MicroRNA‐181b Regulates Ca2+ Influx by Targeting TRPC6 in PASMC from Patients with Idiopathic Pulmonary Arterial Hypertension

Abstract

IntroductionWe have previously showed that upregulated canonical transient receptor potential 6 (TRPC6) contributes to elevated cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH). An increase in [Ca2+]cyt is a major stimulus for pulmonary vasoconstriction and PASMC proliferation. Pulmonary vascular remodeling characterized by excessive PASMC proliferation, and sustained vasoconstriction are two important factors for elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in IPAH patients. However, the molecular mechanisms that regulate [Ca2+]cyt and PASMC proliferation still remain unknown. In this study, we investigate the role of microRNA (miRNA), small non‐coding RNA, which is able to regulate gene expression post‐transcriptionally by inhibiting translation and stability of mRNAs. We hypothesized that decreased miRNA‐181b (miR‐181b) resulted in the enhanced [Ca2+]cyt by upregulating TRPC6 in PASMC isolated from IPAH patients.MethodsPASMC from healthy subjects and patients with IPAH were used for this study. miRNA expression profile was explored using the Human Cancer Pathway Finder miRNA PCR Array. In silico analysis was performed by using standard online software (Targetscan.org) to predict direct targets of miR‐181b. miR‐181b level was quantified by real‐time RT‐PCR in normal and IPAH PASMC. Normal PASMC were transfected with miR‐181b inhibitor while IPAH‐PASMC were overexpressed with miR‐181b. mRNA and protein expression of TRPC6 channels were measured by RT‐PCR and Western blot, respectively. [Ca2+]cyt was measured using fura‐2 AM, a membrane‐permeable Ca2+‐sensitive fluorescent indicator.ResultsmiRNA PCR microarray showed that 5 out of 89 miRNAs were significantly downregulated in IPAH‐PASMC compared to normal PASMC. In silico analysis revealed that out of 5 candidates miR‐181b has the most potential to directly target TRPC6. Downregulated miR‐181b is correlated with increased TRPC6 expression and [Ca2+]cyt in PASMC from IPAH patients compared to PASMC isolated from healthy subjects. Inhibition of miR‐181b in normal PASMC significantly enhanced [Ca2+]cyt and upregulates TRPC6 expression while overexpression of miR‐181b in IPAH‐PASMC inhibits [Ca2+]cyt and decreases TRPC6 level.ConclusionIncreased TRPC6 and elevated Ca2+ influx mediated by downregulated miR‐181b could contributes to excessive PASMC and sustained vasoconstriction in patients with IPAH.Support or Funding InformationThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.