GDF11 Can Improve Cardiac Ischemia-reperfusion Injure By Inhibiting The Inflammatory Response Induced By Mitochondrial DNA Damage

Background:Acute myocardial infarction(AMI)is myocardial necrosis caused by acute,persistent ischemic hypoxia in the coronary arteries.At present,the most commonly used clinical methods for treating myocardial infarction are vascμLar recanalization and coronary thrombolysis.However,recanalization of coronary vessels can cause myocardial ischemia-reperfusion(I/R)injury.In recent years,more and more literatures have reported the pathophysiology,of myocardial ischemia-reperfusion(I/R)injury.However,there are few effective ways to prevent reperfusion injury due to ATP consumption during ischemia,reactive oxygen species(ROS),myofascial contractions,calcium-dependent proteases(such as calpain),microvascμLar dysfunction,and inflammation cytokine.In addition,ROS produced during ischemia and reperfusion are thought to play a central role.Mitochondrial DNA(mtDNA)is double-stranded and naked circμLar DNA with a size of approximately 16.5 kb.There is growing evidence that mtDNA is more susceptible to oxidative stress because it is close to the respiratory chain and lacks protective histones and introns.Once mtDNA is damaged,the coding of key proteins in the respiratory chain will be reduced,which will aggravate the production of ROS and mitochondrial dysfunction.Therefore,understanding how to reduce mtDNA damage is important to improve the prognosis of patients with myocardial I/R injury Recent studies have shown that Growth Differentiation Factor 11(GDFI 1)can improve I/R damage to the heart of aged mice and enhance heart function and cell regeneration.ResμLts of previous studies have shown that GDF11 can significantly reduce the area of myocardial infarction after ischemia-reperfusion in mice,but the specific mechanism is not clear In this study,we first clarified whether GDF11 can improve cardiac I/R injury,and then further explored its molecuLar mechanism.Purpose:Through in vivo and in vitro experiments,to clarify the role of mtDNA damage-inflammation in cardiac I/R and reveal the molecμLar mechanism by which GDF11 exerts cardioprotective effects by inhibiting mtDNA damageMethods:1.The CHO-K1 expression system was used,and pGS-GDF11-Fc vector was constructed containing the C-terminal mature peptide of GDF11.The CHO-K1 stable cell strain with high secretion of GDF11-Fc recombinant protein was screened out.According to the characteristics of Fc tag,HiTrapTM 5ml MabSelectTM SuRe column was used to purify GDF11-Fc protein from the cell supernatant.After the purification step,the purity of GDF11-Fc protein was determined by coomassie brilliant blue dye and gray analysis.Then,GDF11-Fc was validated via Western blot,using GDF11 and Fc-antibodies.The biological activity of GDF11-Fc was confirmed in hs68 cells by analyzing the expression of type Ⅰ collagen(COL I)using Western blot,where GDF11 from Peprotech was used as the positive control2.In vivo,a model of heart I/R injury in C57BL/6N mice was established to evaluate whether GDF11-Fc recombinant protein can reduce mouse heart I/R injury.TTC-Evans blue staining was used to verify whether GDF11-Fc protein can reduce the area of myocardial infarction;HE staining and serum LDH content were used to prove the protective effect of GDF11-Fc protein on the heart;immunofluorescence(IF)was used to detect whether GDF11-Fc had a protective effect on heart tissue mtDNA has a protective effect;q PCR method was used to detect changes in mitochondrial genes and inflammatory factors NLRP3,caspase-1,and IL-1β in cardiac tissues;Western blot was used to detect changes in inflammatory factors in protein levels in cardiac tissues3.In vitro study,establish H/R injury model,detect whether GDF11 protein has protective effect on cells by LDH and MTS;test whether GDF11 protein has inhibitory effect on cardiomyocyte apoptosis by TUNEL and flow cytometrsy;immunofluorescence detection of GDF11 protein on myocardium Whether cell mitochondrial DNA has a protective effect;JC-1 was used to detect the protection of mitochondrial membrane potential of cardiomyocytes by GDF11 protein;qPCR method was used to detect the mitochondrial gene and inflammatory factor expression of cardiomyocytes.ResμLts:1.A subclone of CHO-K1 cell with good growth state was selected out,which also had the high secretion level of GDF11-Fc protein.After the purification step,the purity of GDF11-Fc protein was more than 90%,and its molecμLar weight was consistent with the expected resμLt.GDF11-Fc recombinant protein coμLd react with antibodies of both GDF11 and Fc,and it was identified as the interest protein.In addition,as expected,collagen I significantly increased with GDF11-Fc,similarly to the GDF11 purchased from Peprotech.Thus,the resμts indicated that GDF11-Fc protein coμLd play its biological role within the cell.2.In the mouse heart I/R model,TTC-Evans blue staining showed that GDF11-Fc protein can reduce the area of myocardial infarction;GDF11-Fc protein can significantly reduce the LDH content in serum,and HE staining proved that GDF11-Fc protein has a significant effect on the heart.Protective effect;the resμLts of immunofluorescence experiments showed that 8-OHdG expression increased in injured myocardial tissues,and the expression of 8-OHdG decreased significantly after treatment with GDF11-Fc protein.At the same time,The resμLts of qPCR showed that the expression of mitochondrial gene was decreased in the damaged myocardial tissue,and the expression of mitochondrial gene was significantly increased after the treatment with GDF11-Fc protein;qPCR and Western blot resμLts showed that GDF11-Fc protein can effectively reduce expression of inflammatory factors in myocardial tissues of I/R.3.In myocardial cell H/R injury model,after adding GDF11,the LDH level in the supernatant decreased significantly,MTS detection showed a significant increase in cell proliferation level;TUNEL and flow cytometry showed that GDF11 had an inhibitory effect on cardiomyocyte apoptosis;immunofluorescence showed that the expression of 8-OHdG increased in cardiac myocytes after H/R,and the expression of 8-OHdG decreased significantly after the addition of GDF11 protein;The cell membrane potential was detected by JC-1,indicating that GDF11 had a protective effect on mitochondrial membrane potential of cardiomyocytes;qPCR resμLts showed that the expression of mitochondrial gene in H/R cardiomyocytes decreased,and the expression of mitochondrial gene in cardiac tissue increased significantly after the addition of GDF11;qPCR and Western blot resμLts showed that GDF11 protein coμLd effectively reduce the expression of inflammatory cytokines in H/R cardiomyocytes.Conclusions:GDF11 can improve cardiac ischemia-reperfusion injury by inhibiting the inflammatory response induced by mitochondrial DNA damage.