The K+ channel KCa3.1 promotes contraction in aortic stenosis valve myofibroblast cells

Introduction Aortic stenosis (AS) is characterised by the progressive accumulation of fibrotic extracellular matrix (ECM) and calcific mineral within the aortic valve (AV). These structural changes result in reduced leaflet mobility and left ventricular outflow obstruction. Currently, the only treatment option for AS is replacement of the AV. A non-invasive treatment option is urgently needed to slow down AS progression. Myofibroblasts are the key cells in AV fibrosis owing to their exaggerated ECM production and contractile activity within the valve. KCa3.1 channels are expressed by myofibroblasts and promote pro-fibrotic activity in rodent hearts and several human organs. However, the role of KCa3.1 in AS has not been explored. Methods AVs were collected from patients with severe tricuspid AS undergoing surgical AV replacement. AV myofibroblasts were isolated from the AV tissue and cultured. To evaluate the effect of KCa3.1 channel inhibition on myofibroblast contraction, immunofluorescent staining, qRT-PCR, α-Smooth muscle actin (αSMA) stress fibre formation and a collagen gel contraction assay were employed. The role of KCa3.1 was investigated using stimulation with transforming growth factor β1 (TGFβ1, 10 ng/ml) and treatment with senicapoc (100 nM), a selective KCa3.1 channel blocker that has demonstrated safety and tolerability in two clinical trials. Results Immunofluorescent double staining for αSMA and KCa3.1 revealed colocalization in AS valve cells with myofibroblast morphology, confirming KCa3.1 expression in AS valve myofibroblasts (Figure 1a). To determine the effect of senicapoc on αSMA (ACTA2) expression, myofibroblasts were treated with TGFβ1 +/- senicapoc for 72h (Figure 1b). TGFβ1 significantly increased ACTA2 expression (P=.0078), whereas senicapoc (10 nM) significantly reduced TGFβ1-induced ACTA2 expression (P=.0234, n=7). Quantitative analysis of stress fibres demonstrated that TGFβ1 significantly increased αSMA stress fibres which was significantly reduced in AS valve myofibroblasts treated with the KCa3.1 blocker senicapoc after 72 hours (P=0.0084, n=6) (Figure 1c). AV myofibroblast contraction was significantly increased by TGFβ1 stimulation compared to vehicle control (P<0.0001). Importantly, senicapoc significantly reduced TGFβ1-induced contraction in the AV myofibroblast cells over a 22-hour period (P=0.02, n=8) (Figure 1d). A representative image of the collagen gels after 22 hours of treatment with TGFβ1, with or without senicapoc, is shown (Figure 1e). Conclusions Overall, our findings highlight a critical role for KCa3.1 in the pro-fibrotic signalling of AS valve myofibroblasts. Inhibition of KCa3.1 with senicapoc decreases myofibroblast activation via reducing αSMA mRNA and protein expression, and cell contraction. This finding is particularly relevant, as limiting myofibroblast activation has been shown to mitigate fibrosis. KCa3.1 blockade could be a promising therapeutic target to mitigate fibrosis in AS.