The functional consequences of sodium channel NaV 1.8 in human left ventricular hypertrophy

Abstract

Aims: In hypertrophy and heart failure, the proarrhythmic persistent Na⁺ current (I_NaL ) is enhanced. We aimed to investigate the electrophysiological role of neuronal sodium channel Na_V 1.8 in human hypertrophied myocardium.

Methods and results: Myocardial tissue of 24 patients suffering from symptomatic severe aortic stenosis and concomitant significant afterload-induced hypertrophy with preserved ejection fraction was used and compared with 12 healthy controls. We performed quantitative real-time PCR and western blot and detected a significant up-regulation of Na_V 1.8 mRNA (2.34-fold) and protein expression (1.96-fold) in human hypertrophied myocardium compared with healthy hearts. Interestingly, Na_V 1.5 protein expression was significantly reduced in parallel (0.60-fold). Using whole-cell patch-clamp technique, we found that the prominent I_NaL was significantly reduced after addition of novel Na_V 1.8-specific blockers either A-803467 (30 nM) or PF-01247324 (1 μM) in human hypertrophic cardiomyocytes. This clearly demonstrates the relevant contribution of Na_V 1.8 to this proarrhythmic current. We observed a significant action potential duration shortening and performed confocal microscopy, demonstrating a 50% decrease in proarrhythmic diastolic sarcoplasmic reticulum (SR)-Ca²⁺ leak and SR-Ca²⁺ spark frequency after exposure to both Na_V 1.8 inhibitors.

Conclusions: We show for the first time that the neuronal sodium channel Na_V 1.8 is up-regulated on mRNA and protein level in the human hypertrophied myocardium. Furthermore, inhibition of Na_V 1.8 reduced augmented I_NaL , abbreviated the action potential duration, and decreased the SR-Ca²⁺ leak. The findings of our study suggest that Na_V 1.8 could be a promising antiarrhythmic therapeutic target and merits further investigation.

Keywords: Arrhythmias; Calcium; HFpEF; Late sodium current; Left ventricular hypertrophy; SR-Ca2+ leak; Sodium channels.

Figure 1

Regulation of Na_V1.8 and Na_V1.5 expression in hypertrophy. (A) Western blots were performed utilizing left ventricular human tissue homogenates. (B) Densitometry data of Na_V1.8 and Na_V1.5 show a significant up‐regulation of Na_V1.8 and down‐regulation of Na_V1.5. GAPDH was used as an internal loading control in all blots [NF: n = 12; and left ventricular hypertrophy (LVH): n = 12]. (C) Real‐time quantitative PCR showing the relative mRNA expression of Na_V1.8/GAPDH in left ventricle of human NF (n = 10) and LVH (n = 5). *P ≤ 0.05 and **P ≤ 0.01 vs. NF. Student's t‐test. Data shown as mean ± standard error of the mean and individual values.

Figure 2

(A) Original traces and (B) data showing individual and mean values ± standard error of the mean of I_NaL in human ventricular cardiomyocytes isolated from left ventricular hypertrophy patients (control: n = 8 cells; A‐803467: n = 7 cells; PF‐01247324: n = 7 cells). *P ≤ 0.05. One‐way analysis of variance and Bonferroni's post‐test.

Figure 3

(A) Original action potential recording (0.5 Hz) and (B) data showing individual and mean values ± standard error of the mean of APD₉₀ in left ventricular cardiomyocytes from patients with left ventricular hypertrophy. A‐803467 or PF‐01247324 paced at 0.5 and 1 Hz (n = 5 cells and n = 4 cells, respectively; *P ≤ 0.05 vs. control). Two‐way repeated measures analysis of variance and Bonferroni's post‐test. All the action potential duration (APD) measurements were performed pairwise by wash‐in.

Figure 4

(A) Individual and mean values ± standard error of the mean showing upstroke velocity of action potential in control and drug groups from left ventricular hypertrophy cardiomyocytes. (B) Data showing individual and mean values ± standard error of the mean of action potential amplitude (APA) measurements from isolated human left ventricular hypertrophy cardiomyocytes. (C) Graph shows the resting membrane potential (RMP) of cardiomyocytes measured under control condition and drug treatments (PF‐01247324: n = 5 cells; and A‐804367: n = 4 cells). All measurements were performed pairwise by wash‐in. Paired Student's t‐test was performed.

Figure 5

(A) Representative line scan images and (B) calcium spark frequency (CaSpF) in left ventricular cardiomyocytes from patients with left ventricular hypertrophy under control condition and Na_V1.8 inhibition. Pre‐incubation with either A‐803467 or PF‐01247324 resulted in a significant decrease of CaSpF in hypertrophy compared with control. (C) Mean values of spark duration and (D) spark amplitude of cardiomyocytes. (E) Calculated full SR‐Ca²⁺ leak in left ventricular cardiomyocytes from left ventricular hypertrophy patients (control: n = 145; A‐803467: n = 124 cells; PF‐01247324: n = 91 cells). Data shown as mean ± standard error of the mean. One‐way analysis of variance and Bonferroni's post‐test. *P ≤ 0.05, **P ≤ 0.01.