Expression of skeletal muscle sodium channel (Nav1.4) or connexin32 prevents reperfusion arrhythmias in murine heart

Abstract

Aims: acute myocardial ischaemia induces a decrease in resting membrane potential [which leads to reduction of action potential (AP) V(max)] and intracellular acidification (which closes gap junctions). Both contribute to conduction slowing. We hypothesized that ventricular expression of the skeletal muscle Na(+) channel, Nav1.4 (which activates fully at low membrane potentials), or connexin32 (Cx32, which is less pH-sensitive than connexin43) would support conduction and be antiarrhythmic. We tested this hypothesis in a murine model of ischaemia and reperfusion arrhythmias.

Methods and results: empty adenovirus (Sham) or adenoviral constructs expressing either SkM1 (gene encoding Nav1.4) or Cx32 genes were injected into the left ventricular wall. Four days later, ventricular tachycardia (VT) occurred during reperfusion following a 5 min coronary occlusion. In Nav1.4- and Cx32-expressing mice, VT incidence and duration were lower than in Sham (P < 0.05). In vitro multisite microelectrode mapping was performed in the superfused right ventricular wall. To simulate ischaemic conditions, [K(+)] in solution was increased to 10 mmol/L and/or pH was decreased to 6.0. Western blots revealed Cx32 and Nav1.4 expression in both ventricles. Nav1.4 APs showed higher V(max) and conduction velocity (CV) than Shams at normal and elevated [K(+)]. Exposure of tissue to acid solution reduced intracellular pH to 6.4. There was no difference in CV between Sham and Cx32 groups in control solution. Acid solution slowed CV in Sham (P < 0.05) but not in Cx32.

Conclusion: Nav1.4 or Cx32 expression preserved normal conduction in murine hearts and decreased the incidence of reperfusion VT.

Figure 1

(A) Representative western blots and loading controls showing Cx32 and Nav1.4 (32 and 260 kDa bands, respectively) expression in adenovirus-treated hearts 4 days after adenoviral gene transfection. In each panel, bands from left to right: positive control (liver for Cx32 and skeletal muscle for Nav1.4); LV and RV of mouse injected with Cx32- or SkM1-expressing adenovirus; negative control (LV of mouse injected with empty adenovirus). (B) Confocal images showing (in red) expression of Cx32 or Nav1.4 in LV and RV tissues from a mouse injected with either Ad-Cx32 (left) or Ad-SkM1 (right). Cryosections (10 μm) were cut perpendicular to the apico-basal axis with 1.5 mm steps. LV sections from a mouse injected with empty adenovirus were used as a negative control. The liver and skeletal muscle were used as positive controls for Cx32 and Nav1.4, respectively.

Figure 2

(A) Representative ECG complexes (lead II) obtained in Sham, Nav1.4-, and Cx32-expressing mice before occlusion of the coronary artery. Each trace begins and ends with a P-wave. (B) Examples of lead II ECG during reperfusion in one Sham, one Nav1.4-, and one Cx32-expressing mouse. Arrows indicate the release of ligation. Middle and right panels show ECG 10 and 15 s after beginning reperfusion, respectively. Two fragments of Sham ECG (before and during VT) are shown at a high sweep speed.

Figure 3

(A) Representative AP recorded from the RV free wall of one Sham and one Nav1.4-expressing mouse in Tyrode’ solution containing 4 mmol/L KCl. Note that AP in Nav1.4 mouse has higher amplitude and V_max. (B) Dependence of V_max on MDP. Values were collected from multisite microelectrode mapping at 4 and 10 mmol/L KCl. In each preparation, V_max values were grouped and averaged for each 5 mV step along the abscissa. Each individual experiment was allowed to contribute one point in each step. *P < 0.05 (n = 9 for each group).

Figure 4

Representative maps of conduction times in the RV epicardium of one Sham and one Nav1.4-expressing mouse at 4 mmol/L (A and B) and 10 mmol/L (C and D) KCl. The pacing site is marked with a cross and isochrones are drawn at 3 ms intervals. CV averaged over all recording sites in each preparation is shown at the bottom of each panels. (E) MDP, (F) V_max, and (G) CV show respective mean values for Sham and Nav1.4 groups at 4 and 10 mmol/L KCl. Each preparation contributed one point to the overall average value, and for each preparation, values were averaged over all recording sites. *P < 0.05 vs. 4 mM KCl in the same group and ⁺P < 0.05 vs. Sham at the same potassium concentration (n = 9 for both groups).

Figure 5

Representative maps of conduction times in the RV epicardium of one Sham and one Cx32-expressing mouse at pH in bathing solution = 7.4 (A and B) and 6.0 (C and D). The pacing site is marked with a cross and isochrones are drawn at 3 ms intervals. CV averaged over all recording sites in each preparation is shown at the bottom of each panel. (E) MDP, (F) V_max, and (G) CV show respective mean values for Sham and Cx32 groups at normal and low pH. Each preparation contributed one point to the overall average value, and for each preparation, values were averaged over all recording sites. *P < 0.05 vs. pH 7.4 in the same group, ⁺P < 0.05 vs. Sham at the same pH (n = 8 for both groups).

Figure 6

Effects of high potassium and low pH on MDP (A), V_max (B), and CV (C) in Sham, Nav1.4, and Cx32 groups. In each preparation, values were averaged over all recording sites. Each preparation contributed one point to average value. *P < 0.05 vs. pH 7.4 and 4 mmol/L KCl in the same group and ⁺P < 0.05 vs. Sham at the same potassium concentration and pH (n = 6 for Sham and n = 5 for Nav1.4 and Cx32 groups).