The Effect Of Novel Ion Channel TRPM7 On The Pathogenesis Of Cardiac Fibrosis In Mouse

It is known that all ion channels on cells inducing permeable ions across the cellular membrane are found to be encoded by human genes. Different from the traditional ways of ion channel activation including voltage-gating, receptor operating and mechanosensitivity, the exact protein form, mechanism and function of TRP (transient receptor potential) ion channels, being ubiquitously distributed in mammalia and cloned successfully recently, are still unclear. A few of studies showed that activation of TRP channel is regulated by several of factors as change in osmolarity, stretch force, temperature, pH, intracellular signal molecules, exogenous chemicals and endogenous ligands. TRP proteins show broad tissue distribution like heart, kidney, lung, liver, gut, prostate, nervous system vascular bed and so on. One member of TRP superfamily:melastatin-related transient receptor potential (TRPM) subfamily, some of which is identified to expresse in cardiac tissue. TRPM7 are known with an unique intrinsic kinase domain and ion channe pore region. However, till now the concrete pathophysiological functions and its activation or gating mechanisms of TRPM7 are still elusive.TRPM7 plays important roles in cellular Mg2+ and Ca2+ homeostasis, vessel regulation, cell proliferation and viability and some diseases caused by abnormal calcium and magnesium absorption. A few late reports suggested the exist and the probable electrophysiological features of and TRPM7 in cardiac myocyte recently However,wheather and how the TRPM7 channel and its mediated current be enhanced by specific physiological or pathophysiological stimulus, which is an promising direction in the future, need be explored by futhur studies.With studies going on, it is increasingly recogonized that cardiac fibroblasts (CFs) is not only the skeleton of cardiac tissue, but also the important functional cells which plays pathophysilogical effect on excreting cytokine,transforming and synthetizing collagen regulated for frosis.. Recent studies showed extracellular matrix (ECM), composed mainly by products of CFs, anticipate in alteration of cardiac fibrosis remodeling under pathophysiological conditions, it is realized that development of fibrosis is mainly manifested as cardiac hypertrophy, postinfarction status, heart failure, arrhythmia and sudden cardiac death. Dispointedly, till now, electrophysiological researches have not found any voltage dependent ions channel in CFs. Current up-to-date evidence proclaim mechanic induced potential in mouse cardiac fibroblasts (CFs) tightly correlates with Ca2+ signal. However, we are still confused by which channel inducing Ca2+ influx to trigger the downflow signaling for relative pathophysiological effects.The aims of this study is to explore the specific activation mechanism of TRPM7 under pathophysiological conditions, to investigate the potential molecular pathophysiological mechanism of TRPM7 in cardiac fibrosis in CFs.we conducted this study to make clear whether H+ could effect TRPM7 inward current amplitude and attempt to identify the amino acid residue biding site in TRPM7 pore for cation and if H+ potentiate its inward current in order to comform whether TRPM7 is the molecular basis of TRPM7L current in CFs and responsible for Ca2+ influx;Pro-fibrosis pathological stimulus TGFβis capable of produce pathophysiological effects on collagen metabolism by potentiating TRPM7. The intact experiment is composed of four parts as followings:Part I The study of potentiation of TRPM7 inward current by acidosisObjective To investigate whether acidic pH condition could affect electrop-Hysiological characterization of TRPM7 channel and its potential mechanism.Methods (1) TRPM7 transfected HEK-293 cell and RBL-2H3 cell were cultured; (2) Survey the inward and outward current amplitude in solutions contain different ion ingredient, pH and ion concentration by whole cell patch clamp technique;Results (1) Protons has a significant concentration-dependent Potential effect on TRPM7 inward currents; (2) Protons enhanced TRPM7 inward currents by competing with Ca2+ and Mg2+ for binding sites; (3) Acidic pH conditions decreased the affinity of TRPM7 for Ca2+ and Mg2+ thereby allowing monovalent cations to pass through TRPM7; (4) MIC/MagNuM current amplitude in rat basopHilic leukemia (RBL) cells was significantly potentiated by acidic pH condition.Conclusions (1) Protons enhance TRPM7 inward currents by competing with external Ca2+ and Mg2+ for binding sites in the TRPM7 pore;(2) MIC/MagNuM current amplitude in RBL cells was significantly potentiated by acidic pH condition suggesting that MIC/MagNuM is encoded by TRPM7. Part II Study on expression of TRPM7 in the mouse cardiac fibroblasts(CFs)Objectives To investigate whether TRPM7 is the molecular mechanism of TRPM7L current and TRPM7L is responsible for Ca2+ influx under pathophysiological conditions.Methods (1) Establishment of model of mice cardiac infarction and isolation of CFs; (2) CFs culture and TRPM7 SiRNA infection technique; (3) Confirmation of expression of TRPM7 on CFs by mRNA RT-PCR, polyclonal antibody immunocytochemical approach and conductance analysis of single channel.Results (1) There is a higher level expression of TRPM7 on CFs; (2) All the electrophysiological characterizations of TRPM7 is similar to that of TRPM7 under experimental condition mentioned above; (3) acidic pH condition and TGFβcould enhance the Ca2+influx of CFs as well as infarction; (4) After knock-out TRPM7 gene by SiRNA silencing technique, mRNA level and amplitude of TRPM7L current of TRPM7L has been decresed significantly.Conclusions (1) TRPM7 is the molecular basis of the native TRPM7L in CFs; (2) TRPM7L is responsible for Ca2+ influx under normal physiological and fibrosis-related pathological conditions, such as acidosis and ischemia.Part III Study on the molecular mechanism of cardiac fibrosis potentiated by TRPM7 in myocardial infaction of mouseObjectives To exporle the roles of TRPM7 may play in fibroblasts functions concerning mainly with promotion of fibrosis potentiating by stimulation of TBFβ1.Methods (1) observation of the influences of acidic pH condition, LPA,2-APB and dose-response of different concentration of bi-valence cation on TRPM7 channel between SiRNA silencing group and control group by whole cell patch clamp technique; (2) Ca2+influx under the condition of pathophysiological stimulus such as low pH and TGFβ1 by calcium fluorescence imaging technique; (3) PCR quantification of TRPM7 mRNA level of cell from SiRNA transfected group and control group after TGFβ1 treatment and evaluation level of total collagen mRNA. Results Total collagen context and TRPM7 mRNA in TGFβ1 group is significant higher than control group.Conclusions Strong fibrosis-mediated factors TGFβ1 could inducing abnormal collagen formation in cardiac tissue by up-regulation of Ca2+ influx.