Abstract
Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however its role in the heart is poorly understood. Cardiac fibroblasts are responsible for preserving the structural integrity of the myocardium and play a key role in regulating its repair and remodeling following stress or injury. We investigated expression and function of Piezo1 in cultured human and mouse cardiac fibroblasts. RT-PCR studies confirmed expression of Piezo1 mRNA in cardiac fibroblasts at similar levels to endothelial cells. Fura-2 intracellular Ca2+ measurements validated Piezo1 as a functional ion channel that was activated by the Piezo1 agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium, ruthenium red) and the Ca2+ response was reduced proportionally by Piezo1 siRNA knockdown or in cells from Piezo1+/− mice. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation opposed cardiac fibroblast differentiation; data confirmed by functional collagen gel contraction assays. Piezo1 activation using Yoda1 or mechanical stretch also increased the expression of interleukin-6 (IL-6), a mechanosensitive pro-hypertrophic and pro-fibrotic cytokine, in a Piezo1-dependent manner. Multiplex kinase activity profiling combined with kinase inhibitor studies and phospho-specific western blotting, established that Piezo1 activation stimulated IL-6 secretion via a pathway involving p38 MAP kinase, downstream of Ca2+ entry. In summary, this study reveals that cardiac fibroblasts express functional Piezo1 channels coupled to reduced myofibroblast activation and increased secretion of paracrine signaling molecules that can modulate cardiac remodeling.
Abbreviations
- α-SMA,
- α-smooth muscle actin;
- CDK,
- cyclin-dependent kinase;
- DMSO,
- dimethylsulfoxide;
- ECM,
- extracellular matrix;
- ERK,
- extracellular signal-regulated kinases;
- Gapdh/GAPDH,
- glyceraldehyde-3-phosphate dehydrogenase;
- HEK,
- human embryonic kidney;
- Het,
- heterozygous;
- HUVECs,
- human umbilical vein endothelial cells;
- IL,
- interleukin;
- JNK,
- c-Jun N-terminal kinase;
- MACS,
- magnetic antibody cell separation;
- MAPK,
- mitogen-activated protein kinase;
- MMP,
- matrix metalloproteinase;
- SBS,
- standard bath solution;
- STK,
- Serine-Threonine Kinase;
- WT,
- wild-type.