The Protective Effect And Mechanism Of MYDGF Derived From CMECs On Myocardial Cells After Myocardial Infarction

BackgroundMyocardial infarction(MI)is currently a very harmful ischemic heart disease.ST segment elevation myocardial infarction(STEMI)can significantly improve ischemic symptoms after percutaneous coronary intervention(PCI).But this also poses a new challenge——After ischemia-reperfusion,the sudden opening of blood vessels can cause cell calcium overload and energy metabolism obstacles,an increase in oxygen free radicals,and damage to cardiac microvasculature,further exacerbates myocardial cell death,this injury is clinically known as ischemia-reperfusion injury(I/R).Microvessels in the heart mainly include arterioles,capillaries,and venules.The arterioles(<100 μm in diameter)are located in the ventricular wall,and they respond dI/Rectly to the myocardium’s need for oxygen and nutrients,and strive to match the heart’s blood flow to this demand.Cardiac microvascular endothelial cells(CMECs)are the main components of cardiac microvessels.In a normal heart,there are capillaries next to almost every cardiomyocyte.The number of cardiac endothelial cells is more than three times that of cardiomyocytes,but the ratio of volume(or mass)of cardiac endothelial cells to cardiomyocytes is only 0.04-0.05.It was initially discovered that endothelial cells undergo earlier apoptosis during reperfusion than cardiomyocytes.Recent studies have shown that during I/R injury,vascular endothelial cells(ECs)reduce the damage of cardiac muscle cells(CMs)by secreting protective factors.Myeloid-derived growth factor(MYDGF)is a secreted protein.It has been reported in the literature that MYDGF can protect myocardial cells from ischemia-reperfusion injury in myocardial infarction.According to its amino acid sequence,MYDGF does not belong to any known cytokine or growth factor family,has no sequence homology with other proteins,and has no specific structure.MYDGF receptors or downstream signaling molecules that mediate these effects are also unknown.Therefore,the determination of the structure of MYDGF is helpful for a deeper understanding of its biological function." Crosstalk" between endothelial cells and cardiomyocytes has been shown to protect the heart.The regulation of cardiomyocytes by cardiac protective factors secreted by endothelial cells may be an alternative treatment for reducing I/R damage in cardiomyocytes.However,fewer reports have been made on the interaction between cardiac microvascular endothelial cells and cardiomyocytes.Furthermore,secreted myeloid-derived growth factor(MYDGF)is mainly expressed in bone marrow-derived monocytes and macrophages,and the effect of CMECs-derived MYDGF on myocardial cells after myocardial infarction is unclear.Therefore,this project intends to construct CMECs and CMs co-culture models in vitro,to study the effects and mechanisms of CMECs on cardiomyocytes during I/R injury,and to further explore the role of CMECs-derived MYDGF in cardiomyocytes after myocardial infarction.PurposeExplore the effect of MYDGF derived from cardiac microvascular endothelial cells on cardiomyocytes and specific mechanisms during ischemia-reperfusion injury,and provide new research targets for the treatment of ischemia-reperfusion injury.Methods1.Isolation and identification of rat CMECsThe double-vessel digestion method was used to isolate the microvascular endothelial cells of rat left ventricle.The isolated CMECs were identified by immunofluorescence and angiogenesis experiments.2.CMECs and CMs supernatant replacement modelThe conditioned medium after CMECs H/R was collected and transferred to H9c2.After H9c2 H/R was detected,the LDH changes in the supernatant.3.CMECs and CMs co-culture modelA 3μm Transwell chamber was used to establish a co-culture model.After H/R was co-cultured with CMECs and H9c2,TUNEL and flow cytometry were used to detect apoptosis in the lower chamber H9c2;Western Blotting was used to detect the expression of H9c2 apoptosis-related proteins in the lower chamber;qRT-PCR was used to detect the lower chamber.H9c2 expression of inflammation-related factors.4.Proteomics sequencingThe supernatants of CMECs H8R2 were collected,and the expression of differential proteins in the supernatants was identified and analyzed using the isotope-labeled relative and absolute quantitative(iTRAQ)technology.5.Expression and purification of MYDGF proteinA pcDNA3.1-MYDGF-myc-His(-)B expression vector was constructed,and 293T cells were transfected to express and purify the MYDGF protein.6.The rat heart IlR24h model was constructed and treated with recombinant MYDGF protein.After the experiment,TTC staining was performed to observe the area of myocardial infarction.7.Using AAV9 to overexpress MYDGF in rat heart,and establish I1R24 model,TTC staining,HE,TUNLE,qRT-PCR to detect myocardial tissue damage.Results1.Rat CMECs were successfully isolated.The identification results showed that they could be used in subsequent experiments.2.The supernatant after CMECs H/R can reduce the content of LDH in the supernatant after H9c2 H/R.3.TUNEL and flow cytometry results showed that after H/R co-culture of CMECs and H9c2 cells,the apoptosis rate of H9c2 decreased significantly.Western Blotting results showed that the expression of apoptosis-related proteins Bax and Cleaved-Caspase-3 in H9c2 decreased significantly after H/R co-culture of CMECs and H9c2 cells.qRT-PCR results showed that after co-culture of CMECs and H9c2 cells with H/R,the expression of inflammation-related factors IL-6 and IL-18 in H9c2 decreased significantly.4.iTRAQ results showed that 474 differential proteins were identified in the supernatant after CMECs H/R,of which 169 proteins were up-regulated and 375 proteins were down-regulated The secreted protein MYDGF was further screened,and it was detected after CMECs H/R.Qingzhong was verified.5.Recombinant MYDGF protein can reduce the expression of Cleaved-Caspase-3 and Bax after H9c2 and NRVM H8R2,and promote the phosphorylation of Akt.6.The plasmid stably expressing MYDGF of rat was successfully constructed,MYDGF protein was successfully obtained by using eukaryotic expression system,and purified by nickel column.7.TTC staining,MYDGF protein treatment at rat I1R24 can significantly reduce the area of myocardial infarction.8.AAV9-MYDGF overexpression rats were successfully constr-ucted.After IIR24,the heart damage of AAV9-MYDGF rats was significantly reduced.Conclusions1.CMECs can reduce myocardial cell damage during hypoxia and reoxygenation.2.The expression of MYDGF in CMECs increased significantly during hypoxia-reoxygenation.3.During ischemia-reperfusion,MYDGF derived from CMECs activates the Akt signaling pathway of cardiomyocytes,reduces cardiomyocyte apoptosis,and protects the heart from ischemia-reperfusion injury.