Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model

Abstract

Background: Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain.

Objectives: To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries.

Methods and results: We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice.

Conclusion: Opa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Cardiac morphology and function.

BW (A) and HW/BW ratio (B) in 3- and 6-month-old Opa1+/- (n = 11 and 12 respectively) and WT (n = 6 and 15 respectively) mice. C, D: Echocardiography parameters in 3- and 6-month-old mice,(n = 9/group at 3 months and 10/group at 6 months) whereby C represents left-ventricular end-diastolic diameter (LVEDD), and D depicts fractional shortening (FS). E, F: Examples of electron microscopy images at 12,000x magnification in 3-month-old WT (E) and Opa+/- (F) mice. Values are mean ± SEM. * p<0.05 and ** p<0.01. Non-parametric Mann-Whitney test (HW/BW) and t-test (BW, LVEDD and FS) according to distribution.

Fig 2. Infarct size.

A, B: Examples of left-ventricular sections with TTC staining after 30 minutes of ischemia and 2 hours of reperfusion ex vivo in WT (A) and Opa1+/- (B) mice, and histograms showing AN as a percentage of total LV in WT (n = 11) and OPA1+/- (n = 12) mice (C). D, E: Examples of left-ventricular sections with TTC-staining after 45 minutes of ischemia and 2 hours of reperfusion in vivo in WT (D) and OPA1+/- (E) mice. On the left side, images before TTC staining showing Evans blue coloration of the perfused myocardium and AAR as the non-blue area. On the right side, images after TTC staining showing AN in white. F: Histograms showing AN as a percentage of AAR and AAR as a percentage of total LV area in Opa1+/- and WT mice (n = 8/group). Values are mean ± SEM. * p<0.05 and ** p<0.01. Non-parametric Mann-Whitney test (AN/AAR) and t-test (AAR/LV).

Fig 3. Baseline and post-I/R fission and fusion protein expression.

Opa1, Mfn2, Drp1, and Fis1 expression assessed by means of western blotting at baseline (n = 6/group) and after I/R (n = 3-6/group). GAPDH was used as a loading control. Values are mean ± SEM. * p

Fig 4. Baseline and post-I/R oxidative phosphorylation.

Complex I, II, and IV oxygen consumption were measured in WT and Opa1+/- mouse hearts at baseline and after I/R (n = 6/group). The I/R protocol consisted of 45 minutes of ischemia and 2 hours of reperfusion. The ratio [state 3 rate]: [state 4 rate] is represented by means of the respiratory control index (RCI) in both groups at baseline and after I/R. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Apoptosis assessment and sensitivity to mPTP.

A: Examples of TUNEL images of left-ventricular sections in confocal microscopy (70x magnification) after I/R. Cell nuclei are stained in red (propidium iodide, wavelength = 620 nm). TUNEL-positive nuclei appear in green (fluorescein, wavelength = 460 nm). B: Bax and Bcl-2 protein expressions (n = 4-6/group). C: Percentage of TUNEL-positive nuclei compared to total nuclei (n = 8 in Opa1+/- and 7 in WT). D: Caspase 3 activity amongst Opa1+/- (n = 7) and WT (n = 7) groups. E: mPTP opening sensitivity at baseline and after I/R (n = 6/group). Required calcium overload for mPTP opening in WT and Opa1+/- groups at baseline, after I/R, with or without use of cyclosporine analog (CsA). The I/R protocol included 45 minutes of ischemia and 2 hours of reperfusion. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. $p< 0.05 compared to the same group at baseline + CsA. Non-parametric Mann-Whitney test (TUNEL and Bax/Bcl2 ratio) and t-test (Bax expression and caspase 3 activity).

Fig 6. Calcium transients and SR calcium load in Opa1+/- isolated left-ventricular cardiomyocytes.

A: Typical calcium transients recorded under field stimulation at 1Hz in WT (black) and Opa1+/- (red) isolated left-ventricular cardiomyocytes using fluo-4 calcium dye. B: Mean values of peak calcium transients (WT, n = 32 cells and 4 animals, vs. Opa1+/-, n = 43 cells and 5 animals; *p<0.05). C: Mean values of decay time constant of steady-state Ca2+ transients (WT, n = 32 cells and 4 animals, vs. Opa1+/-, n = 43 cells and 5 animals; *p<0.05). D: Typical example of a caffeine-induced SR Ca2+ release in WT (black) and Opa1+/- (red) left-ventricular cardiomyocytes using fluo-4 calcium dye. E: Mean values of maximum amplitude of caffeine-induced SR Ca2+ release, indicative of SR Ca2+ load (WT, n = 7 cells and 4 animals, vs. Opa1+/-, n = 11 cells and 5 animals; p>0.05). F: Mean values of decay time constant of caffeine-induced SR Ca2+ release, indicative of cytosolic Ca2+ extrusion (WT, n = 7 cells and 4 animals, vs. Opa1+/-, n = 11 cells and 5 animals; *p<0.05). Statistical test was t-test.

Fig 7. Opa1+/- left-ventricular cardiomyocyte AP.

A: Typical AP recorded using whole-cell patch-clamp technique with current clamp at 1Hz in WT (black) and Opa1+/- (red) isolated left-ventricular cardiomyocytes. B: Mean values of AP duration at different percentages of AP repolarization (WT: n = 12 cells and 3 animals vs. Opa1+/-: n = 12 cells and 4 animals; *p<0.05). C, D: Frame C displays 30 seconds of continuous AP recording at a pacing rate of 1Hz early after depolarization, whilst frame D shows a magnified view of the early post-depolarization period. Note that 6/12 Opa1+/- cells present such arrhythmic events, whereas WT myocytes underwent no early subsequent depolarization. Statistical test was t-test.

Fig 8. Dynamic mitochondrial Ca2+ movements during APs recorded using Rhod-2 in conjunction with a whole-cell patch-clamp technique.

A: Typical rhod-2 signal during a steady-state AP recorded at 1Hz in WT (black) and Opa1+/- (red) isolated left-ventricular cardiomyocytes. B: Mean values of peak rhod-2 signal. C: Rate of rise. D: Decay time constant in WT (n = 12 cells and 3 animals) and Opa1+/- cells (n = 12 cells and 4 animals; *p<0.05). Statistical test was t-test.