The Molecular Mechanisms Of RIP3/MLKL Signaling Pathway In Cardiomyocyte Hypertrophy

BackgroundMyocardial hypertrophy is an important manifestation of myocardial remodeling,and it is a series adaptive responses to meet the long-term volume load and pressure load of heart.Myocardial hypertrophy plays an important role in the maintenance of cardiac function under pathological conditions,but it is also a common pathological process of the occurrence and development of many cardiovascular diseases,such as ischemic heart disease and sudden cardiac death.Neurohumoral,energy metabolism imbalance,oxidative stress and other factors can participate in the occurrence and development of myocardial hypertrophy through specific signaling pathways,but the specific regulatory mechanisms of related signaling pathways have not been fully elucidated so far,which brings difficulties to the development of new drugs.The discussion of this problem can provide potential intervention targets for more targeted prevention and treatment of myocardial hypertrophy.Programmed necrosis（necroptosis）,as a caspase-independent cell death mode,has become a research hotspot in recent years.RIP3 is an essential regulatory protein during programmed cell necrosis.RIP3 is a member of the RIP family and is widely expressed in mature tissues such as pancreas,gastrointestinal,lung,liver,kidney,and heart.It has a typical serine/threonine protein kinase domain at its N-terminus,which can regulate the pathophysiological processes of various organs and tissues including the heart by regulating the phosphorylation of downstream target proteins.As a key regulatory protein of programmed necrosis,RIP3 plays an important role in the occurrence and development of heart diseases such as atherosclerosis,ischemia-reperfusion injury,myocardial infarction,myocarditis and heart failure.However,whether RIP3 is involved in the development of cardiac hypertrophy has not yet been reported.Mixed lineage kinase domain-like（MLKL）is the substrate of RIP3.In general,MLKL exists in cytoplasm as an inactive monomer.Activated RIP3 recruits and activates MLKL by phosphorylation of the 357 threonine and 358 serine sites of MLKL.Activated MLKL translocates to cell membranes and organelle membranes,binds with lipids phosphatidylinositol and cardiolipin,destroys membrane integrity,forms perforated channels,and mediates programmed cell necrosis.Studies have shown that the formation of a complex between RIP3 and MLKL is an essential step in the induction of programmed necrosis.However,it is still unclear whether this signaling pathway is involved in the occurrence and development of myocardial hypertrophy.In this study,myocardial hypertrophy was induced by chronic pressure overload,and neonatal rat cardiomyocytes were stimulated with Angiotensinogen II（Ang-II）or phenylephrine（PE）to simulate neurohumoral stress,to observe the expression of RIP3 in hypertrophic myocardium tissue and hypertrophic cardiomyocytes,and to investigate the effects of over-expression or silent RIP3 on myocardial hypertrophy in vivo and in vitro.In addition,we investigated whether the RIP3-MLKL signaling pathway is involved in the process of myocardial hypertrophy and its related signal mechanism.This study provides a new basis for the pathogenesis and drug development of myocardial hypertrophy.Part 1: The expression changes of RIP3 in hypertrophic myocardial tissueand hypertrophic myocardial cells Objective:To observe the expression changes of RIP3,ANP,β-MHC in hypertrophic myocardial tissue caused by aortic constriction,and hypertrophic myocardial cells induced by angiotensin II（Ang-II）or phenylephrine（PE）.Methods:1.The adult Sprague-Dawley（SD）male rats were randomly numbered,and then divided into a sham operation group（Sham group）and aortic banding method model group（AB group）.The general conditions of the rats in each group,such as vitality,diet and drinking water,were observed.The changes of RIP3 m RNA expression in rat myocardial tissue were detected by RT-q PCR at 2 weeks,4 weeks and 6 weeks after model establishment,and the protein expression levels of RIP3,ANP and β-MHC were detected by Western Blot.2.Neonatal rat cardiomyocytes were stimulated with Angiotensinogen II（Ang-II）or phenylephrine（PE）for 24 h,48h and 72 h to simulate the neurohumoral stress and induce cardiomyocyte hypertrophy.RT-q PCR was used to detect the m RNA expression level of RIP3 in hypertrophic cardiomyocytes,the expression of RIP3,ANP and β-MHC were detected by Western Blot.Results:1.Compared with the control group,the expression of RIP3,β-MHC and ANP proteins in the myocardial tissue of the aortic banding surgery group did not change significantly at 2w after operation.The expression of RIP3 in myocardial tissue dramatically increased since the 4th week after surgery,which was tightly associated with the expression level of β-MHC and ANP.And the expression levels of the above proteins increased more significantly at 6 weeks after operation,while the m RNA expression levels of RIP3 had no significant difference between the AB surgery group or the Sham group.2.Compared with the PBS-treated group,the protein expressions of RIP3,β-MHC and ANP in neonatal rat cardiomyocytes were increased after after PE or Ang-II treatment for 48 hours.The expression of RIP3 protein was distinguishably increased since the 72 th hour after treatment,which showed a remarkable correlation with β-MHC and ANP protein levels.And the m RNA level of RIP3 failed to exhibit significant alteration in Ang-II or PE-treated cardiomyocytes.Conclusion:（1）In the model of chronic pressure overload rats established by aortic banding method,the hypertrophy of myocardium appeared obviously in AB group 4 weeks after operation,and the degree of hypertrophy increased with the prolongation of model time;（2）Ang-II or PE treatment for 24 hours can induce cardiomyocyte hypertrophy in newborn rats.As the treatment time increases,the degree of cardiomyocyte hypertrophy increases;（3）RIP3 plays an important role in the process of cardiac hypertrophy,and the change of RIP3 expression is induced by the post-transcriptional level,suggesting that RIP3 may be an important regulatory target for cardiac hypertrophy.Part 2:Regulation of RIP3 in myocardial hypertrophy model in vitro and in vivoObjective:RIP3 was knocked down or overexpressed in vitro and in vivo to explore the regulatory role of RIP3 in myocardial hypertrophy models in vitro and in vivo.Methods:1.Using lentivirus technology,RIP3 expression was modified by sh RNA targeting RIP3（sh-RIP3）to down-regulate the expression level of RIP3 protein in NRCMs,establish primary neonatal rat cardiomyocytes that stably interfere with RIP3,and were treated with PBS,Ang-II,and PE for 24 h.On the other hand,the expression level of RIP3 was up-regulated in NRCMs to overexpress RIP3,and then treated with PBS,Ang-II and PE for 24 h.And then evaluated the response of neonatal rat cardiomyocytes to Ang-II and PE-induced cardiomyocyte hypertrophy after knocked down or overexpression of RIP3.The effect of RIP3 expression level on cell surface area was observed by immunofluorescence technique,and the changes of ANP and β-MHC m RNA expression levels were observed by RT-q PCR.2.The adenovirus vector was introduced into SD male rats to overexpress RIP3,and the rats were randomly numbered,and then divided into Sham group and AB surgical model group.On the other hand,RIP3-specific sh RNA recombinant adeno-associated virus was introduced into rats to reduce the expression level of RIP3 in rat myocardial tissue,and then rats were randomly divided into Sham group and AB surgical model group.The activity,diet drinking water,were observed.The body weight（BW）,heart weight（HW）,lung weight（LW）and tibia length（TL）of the rats in each group were detected 4 weeks after surgery.HW/BW（mg/g）,LW/BW（mg/g）,HW/TL（mg/mm）were measured to reflect the degree of myocardial hypertrophy.The diameter of the myocardium was measured,and the changes of myocardial tissue structure and the degree of myocardial fibrosis were observed by HE staining.Results:1.sh-RIP3 significantly reduced the expression level of endogenous RIP3 protein in neonatal rat cardiomyocytes.In RIP3 knockdown neonatal rat cardiomyocytes,the cell surface area of cardiomyocytes was significantly smaller than that in the scramble group.Compared with the scramble group,the reduction in the surface area of cardiomyocytes in the sh-RIP3 group was more significant after Ang-II and PE treatment.Consistent with these results,the m RNA expression of ANP and β-MHC was significantly decreased in RIP3-knockdown cardiomyocytes following Ang-II and PE treatment.2.Compared with the control group,the overexpression of RIP3 significantly increased the surface area of cardiomyocytes in vitro,and the degree of hypertrophy of cardiomyocytes was more pronounced after stimulation with Ang-II and PE.Correspondingly,in RIP3-overexpressing NRCMs,the upregulation of ANP and β-MHC m RNAs stimulated by Ang-II and PE went a step further.3.In the rats overexpressing RIP3,the HW/BW,LW/BW and HW/TL of rats in the aortic banding surgery group were significantly increased,but there was no significant change in the sham-operated group.Meanwhile,histological analysis showed that the cross-sectional area of cardiomyocytes,the degree of fibrosis and the heart diameter of RIP3-overexpressing rats were significantly larger than those of the control group at 4 weeks after aortic banding surgery.These results suggest that the functional impairment of the heart by AB is further exacerbated by overexpression of RIP3.4.After 4 weeks of AB operation,the HW/BW,LW/BW and HW/TL of the rats in the operation group were significantly decreased,while no significant alterations were observed in the sham operation group.Histological analysis showed that after AB surgery in sh-RIP3 rats,the cross-sectional area of myocardial fibrosis and heart diameter were significantly smaller than those in sh RNA scramble and control rats.Conclusion:1.RIP3 is required for the pathogenesis of Ang-II and PE-induced cardiomyocyte hypertrophy in vitro.Reduction of RIP3 attenuates cardiomyocyte hypertrophy induced in vitro,and overexpression of RIP3 promotes cardiomyocyte hypertrophy in vitro.2.RIP3 may be an important regulatory target of cardiac hypertrophy,and overexpression of RIP3 in cardiomyocytes seems to aggravate but not induce pathological cardiac hypertrophy.Part 3 Research on the signaling pathway of RIP3-induced cardiomyocyte hypertrophyObjective:To explore whether RIP3 interacts with MLKL to affect the influx of calcium ions,thereby promoting the occurrence and progression of cardiac hypertrophy,and to clarify the signaling pathway of RIP3-induced cardiac hypertrophy.Methods:1.we conducted Co-IP experiments using NRCMs to explore whether endogenous RIP3 can interact with MLKL.2.Cell membrane fraction assays were performed using WT and RIP3-/-H9c2 cells.WT and RIP3-/-H9c2 cells were treated with PE and Ang-II for 24 h,and the expression levels of RIP3 and MLKL in the cytoplasm and total cell lysates were observed by Western Blot to determine whether RIP3 interacts with MLKL and is localized to the cell membrane to play a role.3.we performed immunofluorescence using MLKL antibody to directly observe its subcellular localization alteration of WT and RIP3-/-H9c2 cells treated with PE or AngII.4.Fluo4 staining to determine intracellular Ca2+ concentration in WT and RIP3-/-H9c2 cells after treatment with PE or Ang-II.5.The expression of MLKL was downregulated by a sh RNA（sh-MLKL）targeting MLKL in neonatal rat cardiomyocytes infected with lentivirus,and its effect on the surface area of PE or Ang-II treated cardiomyocytes was observed.6.Use calcium channel blocker-lanthanum chloride（La Cl3）and calcium release-activated calcium channel inhibitor 2-Aminoethoxydiphenyl borate（2-APB）to inhibit calcium influx and then the changes of HW/BW,LW/BW,HW/TL and other indexes in RIP3-overexpressing rats at 4 weeks after aortic banding surgery were detected.HE staining was used to observe the degree of myocardial hypertrophy and fibrosis.7.WT and RIP3-overexpressing NRCMs were pretreated with La Cl3 and 2-APB,stimulated with PE,Ang-II and PBS for 24 h,and the degree of cardiomyocyte hypertrophy was observed by immunofluorescence staining.Result:1.As a RIP3 substrate,MLKL directly interacts with endogenous RIP3,and its interaction is enhanced by PE treatment.2.After 24 h stimulation with PE or Ang-II,there was no difference in the protein level of MLKL in the lysates of WT and RIP3-/-H9c2 cells,but there was no MLKL expression on the membrane of RIP3-/-H9c2 cells.And MLKL protein in RIP3-/-H9c2 cells was isolated from the membrane surface by immunofluorescence staining.3.In WT H9c2 cells,the intracellular Ca2+ concentration was significantly increased after 24 h of PE or Ang-II treatment,while in RIP3-/-H9c2 cells,the calcium concentration caused by PE or Ang-II treatment was significantly lower than that of WT H9c2 cells.4.Loss of function of MLKL by sh RNA in the presence of PE or Ang-II treatment largely prevented the cell surface increase induced by RIP3 overexpression.5.After La CI₃ and 2-APB treatment,HW/BW,LW/BW,HW/TL were significantly lower than those in the untreated group.After oe-RIP3,the above indicators decreased more significantly than in the untreated group.Histological analysis showed that myocardial tissue and cardiomyocyte hypertrophy were significantly reduced after La CI₃ and 2-APB treatment compared with PBS treatment.In RIP3-overexpressing rats,La Cl3 and 2-APB treatment also largely abolished the effects of RIP3 overexpression on cardiomyocytes and heart size.6.Immunofluorescence showed that PE or Ang-II stimulated cardiomyocyte hypertrophy,while overexpression of RIP3 further enhanced cardiomyocyte hypertrophy.Interestingly,the degree of cardiomyocyte hypertrophy induced by overexpression of RIP3 was significantly reduced after treatment with La Cl3 or 2-APB.Conclusion:1.RIP3 interacts with MLKL to form a complex and localize to the cell membrane,promoting calcium inflow.2.Loss of function of MLKL by sh RNA in the presence of PE or Ang-II treatment largely prevented the cell surface increase induced by RIP3 overexpression.MLKL is required for RIP3 to fulfill its biological functions.3.Blocking calcium influx reversed the worsening of RIP3-mediated cardiac remodeling,suggesting that calcium influx largely mediates the deleterious role of RIP3 in cardiac remodeling.