Cx43 CT domain influences infarct size and susceptibility to ventricular tachyarrhythmias in acute myocardial infarction

Abstract

Aims: Hearts of mice expressing K258stop in place of connexin43 (Cx43) protein were subjected to acute myocardial infarction in order to assess the importance of Cx43 regulation on infarct size and arrhythmia susceptibility. This mutation K258stop prevents chemical regulation of Cx43 channels, including by low intracellular pH.

Methods and results: Langendorff-perfused hearts of mice harbouring one Cx43 knockout (KO) allele and one K258stop or Cx43 allele (K258stop/KO; Cx43/KO as control) were subjected to 1 h of ischaemia and 4 h of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Inducibility of ventricular tachyarrhythmias (VTs) was tested by applying an endocardial burst-pacing protocol during LAD occlusion. Separately, time course and the extent of acidification-induced closure of gap junction channels were tested by dual-voltage clamp. Infarct volume (as per cent of area at risk) was significantly larger in K258stop/KO hearts compared with Cx43/KO controls (42.2 +/- 3 vs. 30.4 +/- 1.7%, P = 0.004, n = 8 each). During LAD occlusion, K258stop/KO hearts had a higher incidence of pacing-induced VT and a higher frequency of occurrence of spontaneous premature ventricular beats. The occurrence of ventricular arrhythmias was also significantly larger in the K258stop/KO hearts during reperfusion. In separate experiments, we demonstrated reduced sensitivity to acidification-induced uncoupling in cell pairs obtained from K258stop/KO hearts.

Conclusion: Loss of the regulatory domain of Cx43 leads to an increase in infarct size and increased susceptibility to arrhythmias following acute coronary occlusion.

Figure 1

Quantification of infarct size. (A) Representative sections of Cx43/KO (top) and K258stop/KO (bottom) left ventricles after 1 h of LAD occlusion and 4 h of reperfusion. Perfused area: blue; area at risk (AAR): red and white. Bar: 5 mm. AAR volume expressed as percentage of total left ventricular mass (B); infarct volume as percentage of AAR (C). No difference in the AAR, but infarct volume was significantly increased in K258stop/KO hearts (grey) compared with WT/KO (black).

Figure 2

Cycle length and QRS duration. (A) Cycle length (RR intervals; top) and QRS duration (bottom). (B) No significant differences in basic cycle length (BCL) between genotypes. (C) QRS interval steadily decreased in Cx43/KO hearts during reperfusion, but not in K258stop/KO hearts.

Figure 3

Ventricular tachycardia during LAD occlusion and reperfusion. Burst pacing at 20 ms cycle length 1 h after the onset of LAD occlusion. Top: examples showing three burst-pacing events (I, II, III) for representative K258stop/KO (right) and Cx43/KO control hearts (left). Horizontal bars: 5 s; vertical bars: 2.5 V. Bottom: burst-pacing events at expanded time scale. VTs were induced by three sets of burst stimuli in K258stop/KO hearts, and duration of VT episode was longer than that in Cx43/KO hearts.

Figure 4

Ventricular tachycardia susceptibility. (A) Percentage of hearts susceptible to burst-pacing-induced tachyarrhythmias at each time point for Cx43/KO (black columns) and K258stop/KO (grey columns). K258stop-expressing hearts displayed higher inducibility of pacing-induced tachyarrhythmias at 45 min (83.3 vs. 41.6% in WT) and 1 h of LAD occlusion (100 vs. 77.6%). (B) K258stop/KO hearts (grey columns) showed higher number of VT episodes compared with Cx43/KO controls (black columns) at 30 and 45 min of ischaemia. (C) Percentage of hearts displaying spontaneous VTs during each hour of reperfusion. Cx43/KO, black bars; K258stop/KO, grey bars. K258stop-expressing hearts displayed trend towards higher incidence of tachyarrhythmias during second (42.9 vs. 14.3% in control), third (57.1 vs. 14.3%), and fourth hours of reperfusion (57.1 vs. 14.3%). Because of small sample size, difference was not statistically significant (Fisher's exact test). (D) K258stop/KO hearts (grey columns) presented with significantly more episodes of tachyarrhythmias than controls (black columns). To assess this variable, spontaneous VT episodes during the entire reperfusion were counted for each animal and averaged per genotype (1.57 ± 0.95 in Cx43/KO hearts vs. 7.14 ± 2.27 in K258stop/KO hearts; P = 0.043).

Figure 5

PVC during ischaemia and reperfusion. Seven Langendorff-perfused hearts per genotype were monitored for PVCs during LAD occlusion. (A) Percentage of all hearts displaying spontaneous PCVs during each 15 min interval. Cx43/KO (black columns) and K258stop/KO (grey columns). K258stop-expressing hearts displayed a trend towards higher incidence of tachyarrhythmias during the fourth 15 min interval (100 vs. 50%). Because of small sample size, the difference was not statistically significant (Fisher's exact test). (B) K258stop/KO hearts displayed increasingly more episodes of PVC than the hearts of Cx43/KO animals. This trend was significant in the last 15 min of LAD occlusion (Cx43/KO: 2 ± 1.3 vs. K258stop/KO: 16.8 ± 3.8). The same seven hearts per genotype were also monitored for PVCs during the 4 h of reperfusion. (C) Percentage of all hearts displaying PVCs during each hour of reperfusion Cx43/KO (black columns) and K258stop/KO. K258stop-expressing hearts displayed a higher incidence of PVCs during the second, third, and fourth hours of reperfusion. (D) K258stop/KO hearts tended to display more PVCs than the hearts of Cx43/KO animals during reperfusion. This trend was significant during the third and fourth hours of reperfusion.

Figure 6

Acidification-induced uncoupling in pairs of cardiomyocytes. Changes in junctional conductance (G_j) as a function of time after patch break. Cells recorded in dual-patch clamp configuration. Pipettes were filled with an internal pipette solution buffered to pH 7.2 (closed circles) or 6.2 (grey symbols). Notice the progressive reduction in G_j in cell pairs obtained from Cx43/KO hearts (grey triangles), not apparent in cells with the K258stop/KO genotype (grey circles and closed circles; P << 0.001; Student's t-test).