Abstract
Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/− cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.
Highlights
Piezo1 is a mechanosensitive cation channel with widespread physiological importance; its role in the heart is poorly understood
Having demonstrated that cardiac fibroblasts express Piezo1 mRNA, we investigated whether the Piezo1 protein was able to form a functional ion channel
Consistent with the Yoda1-induced increase in intracellular Ca2ϩ being due to influx of extracellular Ca2ϩ through an ion channel, the Ca2ϩ signal was reduced by more than 90% when extracellular Ca2ϩ was absent in human and mouse cardiac fibroblast cultures (Fig. 1, E and F)
Summary
Piezo expression and activity in cardiac fibroblasts mRNA encoding Piezo was detected in cultured cardiac fibroblasts from both mouse and human hearts (Fig. 1, A and B). Concentration–response data for Yoda in murine cardiac fibroblasts revealed a marked effect at 0.3 M, and the maximal response was generated at 10 M; the EC50 of Yoda was estimated to be 0.72 M (Fig. 1G) This was almost identical to the EC50 observed in human cardiac fibroblasts in similar experiments (Fig. 1H) and comparable with that for mouse Piezo heterologously expressed in HEK T-RExTM-293 cells, where the EC50 of Yoda was 0.33 M (Fig. 1I). Piezo1-specific siRNA, which decreased Piezo mRNA expression by 80% in murine cardiac fibroblasts (Fig. 2E), reduced Yoda1-evoked Ca2ϩ entry by a similar level (Fig. 2F), whereas control siRNA was without effect. The Yoda response depended on Piezo and was proportional to its expression level Together, these data establish that Yoda1-induced Ca2ϩ entry in cardiac fibroblasts depends on Piezo expression and that the channel has the expected pharmacological properties
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