Function And Mechanism Of Peptide TAG-23 In Ischemic Heart Disease

Background:Ischemic heart disease(IHD)is a type of cardiovascular disease that seriously endangers human health,which has a high fatality rate and a high incidence.Acute Myocardial Infarction(AMI)is a fast-developed and high mortality rate disease,which has become one of the most dangerous diseases.The prevalence and death rate of ischemic heart disease in our country are still on the rise,which brings a heavy burden to people's lives and has become a major public health problem.At present,the main treatment for AMI is to open the infarcted blood vessel as soon as possible and rebuild blood supply to save the dying myocardium.Although the development of drug therapy,thrombolytic therapy,and interventional stent surgery has alleviated the clinical symptoms of patients to a certain extent,with the aging society and the prevalence of unhealthy lifestyles,the number of patients with ischemic heart disease in our country is still increasing rapidly.Existing technologies cannot meet the increasing demands of patients.Therefore,it is urgent to find more effective treatments.Focusing on the targets for therapeutic intervention has always been the basis for researchers to develop drugs based on the occurrence and development mechanism of ischemic heart disease.At present,the development of a variety of new drugs may become a new sight for the treatment of cardiovascular diseases.For example,m TORC1 inhibitor(Rheb)can protects myocardial ischemia and AMPK agonist(5-amino-4 formamide nucleotide)improves myocardial ischemia by activating autophagy.Since ischemia reperfusion injury is an important pathophysiological basis of ischemic heart disease,some researchers have begun to change their research field in recent years,starting with mitigating the cell damage caused by ischemia-hypoxia,focusing on improving mitochondrial function.Positive progress has been made in drug research and development.For example: Tat-Beclin-1 can reduce the accumulation of polyglutamine,improve the function of mitochondria and protect myocardial ischemia.Tat-DAXXp can reduce the myocardial infarction area by inhibiting ischemia reperfusion injury.Above clues all indicate that the new trends of development of ischemic heart disease from the perspective of mitigating myocardial ischemiareperfusion injury.Peptides have been one of the popular directions in the research and development of biological drugs since 21 st century.Peptide drugs are favored by researchers due to their low toxicity,small immune response,and small molecular weight.In the diagnosis and treatment of cardiovascular diseases,the research on peptides has been widely used in clinical practice.For example,brain natriuretic peptide(BNP)composed of 32 amino acids can be used as a diagnostic biomarker of heart failure.Artificially synthesized brain natriuretic peptide has been successfully marketed as a drug for the treatment of heart failure.It has been found that angiotensin II(Ang II)-derived polypeptide Ang(1-7)can antagonize Ang-II and improve vasodilation.Fibrin-derived polypeptide B?15-42 can not only inhibit the migration of white blood cells,but also can significantly reduce the area of myocardial infarction and scar formation in rats myocardial infarction model.These studies indicate that peptides may have broad prospects in the treatment of ischemic heart disease.Therefore,it may provide new clues for the development of drugs for ischemic heart disease from the perspective of peptides.With the rapid development of technology and transportation,people's lives have been great changed.The prevalence of obesity and cardiovascular disease increase yearly since most people live in a sedentary state of life.Regular exercise is one of the important ways to prevent cardiovascular disease.Most patients with cardiovascular disease are advised to take systematic and regular exercise.In recent years,research on exercise has been continuously updated,and the crosstalk between exercise and cardiovascular has become one of the hottest issues.Studies have confirmed that exercise can induce the expression of mi R-29 a and mi R-29 c.In the model of myocardial infarction,mi R-29 a and mi R-29 c can reduce the expression of collagenase I and collagenase III,thereby exerting anti-fibrotic effects.Exosomes from cardiomyocytes contain HSP70 and HSP90 can promote cardiomyocytes survival.In 2020,the European cardiovascular society issued the first guideline to provide evidence of exercise protocol for various cardiovascular disease patients,which emphasizes patients suffering from cardiovascular diseases conduct investigation,risk assessment and systematic management,and clinicians formulate relevant exercise plans to help patients effectively prevent and recover from cardiovascular diseases.However,the specific mechanism of how exercise protects the cardiovascular system is still unknown.Focus on the crosstalk between exercise and cardiovascular disease may provide new strategies for the prevention and treatment of cardiovascular disease.Part 1 Identification and bioinformatics analysis of TAG-23Objectives:1)Identify the potential functional peptides from the perspective of exercise and provide new clues for drug development of ischemic heart disease.2)Analyze the bioinformatics features of TAG-23 to provide theoretical basis for further functional experiments.Methods:1)Screening the potential peptides from PRIDE proteome X change database(http://www.proteomexchange.org/).The candidate peptides were selected based on the principles of up-regulation after exercise,high conservation,large differences between groups and small differences within groups.2)Isolate and culture primary cardiomyocytes in vitro to construct ischemia-hypoxic reperfusion injury model.Identify the potential functional peptides by CCK8 and LDH measurement.3)Use Peptide Ranker(https://web.expasy.org/protparam/)and Uniprot(http://www.uniprot.org/)to analyze the bioinformatics features of TAG-23Results:1)Based on the principles of high conservation,high difference,and up-regulation after exercise,5 candidate peptides derived from TAGLN,Myosin,LTB4,HMGN2,and Actin were initially obtained.2)Through an in vitro ischemia-hypoxia reperfusion injury model,we found that the TAGLN-derived peptide TAG-23 can promote the cells survival,reduce LDH release and cell damage caused by ischemia reperfusion injury3)TAG-23 is highly conserved in human,rat and mouse.TAG-23 has the characteristics of good lipophilicity and long half-life.TAG-23 can enter the cell and exist in the cell stably.Conclusions:1)TAG-23 is a peptide that promotes cell survival and reduces ischemia hypoxia reperfusion injury.2)TAG-23 is a highly conserved peptide,which can enter the cell and locate in the cytoplasm.3)The good lipid solubility and long half-life of TAG-23 meet the requirement of peptide drug research and development.Part 2 Functional analysis of TAG-23 in vitro and in vivoObjectives:1)Explore the function of TAG-23 in cardiomyocytes.Assess the possibility of TAG-23 to treat ischemic heart disease.2)Explore the function of TAG-23 in mouse myocardial infarction and reperfusion injury.Comprehensively analyze the possibility of TAG-23 to treat ischemic heart disease.Methods:1)Isolate primary cardiomyocytes in vitro and construct ischemia hypoxia reperfusion injury model.The peptide TAG-23 was synthesized in vitro by chemical synthesis,and different concentrations peptide(10?M,20?M,50?M,100?M)were added to the culture medium.Comprehensively evaluate the function of peptide TAG-23 on ischemia-hypoxia reperfusion injury by Tunel assay to assess cell apoptosis,reactive oxygen species by ROS method,and mitochondrial membrane potential by JC-1 detection,oxidative stress,cell damage.2)The mouse myocardial infarction reperfusion injury model was established by ligation of the anterior descending coronary artery,and the function of TAG-23 with different doses(1mg/Kg,5mg/Kg,10mg/Kg)was detected by tail vein injection.TTC Evans Blue assay was used to detect myocardial infarction area.Tunel assay was used to detect cell apoptosis.ELISA was used to detect myocardial injury markers expression(c Tn I,CK-MB).Tissue staining was used to evaluate inflammatory cell infiltration and fibrosis.Results:1)In the primary cardiomyocytes' ischemia-hypoxia reperfusion injury model,the peptide TAG-23 can protect myocardial cells against ischemia-hypoxiareperfusion injury and exerts a protective effect in a concentration-dependent manner.2)The peptide TAG-23 significantly reduces the activation of Caspase3 and PARP,and the expression of the cleaved caspase3 and PARP are significantly weakened.Tunel results indicate that the peptide TAG-23 can significantly reduce the fluorescent signal of apoptosis.ROS results show that the peptide TAG-23 can reduce the production of reactive oxygen species in ischemia hypoxia reperfusion injury,thereby reducing oxidative stress.Under the induction of ischemia-hypoxia reperfusion injury,the mitochondria depolarize with the decrease mitochondrial membrane potential.The peptide TAG-23 can reduce mitochondrial depolarization.3)In a mouse myocardial infarction reperfusion injury model,the peptide TAG-23 can reduce the area of myocardial infarction and reduce myocardial infarction reperfusion injury.4)Tunel results suggest that after the intervention of the peptide TAG-23,the apoptotic signal is significantly reduced,indicating that the peptide TAG-23 can inhibit the apoptosis of cardiomyocytes during reperfusion injury.5)After the treatment of the peptide TAG-23,the expression of myocardial injury markers(c Tn I,CK-MB)decreased,suggesting that the peptide TAG-23 can reduce myocardial infarction reperfusion injury.6)Tissue staining results suggest that the peptide TAG-23 can inhibit myocardial fibrosis,reduce the infiltration of inflammatory cells,and reduce myocardial damage.Conclusions:1)TAG-23 is a peptide that can protect cardiomyocytes and reduce cell apoptosis and oxidative stress from ischemia reperfusion injury.2)TAG-23 can reduce myocardial infarction reperfusion injury,reduce the area of myocardial infarction,inhibit myocardial fibrosis,reduce the infiltration of inflammatory cells,thereby improving heart function.Part 3 Mechanism study of TAG-23 in ischemic heart diseaseObjectives:1)Explore the potential pathway of the peptide TAG-23 and clarify the signal pathway involved in the function of TAG-23.2)Identify the target of the peptide TAG-23 and verify the interaction between TAG-23 and the target.3)Explore the molecular mechanism of TAG-23 involved in ischemia hypoxia reperfusion injury.Methods:1)Analyze the possible pathways of TAG-23 by second-generation sequencing,analyze the potential signal pathways by IPA combined with KEGG,and verify the signaling pathways by Western Blot.2)The peptide Biotin-TAG-23 was synthesized in vitro,and the potential targets of TAG-23 were identified by Pull down,silver staining,and tandem mass spectrometry.3)Verify the binding between TAG-23 and target by co-immunoprecipitation4)Comprehensive analyze PKG-mediated molecular mechanisms by transcription level,translation level,half-life,degradation,and degradation pathways.Results:1)The second-generation sequencing results suggest that TAG-23 may be involved in the regulation of MAPK signaling pathway,Hippo signaling pathway,and T cell receptor signaling pathway.2)Western Blot results found that the peptide TAG-23 can reduce myocardial damage by regulating the MAPK signaling pathway.Further verification found that TAG-23 can reduce cell apoptosis by activating the ERK signaling pathway,while there is no difference between P38 and JNK signaling pathways.3)Two differentially expressed bands were obtained by Pull-down combined with silver staining,and mass spectrometry analysis revealed that 235 proteins may interact with TAG-23.4)Further screening results found that PKG may be the target of TAG-23 based on the principle of(1)remove non-specific binding proteins;(2)high polypeptide repeat fragments;(3)high protein abundance.5)Co-immunoprecipitation experiments were used to further confirm that PKG is the target of TAG-23.The interaction between TAG-23 and PKG can be detected in the H293 cells and H9C2 cells.6)The peptide TAG-23 has no effect on the transcription of PKG,but it can regulate the protein expression of PKG in a concentration-dependent and time-dependent manner at the translation level.7)The peptide TAG-23 can extend the half-life of PKG,reduce the degradation of PKG,and inhibit the ubiquitination of PKG.Conclusions:1)PKG is the direct target of TAG-23.2)The peptide TAG-23 inhibits the ubiquitination of PKG,prolongs the half-life of PKG and reduces the degradation of PKG,thereby activating the ERK signaling pathway to protect the cardiomyocytes from ischemia-hypoxia reperfusion injury.