Modulating cardiac conduction during metabolic ischemia with perfusate sodium and calcium in guinea pig hearts

Abstract

We previously demonstrated that altering extracellular sodium (Na_o) and calcium (Ca_o) can modulate a form of electrical communication between cardiomyocytes termed "ephaptic coupling" (EpC), especially during loss of gap junction coupling. We hypothesized that altering Na_o and Ca_o modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Na_o (147 or 155 mM) and Ca_o (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( W_P), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Na_o and 2.0 mM Ca_o; however, theoretically increasing EpC with 155 mM Na_o was arrhythmogenic, and CV could not be measured. Reducing Ca_o to 1.25 mM expanded W_P, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing W_P with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded W_P, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia.

Keywords: arrhythmia; calcium; conduction; ischemia; sodium.

Fig. 1.

Conduction velocity (CV) during metabolic ischemia and reperfusion. A: representative optical isochrone maps of conduction in hearts perfused with solutions A and D, where solution A slows CV most and solution D slows CV least during simulated ischemia. B–D: percent change from average baseline (−15 and 0 min) for transverse CV (CV_T), longitudinal CV (CV_L), and anisotropic ratio (AR) in hearts perfused with solutions A–D. The yellow shaded region indicates the period of simulated ischemia. Student’s t-tests with Bonferroni correction were applied to determine statistical significance (#P < 0.05 relative to solution A at the same time point). Experimental numbers are shown in Table 2. Soln, solution.

Fig. 2.

Action potential characteristics. A: representative action potentials (left) and expanded action potential upstrokes (right) from hearts perfused with the study solutions. B: summary of the time interval between activation time and 30% and 90% repolarization (APD₃₀ and APD₉₀, respectively) and rise time (RT) during baseline (t = −15 to 0 min; solid boxes) and metabolic ischemia (t = 30 min; open boxes). During ischemia, APD₃₀ significantly decreased with solution C, and RT significantly increased with solutions A, C, and D relative to baseline (n = 6, *P < 0.05). C: temporal responses of APD₃₀, APD₉₀, and RT from hearts perfused with the study solutions. The yellow shaded region indicates the period of simulated ischemia. Student’s t-tests with Bonferroni correction were applied to determine statistical significance (n = 6, *P < 0.05 relative to baseline, t = 0 min). Hearts in arrhythmias during mapping time points were excluded, and only time points that had at least three measurable APD₉₀ values were included in the analysis. Soln, solution.

Fig. 3.

Perinexal width (W_P) during metabolic ischemia and reperfusion. A: representative electron micrographs of perinexi from hearts perfused with solutions A and D. Solution A demonstrated no change in W_P during metabolic ischemia (t = 30 min) and reperfusion (t = 50 min), whereas solution D-perfused hearts demonstrated W_P widening during metabolic ischemia and returned to baseline values during reperfusion. B: summary of W_P values averaged over a distance of 30–105 nm away from the edge of the gap junction plaque. Student’s t-tests with the Bonferroni correction were applied to determine statistical significance (n = 3 hearts/solution with 15 images/heart, *P < 0.05 relative to baseline and #P < 0.05 relative to solution A for a given condition). Base, baseline; Isch, ischemia; Rep, reperfusion; Soln, solution.

Fig. 4.

Connexin (Cx)43 expression and phosphorylation. A: representative Western blots probed for Cx43 phosphorylated at S368 (pCx43-S368), total Cx43, and GAPDH (loading control) are shown during baseline (Base; t = 0 min), metabolic ischemia (Isch; t = 30 min), and reperfusion (Rep; t = 50 min) conditions from hearts perfused with solutions A–D. B and C: summary of pS368-Cx43-to-total Cx43 (B) as well as total Cx43-to-GAPDH (C) ratios normalized to baseline values perfused with solution A. Student’s t-tests with the Bonferroni correction were applied to determine statistical significance (n = 3 hearts/solution, *P < 0.05 relative to baseline). Soln, solution.

Fig. 5.

Effects of albumin and mannitol as modulators of extracellular volume. A: summary of perinexal width (W_P) values averaged over a distance of 30–105 nm away from the edge of the gap junction plaque with solutions containing albumin and mannitol. Metabolic ischemia (t = 30 min) increased perinexal (W_P) expansion with solutions containing albumin and mannitol (n = 3 per group, *P < 0.05 relative to baseline), but perinexal expansion was greater with mannitol (n = 3 per group, #P < 0.05, solution F relative to solution E). B: summary of connexin (Cx)43 phosphorylated at S368 (pCx43-S368), total Cx43, and GAPDH demonstrating that 30 min of ischemia did not alter the pCx43-to-Cx43 ratio between solutions, but total Cx43 during ischemia was reduced with mannitol in solution F relative to solution E (n = 3 per group). C: percent change from baseline in transverse conduction velocity (CV_T), longitudinal conduction velocity (CV_L), and anisotropic ratio (AR) in hearts perfused with solution E (n = 6) and solution F (n = 7) demonstrating that 10 min of ischemia homogeneously decreased conduction but that conduction slowing was greatest with albumin relative to mannitol (#P < 0.05 solution F relative to solution E). D: albumin triangulated action potential morphology more than mannitol at 10-min simulated ischemia, as revealed by the time interval between activation time and 30% repolarization (APD₃₀; #P < 0.05), without significantly changing late repolarization at APD at 90% repolarization (APD₉₀), and the osmolytes did not significantly affect action potential rise time (RT). A.U., arbitrary units; Soln, solution.

Fig. 6.

Arrhythmias. A: Kaplan-Meier curves demonstrating the onset of asystole during simulated ischemia in hearts perfused with the study solutions. Log-rank tests were performed to determine significance in Kaplan-Meier curves (n = 6 hearts/solution, P values noted relative to solution A). B: representative ECGs demonstrating normal-paced rhythm during solution A perfusion (top) and ventricular fibrillation (VF) during solution B perfusion (bottom). C: summary of the number of hearts that were in ventricular tachycardia/VF (solid color) after pacing at 10 and 30 min of ischemia. χ²-Analysis with Bonferroni correction was applied to determine statistical significance (n = 6, #P < 0.05 relative to solution A). Soln, solution.