Mannitol and hyponatremia regulate cardiac ventricular conduction in the context of sodium channel loss of function

Abstract

Scn5a heterozygous null (Scn5a^+/-) mice have historically been used to investigate arrhythmogenic mechanisms of diseases such as Brugada syndrome (BrS) and Lev's disease. Previously, we demonstrated that reducing ephaptic coupling (EpC) in ex vivo hearts exacerbates pharmacological voltage-gated sodium channel (Na_v)1.5 loss of function (LOF). Whether this effect is consistent in a genetic Na_v1.5 LOF model is yet to be determined. We hypothesized that loss of EpC would result in greater reduction in conduction velocity (CV) for the Scn5a^+/- mouse relative to wild type (WT). In vivo ECGs and ex vivo optical maps were recorded from Langendorff-perfused Scn5a^+/- and WT mouse hearts. EpC was reduced with perfusion of a hyponatremic solution, the clinically relevant osmotic agent mannitol, or a combination of the two. Neither in vivo QRS duration nor ex vivo CV during normonatremia was significantly different between the two genotypes. In agreement with our hypothesis, we found that hyponatremia severely slowed CV and disrupted conduction for 4/5 Scn5a^+/- mice, but 0/6 WT mice. In addition, treatment with mannitol slowed CV to a greater extent in Scn5a^+/- relative to WT hearts. Unexpectedly, treatment with mannitol during hyponatremia did not further slow CV in either genotype, but resolved the disrupted conduction observed in Scn5a^+/- hearts. Similar results in guinea pig hearts suggest the effects of mannitol and hyponatremia are not species specific. In conclusion, loss of EpC through either hyponatremia or mannitol alone results in slowed or disrupted conduction in a genetic model of Na_v1.5 LOF. However, the combination of these interventions attenuates conduction slowing.NEW & NOTEWORTHY Cardiac sodium channel loss of function (LOF) diseases such as Brugada syndrome (BrS) are often concealed. We optically mapped mouse hearts with reduced sodium channel expression (Scn5a^+/-) to evaluate whether reduced ephaptic coupling (EpC) can unmask conduction deficits. Data suggest that conduction deficits in the Scn5a^+/- mouse may be unmasked by treatment with hyponatremia and perinexal widening via mannitol. These data support further investigation of hyponatremia and mannitol as novel diagnostics for sodium channel loss of function diseases.

Keywords: basic science research; congenital heart disease; electrophysiology; translational studies.

Figure 1.

Characterizing the Scn5a^+/− mouse heart. A and B: relative to wild type (WT), the Scn5a^+/− mouse expresses ∼60% reduced mRNA and ∼52% reduced voltage-gated sodium channel (Na_v)1.5 protein. Na_v1.5 protein expression is normalized to GAPDH. n = 2 males, 1 female WT; n = 2 males, 1 female Scn5a^+/− hearts, significance determined by Student’s t test, *P < 0.05 compared with WT. C: no significant difference was measured in total connexin-43 (Cx43) expression (normalized to GAPDH) or Cx43 phosphorylation at Ser368 (normalized to total Cx43) between the genotypes. n = 2 males, 1 female WT; n = 2 males, 1 female Scn5a^+/− hearts. P = not significant (n.s.) via one-way ANOVA. D: perinexal width, highlighted in yellow, was not significantly different between WT and Scn5a^+/− hearts under baseline conditions. n = 2 males, 1 female WT; n = 2 males, 1 female Scn5a^+/− hearts, n = 15 images/heart. Significance determined by nested t test; *P < 0.05 compared with WT.

Figure 2.

In vivo ventricular conduction is not significantly different between Scn5a^+/− and wild-type (WT) mice. A: typical example of an ECG trace acquired with the ECGenie system for the WT (black) and Scn5a^+/− (blue) mouse. B: QRS duration is not significantly different between the two genotypes, though QRSJ is significantly longer in the Scn5a^+/− mouse (C). D: RR duration is not different between the WT and Scn5a^+/− mouse. n = 5 males, 1 female WT; 5 males 1 female Scn5a^+/−; significance was determined using Student’s t test, *P < 0.05 compared with WT.

Figure 3.

Conduction velocity (CV) of Scn5a^+/− hearts is not significantly slower than wild type (WT) at baseline, but more sensitive to CV slowing due to mannitol challenge. A: representative activation time maps for Scn5a^+/− and WT ventricles at normonatremia before and after mannitol challenge. Isochrone lines represent 2-ms intervals. Pulse symbol (П) indicates site of electrode placement for pacing. Arrows indicate vectors used for CV calculations. B: neither transverse CV (CVT) nor longitudinal CV (CVL) is significantly slower in the Scn5a^+/− heart relative to WT. n = 8 males, 4 females WT; 6 males, 4 females Scn5a^+/−, significance determined by Welch’s t test, *P < 0.05 compared with WT. C: perfusion with mannitol significantly slowed CVT in WT and CVT and CVL in Scn5a^+/− hearts. D: CVT slows to a greater extent in Scn5a^+/− relative to WT hearts after mannitol challenge. n = 4 males, 2 females WT; 4 males, 1 female Scn5a^+/−. Significance determined by Welch’s t test, *P < 0.05 compared with CV of WT; #P < 0.05 compared with ΔCV of WT.

Figure 4.

Mannitol attenuates hyponatremia-induced conduction velocity (CV) slowing and resolves conduction block in Scn5a^+/− hearts. A: representative activation time maps during baseline, hyponatremia, and combined hyponatremia and mannitol conditions. Isochrone lines represent 2 ms. Pulse symbol (П) indicates site of electrode placement for pacing. Arrows indicate vectors used for CV calculations. B: wild type (WT) does not exhibit severe conduction delay during hyponatremia, but 4/5 Scn5a^+/− exhibit severe conduction delay under these conditions. Significance determined by Fisher’s exact test; *P < 0.05 compared with WT. C: WT and Scn5a^+/− hearts have significantly slowed transverse CV (CVT) in response to hyponatremia, but no significant change in CVT after the addition of mannitol. n = 2 males, 4 females WT; 2 males, 3 females Scn5a^+/−, significance determined by repeated-measures one-way ANOVA with the Geisser–Greenhouse correction (for unequal variability of differences), followed by the Tukey’s multiple comparisons test; *P < 0.05 compared with 152 mM Na⁺ condition.

Figure 5.

Attenuation of conduction velocity (CV) slowing during combined hyponatremia and mannitol challenge is not species specific. A and B: significant reductions in transverse CV (CVT) and longitudinal CV (CVL) are observed after perfusion with 120 mM Na⁺ and flecainide in ex vivo guinea pig hearts. Addition of mannitol does not show additive CV slowing effect in the context of hyponatremia and sodium channel inactivation. n = 6 males, significance determined by one-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05 compared with 145 mM Na⁺ condition.