Abstract
Cardiac fibroblasts play an important role in cardiac matrix turnover and are involved in cardiac fibrosis development. Ca2+ is a driving belt in this phenomenon. This study evaluates the functional expression and contribution of the Ca2+-activated channel TRPM4 in atrial fibroblast phenotype. Molecular and electrophysiological investigations were conducted in human atrial fibroblasts in primary culture and in atrial fibroblasts obtained from wild-type and transgenic mice with disrupted Trpm4 gene (Trpm4−/−). A typical TRPM4 current was recorded on human cells (equal selectivity for Na+ and K+, activation by internal Ca2+, voltage sensitivity, conductance of 23.2 pS, inhibition by 9-phenanthrol (IC50 = 6.1 × 10−6 mol L−1)). Its detection rate was 13% on patches at days 2–4 in culture but raised to 100% on patches at day 28. By the same time, a cell growth was observed. This growth was smaller when cells were maintained in the presence of 9-phenanthrol. Similar cell growth was measured on wild-type mice atrial fibroblasts during culture. However, this growth was minimized on Trpm4−/− mice fibroblasts compared to control animals. In addition, the expression of alpha smooth muscle actin increased during culture of atrial fibroblasts from wild-type mice. This was not observed in Trpm4−/− mice fibroblasts. It is concluded that TRPM4 participates in fibroblast growth and could thus be involved in cardiac fibrosis.
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The dataset generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgments
The authors thank Marie Demion (INSERM U1046, Montpellier, France) for providing the mouse strain used in this study and Claudine Combes for technical assistance in human fibroblast isolation experiments. They also thank Vladimir Saplacan, Dimitrios Buklas, Julien Desgue, and Gerard Babatasi, cardiac surgeons, CHU Caen.
Funding
This work was done with a financial support (RIN ENTRAC) of « Région Normandie », France. Christophe Simard received a fellowship from “Région Normandie,” France. This work was conducted as part of the FHU REMOD-VHF project.
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C. Simard, C. Magaud, and R. Adjlane were in charge of cell isolation and culture. C. Simard and R. Guinamard performed the single-channel patch-clamp recordings. C. Simard, C. Magaud, and Q. Dupas were in charge of the biochemical approaches. L. Sallé, A. Manrique, and P. Bois participated in data analysis and editing of the paper. C. Simard, C Magaud, JF. Faivre, and R. Guinamard were in charge of the design of the experiments and writing of the paper.
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Studies using human atrial appendages were made after the approval of local medical ethics committee (Comité de Protection des Personnes Nord Ouest III, Caen, France, ref.: DC-2013-1967) and patient written informed consent.
Experiments using mouse atrial cells were carried out in strict accordance with the European Commission Directive 2010/63/EU for animal care. The study was also conducted with authorization for animal experimentation #14-98 from the local DDPP (Direction Départementale de la Protection des Populations).
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Supplementary Figure 1.
Expression and localization of smooth muscle actin (α-SMA), CD31 and vimentin in primary culture of human cardiac fibroblasts. A. Expression of CD31 in human umbilical vein endothelial cell line (HUVEC) by immunolabeling. Left panel: omission of the primary antibody; Right panel: detection of CD31 in HUVEC cells. Cells were incubated for 2 h with the secondary antibody (1:250, chicken anti-mouse alexa 488, Thermo Fisher Scientific). B. Immunostaining experiments performed on primary culture of human atrial fibroblasts after 12 days in culture to characterize expression of α-SMA, CD31 (green channels) and vimentin (red channel). Upper panel: example of double immunofluorescence staining of α-SMA and vimentin in primary culture of human cardiac fibroblast. Lower panel: Example of double immunofluorescence staining of CD31 and vimentin in primary culture of human cardiac fibroblast. Note the absence of expression of the endothelial cell marker CD31. DAPI staining (blue) was used to mark the nuclei. Images were obtained using confocal microscopy; Bar = 100 μm. (PNG 193201 kb)
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Simard, C., Magaud, C., Adjlane, R. et al. TRPM4 non-selective cation channel in human atrial fibroblast growth. Pflugers Arch - Eur J Physiol 472, 1719–1732 (2020). https://doi.org/10.1007/s00424-020-02476-0
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DOI: https://doi.org/10.1007/s00424-020-02476-0