| Ischemic heart disease(IHD),commonly induced by coronary artery stenosis,is a condition in which insufficient blood supply(and thus oxygen and nutrients)to the heart muscle.IHD is a leading cause of death worldwide and has been a social and economic burden on communities.There is an urgent need to develop preventive and therapeutic strategies for the treatment of IHD.Clinically,the key step of IHD treatment is to restore blood supply;however,the restoration also increases oxygen free radicals,unbalanced ATP synthesis,calcium overload,and activation of stress pathways,leading to further cardiac dysfunction.This phenomenon is called ischemia reperfusion injury(IRI).Current research has focused on exploring the approaches to avoid reperfusion injury and underlying mechanisms.Slit is an exocrine protein and was first noticed for its roles in guiding migration during the development of axonal processes of the neurons,and subsequently evidence supports this protein involves in various important biological processes.Typically,Slit proteins bind to Robo receptors on the cell membrane,mediating leukocyte chemotaxis,tumorigenesis,formation ofcardiac chambers,and differentiation of stem cells.On the other hand,evidence also show that Slit2 activation is associated with improvement of cellular metabolism,upregulation of calcium signaling,inhibition of leukocyte recruitment,and activation of phosphorylation signals,which are the key protective mechanisms after onset of cardiac ischemia.Thus,we hypothesize that Slit protein mediates signal transduction in myocardial IR and protects the heart.To test this hypothesis,transgenic mice(Slit2-Tg)overexpressing human Slit2(hSlit2)were used,and an ischemia model was induced in isolated mouse hearts to explore the mechanisms underlying Slit2 protective effects in IRI.Three projects were designed in this study: 1)to explore whether Slit2 overexpression affects the physiology,histology,biochemistry,and gene expression of the mouse hearts;2)to establish an IRI model in the isolated mouse hearts,and to explore whether hSlit2 overexpression alters the phenotypes of myocardial IRI;3)to explore the molecular mechanisms underlying the hSlit2 protective effects on myocardial IRI.In the first project,hSlit2 overexpression in the hearts was evaluated by semi-qPCR and Western blotting,and the expression of mouse-specific Slit isoforms was evaluated by qPCR.The cardiac function and tissue structure of Slit2-Tg mice were respectively detected by small animal ultrasound system and HE staining.In the second project,isolated mouse hearts prepared by a Langendorff system was employed to determine the effects of hSlit2 overexpression on cardiac recovery after IR.The area of infarction wasdetermined by TTC staining.The tissue changes were detected by HE staining,and the damage of subcellular structure was examined by an electron microscope.In the third project,Western blotting and Pro-Q staining were employed to investigate the intracellular signaling of hSlit2 overexpression and myofilament phosphorylation levels,respectively,in IRI.The expression levels of specific PKC isoforms and protein phosphatases were also been determined by Western blotting.The study first revealed that compared with C57BL/6J mice,hSlit2 gene was highly expressed in the hearts of Slit2-Tg mice,but did not significantly change cardiac function and tissue structure.Secondly,we found that hSlit2 overexpression exerted protective effects on myocardial IRI in elevating the hemodynamic parameters,reducing infarction area,alleviating mitochondrial edema,and maintaining the myofilament array and structural integrity after reperfusion.Finally,our study indicated that compared with C57BL/6J mice,the phosphorylation levels of site Ser19 on MLC2 in Slit2-Tg mice were upregulated after reperfusion(1.586 ± 0.190 vs.1.143 ± 0.028,P<0.05),whereas the phosphorylation levels of site Ser43 on TnI were downregulated(0.837 ± 0.077 vs.1.098 ± 0.080,P<0.05).In addition,compared with C57BL/6J,PKC? expression was downregulated in Slit2-Tg mice(1.369 ±0.072 vs.0.415 ± 0.043),and protein phosphatase PP1?(1.369 ± 0.072 vs.0.809 ± 0.094,P<0.01)and PP2A(1.388 ± 0.123 vs.1.09 ± 0.047,P<0.05)were upregulated.Based on the above findings,three main conclusions were drawn: 1)the morphology,structure,and function of the hearts of Slit2-Tg are comparable to that of C57BL/6J mice;2)Slit2 overexpression significantly improves systolic function after reperfusion and maintains the structural integrity of the heart;3)Compared with C57BL/6J mice,the Slit2-Tg mouse hearts resist to injury insults after reperfusion,associated with upregulation of protective signals PP1,and PP2 A and downregulation of the detrimental signal PKC?.This study uncovered the protective mechanisms of Slit2 in myocardial IRI,indicating that Slit2 can be a potential therapeutic target of IHD. |
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