 WT hearts. ST-T-wave alternans was not seen in CypD KO hearts (n=5) and in only 1 of 6 WT hearts treated with CsA. Consistent with these results, WT hearts exhibited a significantly higher average arrhythmia score than CypD KO (p<0.01) hearts subjected to FCCP treatment or chemical ischemia-reperfusion (p<0.01). In conclusion, CypD deficiency- induced mPTP inhibition attenuates CaWs and Ca²⁺ alternans during mitochondrial depolarization, and thereby protects against arrhythmogenesis in the heart.

Keywords: Arrhythmias; Calcium; Cyclophilin D; Heart; Mitochondria; mPTP.

Figure 1. FCCP Depolarized the ΔΨ_m to the Same Extent in WT and CypD KO Myocytes

FCCP (A, 100 nM; B, 1 μM; C, 20 μM) was used to depolarize Δψ_m. The fluorescence in the presence of 30 μM FCCP was set as 100% dissipation (not shown). (A-a, B-a, C-a) Snapshots of TMRM fluorescence at baseline (0), 6, and 10 minutes in the presence of FCCP are shown. (A-b, B-b, C-b) Summary data showing a decrease in TMRM fluorescence, * p < 0.05, ** p < 0.01 compared to the respective baseline value.

Figure 2. FCCP-Induced mPTP Opening was Attenuated in CypD KO Myocytes

(A-a, B-a) Snapshots of calcein fluorescence at baseline (0 min), 6 min, and 10 min after the treatment with FCCP at 1 and 20 μM, respectively. (A-b, B-b) Summary time course data showing the decrease in calcein fluorescence in the presence of FCCP, * p < 0.05, ** p < 0.01 compared to baseline within each group; # p < 0.05, ## p < 0.01 compared to WT.

Figure 3. FCCP-Induced Mitochondrial Ca²⁺ Release was Reduced in CypD KO Myocytes

FCCP-Induced mitochondrial Ca²⁺ release was evaluated by the elevation of basal Ca²⁺ levels in Ca_o-free Tyrode’s solution after SR Ca²⁺ was depleted by 10 mM caffeine (Caff) and 1 μM thapsigargin (Tha). (A) Less mitochondrial Ca²⁺ release by 1 μM FCCP was observed in CypD KO myocytes compared to WT. (B) FCCP at high concentration (20 μM) induced same mitochondrial Ca²⁺ release in both CypD KO and WT myocytes, indicating equal mitochondrial Ca²⁺ content. (C) Summarized data, ** p < 0.01.

Figure 4. FCCP-Induced CaW Rate Increase was Attenuated in CypD KO Myocytes

(A) A representative Ca²⁺ fluorescence trace showing the effect of FCCP (50–100 nM) on spontaneous CaWs in a WT ventricular myocyte. (B) The same as A, except in a CypD KO ventricular myocyte. (C) Data summary, * p < 0.05, compared to control within each group; ## p < 0.01 between CypD KO and WT.

Figure 5. Lower Incidence of FCCP-Induced Ca²⁺ Alternans was Observed in CypD KO Myocytes

(A–C) Representative traces of control (top), treated with FCCP 50 nM (middle), and expanded area of interest (bottom), for each group: WT (A), CypD KO (B), and WT pretreated with CsA 1 μM (C). Cells were field stimulated using a programed protocol as indicated in B. The pacing cycle length decreased progressively from 300 to 80 ms (B). (D) Summarized Data. *p < 0.05, ** p < 0.01 compared to the WT group.

Figure 6. Lower Incidence of FCCP-Induced ST-T Wave Alternans was Observed in CypD KO Mouse ex-vivo Hearts

(A) Pseudo-Lead II ECG signals recorded from Langendorff-perfused WT or CypD KO hearts in the absence (Ctl) and presence of 30 nM FCCP (FCCP). Arrows indicated alternating ST level alternans. (B) Overlapped ECG waveforms; a and b represent individual complexes appearing in the ECG traces as indicated in A. (C) Summarized Data showing incidences of ST alternans in WT and CypD KO hearts. *p <0.05 by Fisher’s exact test.

Figure 7. FCCP and Chemical I/R Induced Arrhythmias in ex-Vivo Hearts and Relevance to mPTP

(A–C) Pseudo-Lead II ECG signals recorded from WT mice treated with FCCP (A), or FCCP + CsA (B), and CypD KO mice treated with FCCP (C). Programmed stimulations were applied to evaluate the vulnerability to arrhythmias in each case. (E–G) Pseudo-Lead II ECG signals recorded from WT mice treated with a chemical ischemia solution (E), or a chemical ischemia solution + CsA (F), and CypD KO mice treated with a chemcical ischemia solution (G). Programmed stimulations were applied 10 minutes after reperfusion, and every 10 minutes thereafter to evaluate the vulnerability to arrhythmias in each case. The top panels show baseline ECG before treatment. A representative arrhythmia event is shown in each case. (D, H) Summarized arrhythmia scores for each group, * p < 0.05, **p < 0.01, compared to the WT.