BS59 The k+ channel KCa3.1 as a novel target for aortic stenosis

• Published in: Heart (British Cardiac Society) Vol. 110; no. Suppl 3; pp. 286 - 287
• Main Authors: Whitfield, Molly, Aslam, Saadia, Biddle, Michael, Goncalves De Sousa, João, Taveira, Daniel, Duffy, Mark Stephen, Hall, Alfie J, Acharya, Metesh, Mariscalco, Giovanni, McCann, Gerry, Bradding, Peter, Roach, Katy M, Singh, Anvesha
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group Ltd and British Cardiovascular Society 01.06.2024; BMJ Publishing Group LTD
• Subjects: Aortic Stenosis; Basic science; Collagen; Extracellular matrix; Fibroblast; Fibroblasts; KCa3.1; Proteins; Smooth muscle
• ISSN: 1355-6037; 1468-201X
• DOI: 10.1136/heartjnl-2024-BCS.284

Introduction & AimAortic stenosis (AS) is characterised by the progressive accumulation of fibrotic extracellular matrix and calcific mineral within the aortic valve (AV). These structural changes result in reduced leaflet mobility and left ventricular outflow obstruction, which if left untreated is associated with high mortality. The only treatment option for AS is replacement of the AV, a costly and risky procedure. A non-surgical treatment option is urgently needed to prevent AS progression. Myofibroblasts are the key cells in AV fibrosis owing to their exaggerated extracellular matrix production and contractile activity within the valve, which also promotes the activation of osteogenic signalling. 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. There is a KCa3.1 channel blocking compound (senicapoc) that was safe and well-tolerated in clinical trials for sickle cell anaemia. We aimed to examine the expression and function of the KCa3.1 ion channel expression in human AS.MethodsAVs were collected from patients with severe tricuspid AS undergoing surgical AV replacement. AV fibroblasts were isolated from the AV tissue and cultured. Immunohistochemical staining, qRT-PCR and patch clamp electrophysiology were used to determine KCa3.1 channel expression. α-smooth muscle actin (αSMA) and collagen type 1 expression were used to assess the myofibroblast phenotype. The function of KCa3.1 was examined using TGFβ1 (10 ng/ml) stimulation and the selective KCa3.1 channel blocking compound senicapoc (100 nM).ResultsIHC/IF staining confirmed KCa3.1 protein expression in both AV fibroblasts and tissue (figure 1). Patch clamp electrophysiology revealed functional KCa3.1 ion channels that were blocked by senicapoc (n=5). RT-PCR confirmed basal expression of KCa3.1 mRNA (KCNN4) in AV fibroblasts that was increased by TGFβ1 stimulation. Furthermore, senicapoc reduced TGFβ1-induced αSMA and collagen type 1 mRNA expression in AV fibroblasts (n=6 and n=5, respectively). Cell migration determined by the wound healing assay was not affected by KCa3.1 channel blockade (n=4). Preliminary immunofluorescence revealed senicapoc also reduced TGFβ1-induced αSMA protein intensity and cell area in AV fibroblast donors (n=3).ConclusionsWe demonstrate for the first time that the KCa3.1 ion channel is expressed in cultured AS valve fibroblasts and valve leaflets, and may therefore contribute to the development of AS. Moreover, TGFβ1-induced upregulation of the channel suggests the biological effects of TGFβ1 may be linked with KCa3.1 channel activity. Importantly, senicapoc reduced protein and mRNA expression of the key fibrotic proteins collagen type 1 and αSMA within the AS valve fibroblasts. Blocking KCa3.1 may represent an unexploited therapeutic target in reducing pro-fibrotic myofibroblast activity in AS.Abstract BS59 Figure 1KCa3.1 is expressed in aortic stenosis valve tissue. Immunofluorescent staining of the K+ Channel KCa3.1 (red) and α-Smooth Muscle Actin (green) in sectioned aortic stenosis valve tissue. KCa3.1+ cells were found throughout the tissue and colocalised with a number of αSMA+ cells, likely myofibroblasts (yellow). DAPI was used for nuclei staining (blue) and isotype controls were negative. (x200)Conflict of InterestNone