Piezo1 Mediates Smooth Muscle Cell Proliferation In Idiopathic Pulmonary Arterial Hypertension

【Background】Pulmonary hypertension is a rare but fatal cardiopulmonary disorder characterized by elevated pulmonary vascular resistance（PVR）and pulmonary arterial pressure.Sustained pulmonary vasoconstriction and pulmonary arterial remodeling determine the pathological and pathophysiological changes of pulmonary vasculature during pulmonary hypertension.It has been widely demonstrated that the enhanced intracellular free calcium concentration([Ca²⁺]_i)caused by both intracellular calcium release from the internal calcium stores and extracellular calcium influx through the substantial types of plasma membrane localized calcium channel plays essential roles in driving the pathogenic progression of pulmonary hypertension.Emerging studies have reported a mechanosensitive Piezo Type Mechanosensitive Ion Channel Component 1（Piezo1）plays essential roles in regulating the vascular tone through mechanistic actions on intracellular calcium homeostasis.However,the specific roles of Piezo1 in pulmonary vessels remain incompletely understood.【Objectives】We aim to investigate whether and how Piezo1 mediates cell proliferation through regulating the intracellular calcium homeostasis in human pulmonary arterial smooth muscle cells（PASMCs）under normal and idiopathic pulmonary arterial hypertension【Methods】1.Cultured human PASMCs isolated from both Normal donors and idiopathic pulmonary arterial hypertension（IPAH）patients were used as cell models.2.Fura-2 based intracellular calcium imaging was performed to measure the intracellular free calcium concentration([Ca²⁺]_i).3.By using immunofluorescence staining strategies,we assessed the location of Piezo1 at the subcellular organelles.4.By using live imaging and isolated vessel tension measurement,we studied the effects of Piezo1 activation on the contraction of PASMCs.5.By using both 5-ethynyl-2-deoxyuridine incorporation or CCK8 assay,we studied the effects of Piezo1 activation on the proliferation of PASMCs.【Results】1.First,activation of Piezo1 induces dramatic increase of the[Ca²⁺]_i by inducing:1)the intracellular calcium release from intracellular calcium stores in organelles including ER/SR,Mitochondria,Nucleus that express Piezo1,defining as the activity of intracellular Piezo1（Intra-Piezo1）.The intracellular calcium store depletion and increased[Ca²⁺]_i independent of Ry R and IP3R then triggers SOCE.2)the extracellular calcium influx through the plasma membrane（PM）Piezo1 channel,defining as the activity of PM-Piezo,which is independent of PM calcium channels(e.g.store-operated Ca²⁺channels and L type voltage-dependent calcium channel)and Caveolin-1.2.Piezo1 activation-induced increase of[Ca²⁺]_i is also linked to the accelerated contraction and proliferation of PASMCs3.Yoda1 induces dose-dependent vasocontraction in endothelium-denuded rat intrapulmonary arteries.4.Significant upregulation and increased activity of Piezo1 were observed in IPAH-PASMCs versus Normal-PASMCs,contributing to the increased[Ca²⁺]_i and excessive proliferation of idiopathic PAH-PASMCs.【Conclusion】In summary,Piezo1 mediates the increase of[Ca²⁺]_i by triggering both intracellular calcium release and extracellular influx.The enhanced Piezo1 expression and activity accounts,at least partially,for the abnormally elevated[Ca²⁺]_i and proliferation in IPAH-PASMCs.