Wnt signalling suppresses voltage-dependent Na+ channel expression in postnatal rat cardiomyocytes

摘要

Wnt signalling plays crucial roles in heart development, but is normally suppressed postnatally. In arrhythmogenic conditions, such as cardiac hypertrophy and heart failure, Wnt signalling is reactivated. To explore the potential role of Wnt signalling in arrhythmogenic electrical remodelling, we examined voltage-dependent ion channels in cardiomyocytes. Treatment of neonatal rat ventricular myocytes with either recombinant Wnt3a protein or CHIR-99021 (CHIR, a glycogen synthase kinase-3β inhibitor) caused a dose-dependent increase in Wnt target gene expression (Axin2 and Lef1), indicating activation of the Wnt/β-catenin pathway. Cardiac Na⁺ current (INa) density was reduced by Wnt3a (−20 ± 4 vs. control −59 ± 7 pA pF⁻¹, at −30 mV) or CHIR (−22 ± 5 pA pF⁻¹), without changes in steady-state activation, inactivation or repriming kinetics. Wnt3a and CHIR also produced dose-dependent reductions in the mRNA level of Scn5a (the cardiac Na⁺ channel α subunit gene), as well as a 56% reduction (by Wnt3a) in the Nav1.5 protein level. Consistent with INa reduction, action potentials in Wnt3a-treated neonatal rat ventricular myocytes had a lower upstroke amplitude (91 ± 3 vs. control 137 ± 2 mV) and decreased maximum upstroke velocity (70 ± 10 vs. control 163 ± 15 V s⁻¹). In contrast, inward rectifier K⁺ current and L-type Ca²⁺ channels were not affected by Wnt3a treatment. Taken together, our data indicate that the Wnt/β-catenin pathway suppresses INa in postnatal cardiomyocytes and may contribute to ion channel remodelling in heart disease.Key points class="unordered" style="list-style-type:disc"> Wnt signalling is activated in arrhythmogenic heart diseases, but its role in the regulation of cardiac ion channel expression is unknown. Exposure of neonatal rat ventricular myocytes to Wnt3a, an activator of canonical Wnt signalling, decreases Scn5a mRNA, Nav1.5 protein and Na⁺ current density. Wnt3a does not affect the inward rectifier K⁺ current or L-type Ca²⁺ channels. The Wnt pathway is a negative regulator of cardiac Na⁺ channel expression and may play a role in altered ion channel expression in heart disease. class="head no_bottom_margin" id="__sec2title">IntroductionIon channels are critical for the rhythmic contraction of the heart (Marban, ; Schram et al. ). Alterations in ion channel expression and function are common in both ischaemic and non-ischaemic heart disease, contributing to impaired contractility and electrical instability (Tomaselli & Marban, ). In particular, cardiac Na⁺ current (INa) density is reduced in human heart failure (Valdivia et al. ; Shang et al. ), a canine model of heart failure (Maltsev et al. ) and a mouse model of dilated cardiomyopathy (Hesse et al. ). Dysfunctional Na⁺ channels due to mutations of Scn5a (the gene for cardiac INa α subunit) in patients are associated with increased susceptibility to heart failure and atrial fibrillation (Olson et al. ; Ge et al. ). Reduction of INa compromises contractility (Koesters et al. ), causes slow conduction (Sato et al. ) and induces ventricular arrhythmia (Papadatos et al. ). Thus, INa represents a therapeutic target, but the mechanisms underlying INa reduction in heart disease remain unknown.The Wnt signalling pathway is evolutionarily conserved in the animal kingdom (Cadigan & Nusse, ). Wnt ligands are secreted soluble proteins that bind to cell membrane receptors and activate intracellular cascades. In the canonical pathway, Wnt receptor activation inhibits GSK-3β, a key mediator of β-catenin degradation; β-catenin then accumulates in the cytosol and translocates into the nucleus where it regulates the transcription of a variety of genes, including Axin2 and Lef1 (). The canonical Wnt/β-catenin pathway is active during embryonic development and critically regulates cardiogenesis (Gessert & Kuhl, ). After birth, the pathway is suppressed in normal hearts, but is reactivated in various disease conditions (Dawson et al. ), including myocardial infarction (Oerlemans et al. href="#b18" rid="b18" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434897320">2010), cardiac hypertrophy and heart failure (Haq et al. href="#b6" rid="b6" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434897312">2003; Malekar et al. href="#b15" rid="b15" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_434897327">2010).href="/pmc/articles/PMC4358676/figure/fig01/" target="figure" rid-figpopup="fig01" rid-ob="ob-fig01">target="object" href="/pmc/articles/PMC4358676/figure/fig01/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC4358676/figure/fig01/" target="figure" rid-figpopup="fig01" rid-ob="ob-fig01">Figure 1Wnt3a and CHIR activate β-catenin signalling in NRVMsA, simplified diagram of the canonical Wnt/β-catenin signalling pathway. Wnt ligands bind to and activate the receptor complex (Fzd and Lrp5/6) leading to inhibition of GSK-3β, a key mediator of β-cat degradation; β-cat then accumulates in the cytosol and translocates into the nucleus where it regulates the transcription of a variety of genes, including Axin2 and Lef1. Wnt3a and CHIR used to activate the pathway were shown in red. B, expression of Wnt receptor genes in NRVMs, n = 3. C, expression of Wnt ligand genes in NRVMs, n = 3. D, expression of Wnt inhibitor genes in NRVMs, n = 3. E and F, mRNA of Axin2 and Lef1 in NRVMs after treatment with Wnt3a (0.1, 0.3 or 1.0 μg ml⁻¹) or CHIR (3 μm) for 48 h. *P < 0.05 vs. 0 μg ml⁻¹ Wnt3a group, n = 3. β-cat, β-catenin; CHIR, CHIR-99021; NRVMs, neonatal rat ventricular myocytes.