Atrial fibrillation (AF) is associated with atrial dilatation, structural and mechanical remodelling and inflammation. The interplay between mechanical stimuli and inflammation is not fully understood. The stretch-activated ion channel Piezo1 is expressed at higher levels in atrial fibroblasts (FB) isolated from AF patients compared to those in sinus rhythm patients.1 Piezo1 influences the expression and secretion of interleukin-6 (IL6)2, suggesting it may mediate mechanically-induced inflammation in AF. This study aimed to investigate functional effects of Piezo1–dependent cytokines from FB on immune cell recruitment and endothelial cell properties.
We combined transient transfection of immortalised human atrial FB (HAF) to overexpress or downregulate Piezo1 expression with bulk RNA sequencing, qPCR and ELISA to examine the influence of Piezo1 expression levels on cytokine expression by HAF. Co-culture assays were used to assess the interplay between FB, endothelial cells and THP-1 monocytes. The effect of selected cytokines on endothelial cell mechanical properties was assessed by nanoindentation.
RNA sequencing revealed reduced expression of 12 cytokines in HAF with Piezo1 knock-down, compared to control HAF. Of those CC-chemokine ligand 2 (CCL2) is of particular relevance for immune cell recruitment from the circulation. With ELISA, we showed that Piezo1 overexpression leads to elevated secretion of CCL2. To assess the functional relevance of this finding, we used a transwell migration assay with THP1 cells in the upper and HAF cells in the lower compartment. Piezo1 overexpression in HAF led to a 34% larger number of trans-migrated THP1 cells within 4 days of co-culturing compared to no overexpression (N=3, p<0.0001), an effect not detected after CCL2 neutralisation using an antibody (N=3, p<0.0001).
We then assessed potential effects of the Piezo1-dependent HAF secretome on endothelial cells. To do so, we exposed human umbilical vein endothelial cells (HUVEC) for 4 h to culture media primed by control HAF or HAF with Piezo1 overexpression. After media removal, we added untreated THP1 monocytes and, after 2 h, removed non-adherent cells. Pre-exposure of HUVEC to medium of Piezo1 overexpressing HAF lead to a 50% higher level in the number of HUVEC-adherent THP1 cells, compared to exposure to control HAF medium (N=3, p=0.0086).
Extravasation into tissues requires immune cell migration past endothelial monolayers, which may be affected by mechanical changes in endothelial cell properties. We therefore investigated mechanical properties of HUVEC after 48 h treatment with IL6 or CCL2 using nanoindentation. HUVEC stiffness was 16.5% lower after IL6-exposure (N=4, p=0.0067), which could be indicative of impaired barrier function of endothelial cells.3 In contrast, CCL2 treatment did not lead to a significant change in HUVEC cell stiffness (N=1, p=0.0265, pilot observation).
Our data demonstrate that Piezo1 overexpression in HAF increases the secretion of inflammatory cytokines including IL6 and CCL2. CCL2 enhances monocyte chemotaxis and adhesion to endothelial cells, and IL6 reduces endothelial cell stiffness. In view of AF-related upregulation of Piezo1 in FB, these mechanisms may contribute to immune cell recruitment and atrial inflammation.
1Jakob D et al. JMCC 2021/158: 49-62.
2Emig R et al. Cells 2021/10: 663.
3Kefala R et al. Sci Rep 2025/15:8064.
