The Regulation Mechanism Of TMEM16A Involved In Pulmonary Arterial Hypertension Induced By High Blood Flow

Objective:To identify the expression and characteristics of TMEM16A in pulmonary smooth muscle cells of rats, and explore the electrophysiological characteristics of CaCC, which is probably mediated by TMEM16A.Methods:SD rats were randomly divided into normal, sham and shunt groups. Abdominal aorta-inferior vena cava fistulization shunting operations were carried out to establish animal model of pulmonary arterial hypertension induced by high pulmonary blood flow. Pulmonary artery pressures were measured 11 weeks after the operations. HE staining and immunohistochemistry were used to observe the pathological change and the distribution of TMEM16A; qRT-PCR and western blot were used to measure the expression of TMEM16A mRNA and protein of pulmonary smooth muscle cells in different groups respectively. Whole cell patch clamp technology was used to record the electrophysiological characteristics of CaCC.Results:Compared with normal and sham groups, the pulmonary artery pressures in shunt group significantly increased after 11 weeks’shunting (P＜0.01), indicating the animal model of pulmonary arterial hypertension induced by high pulmonary blood flow was successfully established. HE staining showed the pulmonary arterioles in shunt group presented with thicken vessel walls and stenosis lumen. Immunohistochemistry showed TMEM16A mainly located in the pulmonary artery cells’membrane. qRT-PCR showed TMEM16A mRNA expression in shunt group significantly increased compared with normal and sham groups (P<0.01), while there was no statistical difference between normal and sham groups (P>0.05). Western blot showed TMEM16A protein expression in shunt group significantly increased compared with normal and sham groups (P<0.01), while there was no statistical difference between normal and sham groups (P>0.05). The stable CaCC currents of pulmonary smooth muscle cells were recorded, presenting outward rectification and calcium sensitivity. The current densities of pulmonary smooth muscle cell in shunt group were significantly increased compared with normal and sham groups, and Ⅰ-Ⅴ curves moved upwards (P<0.01), while there was no statistical difference between normal and sham groups (P>0.05).Conclusions:(1) TMEM16A was highly expressed in pulmonary smooth muscle cell of pulmonary arterial hypertension rats induced by high pulmonary blood flow, may contributing to the pathogenesis of pulmonary arterial hypertension. (2) The current and current density increased during the process of pulmonary arterial hypertension induced by high pulmonary blood flow, probably contribute to the mechanisms of PAH.Objective:In order to explore whether TMEM16A mediates CaCC in pulmonary smooth muscle cell and the electrophysiological change, lentiviral vector, RNA interfere technology were used to knockdown the expression of TMEM16A, laying the foundation to clarify the mechanism of TMEM16A involved in pulmonary arterial hypertension induced by high pulmonary blood flow.Methods:The same animal model of pulmonary arterial hypertension induced by high pulmonary blood flow was established as part 1, as well as group dividing, pulmonary artery pressure measuring and pulmonary smooth muscle cell culture. RNAi technology was used to prepare the lentivirus and package, followed with cell transfection specific to target gene TMEM16A. qRT-PCR and western blot were used to evaluate the interfering efficiency. After screened out the most efficient vector, qRT-PCR and western blot were used to measure TMEM16A mRNA and protein expression pre and after transfection. Whole cell patch clamp was used to test the electrophysiological change pre and after cell transfection.Results:Lentivirus with green fluorescence protein was successfully transfected into pulmonary smooth muscle cells after lentivirus preparing, package and transfection. qRT-PCR and western blot were used to screen out the most efficient interfering vector (83%). TMEM16A mRNA expressions after transfection decreased significantly in normal, sham and shunt groups (P<0.01). TMEM16A protein expressions also decreased significantly after transfection in all groups (P<0.05). CaCC currents in all groups were decreased, combined with significantly decreased CaCC current densities (P＜0.01). CaCC current densities in shunt group after transfection increased significantly compared with normal (P<0.01) and sham groups (P<0.05), while there was no statistical difference between normal and sham groups after transfection (P>0.05).Conclusions:(1) The lentivirus vector and the most efficient RNA interfering vector were successfully established. (2) The transfection decreased TMEM16A mRNA and protein expression efficiently, as well as cell membrane currents and current densities, which was identified by qRT-PCR and western blot. (3) TMEM16A mediated CaCC was involved in the pathogenesis of pulmonary arterial hypertension induced by high pulmonary blood flow. transfection decreased significantly in normal, sham and shunt groups (P<0.01). TMEM16A protein expressions also decreased significantly after transfection in all groups (P<0.05). CaCC currents in all groups were decreased, combined with significantly decreased CaCC current densities (P＜0.01). CaCC current densities in shunt group after transfection increased significantly compared with normal (P<0.01) and sham groups (P<0.05), while there was no statistical difference between normal and sham groups after transfection (P>0.05).Objective:We have down-regulated the expression of TMEM 16A in part 2, providing an important function-reverse researching method to study the role of this target gene in pathogenesis of pulmonary arterial hypertension. So we continue to adopt this technology, exploring the role of TMEM16A to proliferation of pulmonary smooth muscle cells and the potential signal pathway.Methods:The same animal model of pulmonary arterial hypertension induced by high pulmonary blood flow was established as part 1, as well as group dividing, pulmonary artery pressure measuring and pulmonary smooth muscle cell culture. The RNA interfering vector established in part 2 was used to study the function of pulmonary smooth muscle cell. MTT assay was used to measure the proliferation of pulmonary smooth muscle cells, and western blot was used to measure the expression of PCNA、P-ERK and ERK proteins.Results:Pulmonary smooth muscle cells began to enter into logarithmic growth phase 48 hours after cell inoculating. The proliferation rate in shunt group significantly increased compared with normal and sham groups (P<0.01), while there was no statistical difference between normal and sham groups (P>0.05). After cell transfection specific to TMEM16A, the proliferation rates in all groups were significantly decreased (P＜0.01). The expression of PCNA in shunt group pre transfection significantly increased compared with normal and sham groups (P<0.01), while there was no statistical difference between normal and sham groups (P>0.05). After cell transfection specific to TMEM16A, the expressions of PCNA in all groups significantly decreased (P<0.01). The expression of P-ERK/ERK in shunt group pre transfection significantly increased compared with normal and sham groups (P<0.05), while there was no statistical difference between normal and sham groups (P>0.05). After cell transfection specific to TMEM16A, the expressions of P-ERK/ERK in all groups significantly decreased (P<0.05).Conclusions:(1) TMEM16A contributes to proliferation of pulmonary smooth muscle cells in rats, involving in pathogenesis of pulmonary arterial hypertension induced by high pulmonary blood flow. (2) TMEM16A probably contributes to proliferation of pulmonary smooth muscle cells in rats through MAPK/ERK signal pathway.