Study On The Role Of UQCRC1Overexpression In Cardioprotection And Its Signal Pathways

Although a great deal of effort has long been sought to provide better protection forpatients with cardiovascular disease against perioperative myocardial ischemia/reperfusioninjury, it is always difficult to achieve the goal. There is increasing evidence that upregulationof ubiquinol-cytochrome c reductase core protein1(UQCRC1) may take part incardioprotection. However, the specific role of UQCRC1and its underlying mechanismsstill remain unclear.Objective:To investigate the role of UQCRC1in cardioprotection and its possible mechanisms ofprotection against ischemia/reperfusion injury in cardiomyocytes, so as to provide a noveltarget for developing specific cardioprotective drugs.Methods:Overexpression of UQCRC1gene and protein in H9c2cardiac cells was establishedthrough constructing a recombinant adenovirus vector containing UQCRC1. Thecardioprotective role of UQCRC1overexpression and its possible signal pathways in H9c2cardiac cells were investigated by utilizing exogenous Zn2+, Zn2+-selective chelatorN,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), signal pathways specificinhibitors such as inhibitor of phosphatidylinositol3-kinase (PI3K) LY294002and inhibitorof extracellular-signal-regulated kinase (ERK) PD98059before oxygen-glucose deprivation(OGD) injury or hydrogen peroxide damage, followed by detection of cell viability, cellapoptosis rate, mitochondrial membrane potential, mitochondrial free Zn2+concentration andthe expression of relative signal proteins as well as apoptosis-related proteins with thosetechniques such as fluorescence labeling, immunofluorescence staining, western blotting andso on.Results: 1. Both the recombinant adenovirus vector containing UQCRC1and overexpression ofUQCRC1in H9c2cardiac cells were successfully constructed.2. Overexpression of UQCRC1protected H9c2cardiac cells against OGD andperoxidation injury. On one hand, UQCRC1overexpression markedly upregulated theexpression of antiapoptotic protein Bcl-2, downregulated the expression of proapoptoticprotein Bax, decreased the expression of active caspase3and increased cell viability afterOGD injury. On the other hand, UQCRC1overexpression obviously attenuated cellapoptosis and loss of mitochondrial membrane potential both induced by peroxidationinjury.3. Exogenous Zn2+significantly enhanced the protective effects of UQCRC1overexpression, while the Zn2+-selective chelator TPEN completely abolished the protectiveeffects. Furthermore, upregulation of UQCRC1leaded to lower concentration of free Zn2+inmitochondria, while downregulation of UQCRC1resulted in higher concentration of freeZn2+in mitochondria. Taken together, Zn2+is an essential cofactor for the cardioprotectiverole of UQCRC1overexpression, while UQCRC1is a key binding site for Zn2+inmitochondria.4. UQCRC1overexpression upregulated the expression of antiapoptotic protein Bcl-2,downregulated the expression of proapoptotic protein Bax, inhibited the activation ofcaspase3apoptotic signal pathway, increased cell viability, maintained mitochondrialmembrane potential and reduced cell apoptosis after OGD or hydrogen peroxide injurythrough activating PI3K/Akt/GSK-3β signal pathway.(1) Inhibition of PI3K completely abolished the protective role of UQCRC1overexpression in cell viability, cell apoptosis and mitochondrial membrane potential afterOGD or hydrogen peroxide injury in H9c2cardiac cells.(2) After OGD injury, UQCRC1overexpression dramatically increased thephosphorylation of PI3K downstream signal molecules including protein kinase B (PKB,also named Akt) and glycogen synthase kinase-3β (GSK-3β), which was completelyabolished by inhibition of PI3K.(3) UQCRC1overexpression regulated the expression of apoptosis-related proteinsafter OGD injury to reduce cell apoptosis. UQCRC1overexpression decreased activecaspase3expression. Moreover, UQCRC1overexpression upregulated the expression ofBcl-2protein, downregulated the expression of Bax protein and reduced the Bax/Bcl-2ratio. Inhibition of PI3K completely abolished the effect of UQCRC1overexpression on activecaspase3expression, also abolished the effects both on upregulation of Bcl-2expressionand on downregulation of Bax expression.5. UQCRC1overexpression protected cardiomyocytes partially through activatingERK/GSK-3β signal pathway.(1) Inhibition of ERK partially abolished the protective effects of UQCRC1overexpression on OGD and hydrogen peroxide injury in H9c2cardiac cells. Inhibition ofERK partially abolished the protective role of UQCRC1overexpression in cell viability, cellapoptosis and mitochondrial membrane potential after OGD or hydrogen peroxide injury.(2) Inhibition of ERK partially abolished the effect of UQCRC1overexpression on thephosphorylation of GSK-3β.(3) Inhibition of ERK partially abolished the effect of UQCRC1overexpression onactive caspase3expression, also partially abolished the effects both on upregulation ofBcl-2expression and on downregulation of Bax expression.Conclusions:1. Overexpression of UQCRC1protects H9c2cardiac cells from ischemia/reperfusioninjury and peroxidation injury.2. Zn2+is an essential cofactor for the cardioprotective role of UQCRC1overexpression,while UQCRC1is a main binding site for Zn2+in mitochondria.3. UQCRC1overexpression can protect H9c2cardiac cells from injury throughactivating PI3K/Akt/GSK-3β signal pathway and partially through activating ERK/GSK-3βsignal pathway.4. Cardioprotective role of UQCRC1and its signal pathways in H9c2cardiac cellshave been demonstrated for the first time. UQCRC1may be another novel protectiveprotein against ischemia/reperfusion injury and peroxidation injury, which may provide anew target for developing specific cardioprotective drugs.