Role of cardiac mitofusins in cardiac conduction following simulated ischemia-reperfusion

Abstract

Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.

Figure 1

Mfn1 and Mfn2 expression in the Mfn-DKO hearts. (A) Representative Western blot image (cropped) of Mfn1 and Mfn2 expression in the WT control versus Mfn-DKO hearts. Original gel images are presented in Supplementary Fig. 1. (B) Densitometry analysis of Mfn1 and Mfn2 expression in the Western blot images; n = 3 hearts per group, *p < 0.05 when compared against WT.

Figure 2

Functional characterization of isolated cardiomyocytes from mouse whole hearts using (OptioQUANT) platform. (A) Representative waveforms of action potential obtained from the wild-type (black trace) and Mfn1&2 KO (red traces) mouse cardiomyocytes. Dots represent AP points at 30%, 50%, 70% and 90% repolarization after depolarization. (B) Histograms (bottom) of wild type and mfn1&2 KO mouse cardiomyocytes show substantial variability and non-identical distribution of APD₉₀. The empirical cumulative distribution plot (upper) shows prolonged action potential durations in the transgenic KO mouse cardiomyocytes. The blue dotted line illustrates median value. (C) Bar graph summarizing the APD at 30%, 50%, 70% and 90% of repolarization in WT (n = 77) and DKO (n = 152) cells. APD₃₀: 0.015 ± 0.002 vs 0.015 ± 0.001; APD₅₀: 0.024 ± 0.002 vs 0.033 ± 0.002; APD₇₀: 0.044 ± 0.003 vs 0.066 ± 0.002 and APD₉₀: 0.076 ± 0.004 vs 0.113 ± 0.003. All APD values in seconds. *p < 0.001 determined by Student's t-test. *eCDF: empirical cumulative distribution function; APD action potential duration.

Figure 3

Conduction velocities (CV) of the atrial and ventricular tissue. Baseline CV of the (A) left atrial and (B) left ventricle of the wild-type (WT) versus Mfn-DKO hearts, *p < 0.05 when compared against WT. Changes in the CV during simulated ischemia (sI) and reperfusion (R) of the (C) left atrial tissue and (D) left ventricular tissue of the WT versus Mfn-DKO hearts, *p < 0.05 when compared against DKO; ^†p < 0.05 when compared against the lowest CV at the end of ischemia. Data are presented as mean ± SEM.

Figure 4

Lateralization of Cx43 in the Mfn-DKO hearts. Cross-sections of left ventricle from 8-weeks-old males, (A) WT control versus (B) Mfn-DKO, cardiac-specific, transgenic mice, immuno-stained with anti-Cx43 (brown). Examples of lateralised regions of Cx43 are delineated with red arrows. The negative controls using normal rabbit IgG and secondary antibody solely, without primary antibody, are represented by (C,D) respectively. Scale bar = 20 µm (E) Quantitative analysis of Cx43 lateralization. Images are representatives of 3 hearts for each group. Data are presented as mean ± SEM; *p < 0.05 when compared against WT.

Figure 5

Cx43 protein expression in the Mfn-DKO versus WT hearts. (A) Representative immunoblots (cropped) of total Cx43 and phosphorylated Cx43 from 3 different WT versus Mfn-DKO hearts. Original gel images are presented in Supplementary Fig. 2. (B) Quantification of band intensity for total Cx43, normalized to GAPDH expression, *p < 0.05 when compared against WT. (C) Quantification of band intensity for phosphorylated Cx43, normalized to total Cx43.

Figure 6

Schematic of murine atrial and ventricular isolation for MEA studies. Murine atrial tissue were placed epicardial surface down whereas transverse cross-section of the ventricle was emplaced onto the MEA for electrophysiological studies. Tissues were weighed down by a weighing harp. Representative electrograms recorded from a ventricular tissue slice using the MEA, with the proximal electrode (E1) and progressively distal electrodes (E2 and E3) to the paced stimulus. STIM indicates site of pacing stimulus, REF indicates the reference electrode position of the MEA. AT indicates activation time measured from stimulus onset to the steepest negative deflection of the electrogram. Created with BioRender.com”.