| Background and Objective:Reperfusion therapy is the first choice for patients with acute myocardial infarction(AMI).Timely and effective reconstruction of coronary blood flow and rescue of ischemic myocardium are the most effective approaches to limit the area of myocardial infarction and improve cardiac function.However,after the blood supply of infarcted vessels resumes,AMI patients are often confronted with adverse cardiac events such as no reflow,myocardial stunning,reperfusion arrhythmia and irreversible cardiomyocyte death,resulting in myocardial ischemia / reperfusion injury(IRI),which severely restricts the benefit from clinical treatment.The mechanisms involving into IRI injury are complicated and multifactorial.Studies have shown that m6 A modification of RNA and ferroptosis playing an important role in IRI injury.Hence,for clinical practices,it is of great significance to explore the underlying causes behind such pathogenesis and find effective therapeutic targets.As a post transcriptional modification,m6 A modification of RNA significantly increases the level of m6 A in ischemic heart disease and ischemia-reperfusion injury,suggesting that m6 A modification strongly assists myocardial protection;however,its role and mechanism in myocardial IRI injury are unknown.The demethylase ALKBH5 is the key regulator of methylation modification.Studies have confirmed that ALKBH5 playing a critical role in cardiomyocyte proliferation and myocardial repairment,but the regulatory mechanisms of ALKBH5 in the processes of acute myocardial infarction and myocardial IRI injury have not been clarified.In addition,studies have confirmed that ferroptosis,which is associated with ferroptosis suppressor protein 1(FSP1),is closely related to cardiovascular diseases.In this thesis,the relationship between the expression of kbalp1 and myocardial ischemia-reperfusion in rats was examined firstly.We then found that FSP1 is an important methylation modification target for ALKBH5 to activate ferroptosis based on cell experiments.Finally,it is clarified that ALKBH5 mediates the molecular mechanism of FSP1 methylation modification to activate iron death signal pathway.These findings will provide new perspectives and targets for the prevention and treatment of ischemia-reperfusion myocardial injury.Methods:1.The mouse model of cardiac ischemia-reperfusion injury was established,and the methylation level of m6 A in myocardial RNA was detected by colorimetry;q RT-PCR was used to detect the expression levels of methyltransferase(MELLTL3 and MELLTL14)and demethylase(ALKBH5and FTO)in myocardial tissue of IRI group;RNA-Seq was used to determine the expression of m6 A key modifying enzyme in IRI myocardial tissue;The expression of total m6 A in myocardial tissue of IRI group was detected by dot blot;The expression of ALKBH5 protein in myocardial tissue of IRI group was identified with Western blot.2.Primary cardiomyocytes of SD suckling rats aged 1-2 days were isolated,cultured and identified by immunofluorescence.The viability of cardiomyocytes and the changes of reoxygenation cell viability after 4 hours of hypoxia were detected by CCK-8;The methylation level of m6 A in cardiomyocyte RNA was detected by colorimetry;The changes of total m6 A level of cardiomyocytes in H/R group were detected by dot blot;The expressions of ALKBH5 m RNA and protein in cardiomyocytes of H/R group were detected by q RT-PCR and Western blot.3.After ad-ALKBH5 was transfected into cardiomyocytes for 36 hours,they were treated with H/R firstly,and then sent for testing the expressions of ALKBH5 m RNA and protein by q RT-PCR and Western blot.The changes of cardiomyocyte viability were detected by CCK-8.In addition,we detected the changes of cardiomyocyte mitochondrial membrane potential by tmre staining.4.Ad-ALKBH5 was injected into the apex to overexpress mice;After 36 hours,the mice were treated with IRI,and the changes of cardiac function were detected by transthoracic color Doppler ultrasound.Further,the changes of c Tn I content and LDH activity in mouse serum were detected.After overexpression of ALKBH5,the changes of left ventricular ejection fraction and left ventricular short-axis shortening rate were observed,and the changes of myocardial infarction area were detected by TTC / Evans blue staining.5.The expression of ALKBH5 in overexpressed cardiomyocytes,followed by hypoxia reoxygenation.The changes of ROS intensity of cardiomyocytes were detected by dcf-hc staining and FACS.In addition,the changes of iron death markers MDA,ferritin and divalent iron in cardiomyocytes at H/R,and the changes of GSH level in serum of mice were detected by ELISA.6.After ad-ALKBH5 was transfected into cardiomyocytes for 36 hours,H/R treatment was performed.RNA-seq was used to detect the changes of iron metabolism related signal pathways,and the specific mechanism of whether ALKBH5 promoted the expression of FSP1 protein by regulating the m6 A modification of FSP1 was elucidated by the methods of West blot and s RAMP prediction.Results:1.After 24 hours of ischemia reperfusion injury,the total level of m6 A was significantly increased and the expression of ALKBH5 was significantly decreased.2.The level of m6 A methylation in myocardial tissue RNA was detected by colorimetric method,showing significant increase of the total m6 A level in myocardial tissue of IRI group;Qrt-pcr results showed that the expression levels of methyltransferases METTL3 and METTL14 were significantly increased in IRI group,while the expression level of demethylase ALKBH5 was significantly decreased,and the expression level of FTO was not significantly changed.Dot Blot showed that the total m6 A level in the IRI group was significantly increased.Western Blot analysis showed that ALKBH5 protein level in IRI group was significantly decreased.3.CCK-8 assay and TMRE staining showed that ALKBH5 overexpressed significantly alleviated H/R induced myocardial cell injury.4.Ad-ALKBH5 was injected into the apex of the heart,and after 36 hours the mice were then treated with IRI.The results of cardiac color ultrasound showed that ALKBH5 was overexpressed,with significantly increased LVDP and + DP/DT,and decreased LVEDP and-DP/DT along the time.These significantly improved the systolic and diastolic functions of the left ventricle of mice.ELISA results showed that the content of c Tn I in serum of ALKBH5 overexpressed mice increased.Meanwhile,ALKBH5 overexpression significantly decreased LDH activity.TTC/Evans Blue staining was used to determine the size of myocardial infarction and it was found that overexpression of ALKBH5 reduced IRI induced myocardial infarction.5.Overexpression of ALKBH5 inhibits H/R-induced iron death.6.RNA-seq analysis showed that H/R activated the iron death signal pathway of cardiomyocytes,and the overexpression of ALKBH5,which significantly reverse its induced iron death;q RT-PCR showed that the expression of FSP1 decreased during H/R,and overexpression of ALKBH5 promoted the expression of FSP1;Western blot results also showed that the expression of FSP1 decreased during H/R;Overexpression of ALKBH5 promoted the expression of FSP1;SRAMP prediction showed that there were many m6 A methylation sites in the m RNA sequence of FSP1 gene,of which962 BP site was highly conservative;The m6 A modification of FSP1 in cardiomyocytes was detected by me rip.The m6 A modification of FSP1 increased during H/R,and overexpression of ALKBH5 inhibited the m6 A modification of FSP1;The attenuation rate of FSP1 m RNA was analyzed by transcription inhibition test.The result showed that overexpression of ALKBH5 could significantly reduce the attenuation of FSP1 m RNA.Conclusion:In myocardial ischemia-reperfusion injury,overexpression of ALKBH5 is effective in preventing methylation modification of FSP1 and increasing its expression,thus inhibiting the signaling pathway of iron death and ultimately protecting myocardial cells. |
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