Cardiac expression of skeletal muscle sodium channels increases longitudinal conduction velocity in the canine 1-week myocardial infarction

Abstract

Background: Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs.

Objective: The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression.

Methods: Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression.

Results: Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P <.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P <.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes.

Conclusion: Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4.

Figure 1

A. The dV/dt of the upstroke of the cardiac action potential recorded from isolated epicardial tissue slabs excised from the injected sites of the 1 week old myocardial infarction as a function of the maximum diastolic potential (MDP). Tissues were superfused with solutions containing various potassium concentrations to depolarize the cell. Differences between the two groups of animals are statistically significant at −85 mV and at more depolarized potentials B and C. Respective averaged data of maximum diastolic potential (MDP) and conduction velocity (CV) in control and Nav1.4 animals, during superfusion with [K⁺] 4 and 7 mmol/L. Although superfusion with elevated [K⁺] caused a similar decrease of MDP, conduction was significantly faster in the Nav1.4-expressing tissue than in the non-Nav1.4-expressing control tissue at 7 mmol/L [K⁺]. Unfilled symbols: GFP-injected controls (n=18). Filled symbols: Nav1.4 (n=16). * indicates P<.05.

Figure 2

Representative activation maps superimposed on photographic images of tissue slabs excised from the infarct zones of Nav1.4 dogs (right panels) and GFP-injected control dogs (left panels). Panels A and C: activation patterns of the tissue superfused with [K⁺] 4 mmol/L. Panels B and D: superfusion with [K⁺] 7 mmol/L. Dots indicate microelectrode impalement sites. Numbers are activation times relative to the stimulus artifact. St: Stimulus site. EG: reference electrogram site. Horizontal scale 2 mm; interval between isochrones 5 ms.

Figure 3

Representative activation maps from a control (GFP-injected) animal and a Nav1.4 injected animal. Each panel shows the electrode grid (dots, 0.5 mm interelectrode distance) and the site of stimulation (symbol ∏). Lines indicate 5 ms isochrones, colors are isochronal classes). Dotted lines show where conduction velocities were calculated. Close to the stimulus site, electrograms incorporated a stimulus artifact that was too large to allow identification of the moment of local activation. Note that longitudinal but not transverse conduction velocity is increased in the Nav1.4 expressing site but not in the control site. Normal zone (non injected) maps from the same animals are shown in the top panels.

Figure 4

Averaged longitudinal and transverse conduction velocities (V_L and V_T). In the noninjected normal zones longitudinal and transverse conduction velocities are not statistically different. In the Nav1.4-expressing infarcted zones the longitudinal but not the transverse conduction velocity is significantly increased. * indicates P<.05.

Figure 5

Confocal immunofluorescent labeling of Nav1.4 (red). Panel A, Positive control: a skeletal muscle section immunostained with Nav1.4 antibody. Panel B, Negative control: a left ventricular section without Nav1.4 injection, immunostained with Nav1.4 antibody. Panel C. Representative images of left ventricular sections at the injection site immunostained with Nav1.4 antibody. The antibody stained intensely at the longitudinal end of the cardiomyocytes in the Nav1.4 injected sections. Panel D, enlargement of section in panel C. DAPI (blue) was used as a nuclear stain. Scale bars are 20 microns in Panels A–C and 5 microns in Panel D.

Figure 6

Confocal co-immunofluorescent labeling of Nav1.4 and Cx43 from same dog as in Figure 5. Representative images of left ventricular sections at the Nav1.4 injection site co-immunostained for Nav1.4 (red) and Cx43 (green) antibody. Note the co-localization of Nav1.4 and Cx43 protein in the overlay (yellow).