TRPV4 Is Involved In Irisin-induced Endothelium-dependent Vasodilation

BackgroundIrisin,an exercise-induced myokine,induces conversion of white into brown adipocytes,promotes mitochondrial energy expenditure and biogenesis.Studies show that Irisin has protective effects on endothelial function in animals,including human.Defects in Irisin signaling pathways can cause endothelial dysfunction in obese patients and diabetics.However,the mechanisms underlying the effects of Irisin on endothelial function have not been elucidated.Transient receptor potential vanilloid subtype 4（TRPV4）channel is one of the most important Ca²⁺permeable cation channels in vascular endothelial cells.This study sought to explore the relationship between Irisin-induced vasodilation and TRPV4 channel-mediated extracellular calcium influx.Objective1.To explore the role of TRPV4 channel in Irisin-mediated calcium influx2.To investigate whether TRPV4 channel is involved in the regulation of Irisin-induced vasodilation of mesenteric artery in SD ratsMethod1.Concentration Measurement of Intracellular CalciumIrisin was used to treat primary cultured rat mesenteric arterial endothelial cells（MAECs）to induce calcium influx,intracellular Ca²⁺concentration was detected by calcium imaging fluorescence microscopy.2.Perfusion SurgerySD rats were euthanized with CO₂ and fixed to the surgical plane.A tissue clipper was used to cut the ribs on both sides of the chest wall.After the diaphragm was cut,the entire chest wall was opened and fixed with a hemostatic forcep.At the same time,the heart was fully exposed to the visual field.The liver was carefully separated from the diaphragm.At the apex of the left ventricle,the infusion needle was carefully inserted while the heart was clamped with hemostatic forceps to hold the infusion needle.An iris scissor was used to incise an incision in the animal’s right atrium.200 mL of PBS was slowly perfused and clearance of blood from the liver was a good indicator of perfusion.3.Culture of rat mesenteric artery endothelial cellsAfter the Sprague-Dawley Rats were perfused,and the mesenteric artery was isolated in sterile environment,the mesenteric artery was dissected and then digested with 0.02%collagenase type I in PBS for 45 minutes at 37°C constant temperature water bath.Digested mesenteric artery were centrifuged at 1600 rpm for 5 minutes,the supernatant was discarded,and the cells which deposited at the bottom of the centrifuge tube were resuspended in DMEM contain 10%FBS,100 U/mL penicillin,and 100 ug/mL streptomycin.After incubated at 37°C、5%CO₂ incubator for 1 h,the medium was replaced with fresh DMEM to remove non-adherent cells.The remaining adherent endothelial cells were cultured at 37°C、5%CO₂ incubator for 3–5 days before experiments.4.Mesenteric artery tension measurementSegments of rats mesenteric artery were dissected in a Petri dish filled with ice-cold Krebs solution oxygenated with 95%O₂ and 5%CO₂.The isolated mesenteric arterial segments were transferred to a bath which contained fresh Kirschner’s solution and mixed oxygen.One end of the segment was fastened to a hook on the bottom of the tension transducer by using surgical silk sutures.The other end was equidistant connected to the tension sensor with the signal amplification and acquisition system.Isometric vascular tone was recorded and analyzed by the DMT 620M in vitro microvascular tone measurement system.Result1.Increase of intracellular calcium concentration in mesenteric arterial vascular endothelial cells of primary cultured SD rats were induced by Irisin-induced calcium influx.2.Irisin could induce vasodilation of mesenteric arteries in SD rats,and this relaxation was endothelium-dependent.3.Irisin had no effect on endothelium-independent vasodilation.4.Pretreatment with TRPV4 channel inhibitor fully abrogated Irisin-induced vasodilation.Conclusion1.Irisin induced extracellular Ca²⁺influx into the endothelial cells via TRPV4 channel.2.Irisin induced endothelium-dependent vasodilation through the TRPV4 channel.