| Myocardial ischemia-reperfusion injury is reversible ischemic injury of myocardial cells transformed into irreversible when restoration of blood supply to myocardial cells. Now myocardial reperfusion injury become an obstacle for coronary thrombolysis and coronary artery bypass surgery application. As we know, the main mechenism of myocardial ischemia reperfusion injury are energy metabolism, free oxygen radicals and calcium overload formation. Energy metabolism is the initial factor, free oxygen radicals is an important part of ischemia-reperfusion injury, and calcium overload is a irreversible pathway in the final injury of ischemia-reperfusion.Clinical and experimental studies have demonstrated that, calcium overload involved in myocardial ischemia-reperfusion injury, and plays a leading role in the pathogenesis of myocardial ischemia reperfusion injury. Calcium overload is induced by intracellular Ca2+equilibrium disorder. As a second messenger, calcium plays important roles in cell communication and injury. In resting state, the extracellular concentration of free Ca2+is0.1-10mmol/L, while the intracellular concentration is only0.1μmol/L, mainly distribute in the nucleus, mitochondria, endoplasmic reticulum/sarcoplasmic reticulum and the plasma membrane. When small amounts of extracellular Ca2+into the intracellular or calcium store release slightly increased, cytoplasmic Ca2+may significant increase, leading a series of physiological and biochemical reactions, such as cell structural damage, apoptosis, death and cell degeneration, etc., So maintaining intracellular calcium homeostasis is very important.According to the present study that calcium overload occurs mainly in the reperfusion period. Calcium overload can lead to ventricular diastolic dysfunction, even caused diastolic heart failure; calcium overload can cause acidosis and arrhythmia; Strong contraction band, muscle fiber rupture and necrosis appear during reperfusion; The protease and calcium-dependent phospholipase can be activated by calcium overload and undermine the structural integrity of the biofilm, and produce lysophosphatidic in the membrane phospholipid decomposition process into the mitochondria to inhibite ATP synthesis; In addition, a large number of Ca2+in the form of calcium phosphate deposits in the mitochondria, damage the mitochondrial oxidative phosphorylation. Sjaastad’s studies showed that survival cells after myocardial infarction more prone to take place calcium overload, and therefore more vulnerable to sufferischemia-reperfusion injury.Intracellular calcium is an important second messenger in circulatory system, can regulate diverse cellular functions and maintain normal cells state, it is also a media for information exchange between cells and with the outside world. In the normal state, redundancy calcium can be pumped out by mitochondrial membrane potential proton pump electronic intracellular transport system to maintain normal intracellular calcium storage levels. However, in certain pathological cases, such as myocardial ischemia-reperfusion injury (MRI), stable state was broken up, resulting in the accumulation of intracellular calcium up to calcium overload, eventually leading to cell death. ObjectiveBased on the above data, calcium overload is one of the main mechanism of MRI, and Wnt5a/Frizzled-2signaling pathway is related with intracellular calcium, then is there any connection between them? Whether Wnt5a/Frizzled-2signaling pathways activted after myocardial ischemia reperfusion injury, and involved in this Pathological process?Methods and ResultsAccording to our hypothesis, in vitro and in vivo models were used to vertify Wnt5a/Frizzled-2signaling patyway existed in myocardial cells and involved in the mechanism of myocardial ischemia reperfusion injury.Part one:To determine whether Frizzled-2and Wnt5a were expressed in H9c2cells, we examined their expressions in non-transfected (normal) H9c2cells by Q-PCR and Western blot. We found that Frizzled-2and Wnt5a were weakly expressed through both Q-PCR and Western blot detection. Next, to examine the roles of Frizzled-2in Wnt5a signaling and calcium inflow, we transfected rat H9c2cells with synthesized frizzled-2plasmid and then analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot. When the cells were transfected, we observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately12-fold and10-fold, respectively, in comparison to that in non-transfected cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately8-and3-fold, respectively, when compared with that in non-transfected cells (P<0.001). In addition, it is known that phosphorylation of CaMK II indicates the action of the frizzled-2mediate Wnt5a signaling pathway [35], and we found that p-CaMK Ⅱ was also significantly increased4-fold after the transfection (P<0.001;), compared to non-transfected cells.To identify whether activation of the Wnt5a/Frizzled-2pathway regulates Ca2+ release from intracellular stores, we examined whether changes in intracellular Ca2+inflow was affected by frizzled-2-transfection, indicated by the intensity of fluorescence of fluo-3/AM under confocal microscope after transfection. We found that the intensity was significantly increased2.5-fold after the transfection (P<0.001) when compared to the normal cells. This indicated that intracellular Ca2+inflow was increased in frizzled-2-transfected H9c2cells.As described above, we showed that the expression of Wnt5a was closely associated with the expression of the Frizzled-2receptor, and that cells transfected with frizzled-2plasmid dramatically increased intracellular Ca2+inflow. Nevertheless, we could not draw a conclusion that Frizzled-2transduces binding of Wnt5a and leads to the intracellular Ca2+release. To resolve this problem, a special inhibitor called stealth RNAi was used to block the expression of the frizzled-2gene. Compared to the treatment without stealth RNAi, we found that the treatment with stealth RNAi significantly inhibited both gene and protein expression of Frizzled-2in transfected H9c2cells nearly to the control level, as demonstrated by Q-PCR and Western blot (P<0.001). Moreover, the expressions of the Wnt5a gene and protein were also markedly downregulated in transfected H9c2cells following the suppression of the frizzled-2gene by the treatment of stealth RNAi (P<0.001), compared to that without this treatment. To examine whether the expression of p-CaMKⅡ was blocked by the inhibition of frizzled-2signaling, we detected its expression by Western blot after application of stealth RNAi. We found that the treatment of stealth RNAi significantly inhibited the expression of p-caMK Ⅱ by1.5-fold in frizzled-2transfected H9c2cells, indicating that the activation of the Wnt5a/Frizzled-2pathway was suppressed by blocking frizzled-2.To examine whether intracellular Ca2+inflow was affected by blocking frizzled-2signaling, we measured the intensity of fluorescence of fluo-3/AM under confocal microscope after application of stealth RNAi. We found that stealth RNAi dramatically decreased the intensity of fluorescence in frizzled-2transfected H9c2cells, compared to that without the treatment. Taken together, we can draw a conclusion that the Wnt5a/Frizzled-2pathway exists in rat H9c2cells and activates Ca2+release from intracellular stores.Part two:Ca2+overload is known to be an important mechanism after I/R. In the adult heart and blood vessels, Wnt signaling activity is quite low under normal conditions. However, this pathway is reactivated during the pathological cardiac remodeling induced by pressure overload, in injured arteries and after myocardial infarction. In an initial study by Blankesteijn, et al, frizzled-2mRNA levels were found to be gradually upregulated in the first10days after myocardial infarction in the rat. Therefore, we used a rat I/R model to test whether the Wnt5a/Frizzled-2signaling pathway relative elements upregulate in injured cadiocyte. Firstly, we detected the expression of Frizzled-2and Wnt5a in normal rat H9c2cells on both the gene and protein level. We found that gene and protein expressions of both Frizzled-2and Wnt5a were expressed in normal cadiocyte. However, after I/R, gene expressions of frizzled-2and Wnt5a were significantly elevated approximately8-and3-fold, respectively, in the cadiocyte, when compared with that in normal H9c2cells (P<0.001and P<0.05, respectively). Protein expressions of Frizzled-2and Wnt5a were also elevated by4-and6-fold in cadiocyte, respectively, when compared to that in normal cadiocyte (P<0.001).While p-CaMKII was expressed at a very low level in normal cadiocyte, however, its expression was markedly increased approximately7-fold in the cadiocyte after I/R (P<0.001).Because the mechanism of intracellular Ca2+accumulation after I/R are complicate, and hard to examine whether Wnt5a/Frizzled-2pathway contributes to the Ca2+accumulation after in vivo. So we choose to verify our hypothsis in pure populations of these cells.Part three:we used H/R to mimic I/R in vivo [35,36]. Following H/R, We analyzed the expression of Frizzle-2and Wnt5a collected from Q-PCR and Western blot, observed that gene expressions of both frizzled-2and Wnt5a were significantly increased approximately8-fold and3-fold, respectively, in comparison to that normal cells (P<0.001). Moreover, protein expressions of both Frizzled-2and Wnt5a were significantly increased approximately7-and2-fold, respectively, when compared with that in normal cells (P<0.001).We found that the Ca2+intensity was significantly increased2-fold after H/R (P<0.001) when compared to nomal cells. This indicated that intracellular Ca2+inflow was increased in H/R treatment H9c2cells.In addition, we found that p-CaMK II was also significantly increased4-fold after the H/R (P<0.001), compared to non-transfected cells.To determine the interaction between H/R-induced activation of Wnt5a/Frizzled-2signaling and accumulation of intracellular Ca2+, we suppressed the expression of the frizzled-2receptor gene by transfect with stealth RNAi before H/R, and detected the effects of stealth RNAi on gene and protein expression of Frizzled-2and Wnt5a after H/R. we observed a significant inhibitory effect on gene expression of frizzled-2in the transfected H9c2cells, compared with that in cells without transfect with stealth RNAi, as demonstrated by the fact that gene expression of frizzled-2was inhibited nearly6-fold. Surprisingly, the expression of the Wnt5a gene was also downregulated nearly3-fold (P<0.001). In addition, protein expressions of Frizzled-2and Wnt5a in transfected cells was significant decreased when compared to cells without transfection (P<0.001). Encouraging, the intensity of Ca2+fluorescence was significantly decreased in transfected cells after the H/R, when compared to those without transfection(P<0.001).Moreover, p-CaMKII in transfected cells was also inhibited by nearly3-fold, compare to only H/R treatment cells (P<0.001).Conclusion and SignificanceAccording to the results from this study we can draw the following conclusions:(1) Wnt5a/Frizzled-2signaling pathways exist in mammals myocardial cells;(2) Expression of Frizzled-2and Wnt5a were upregulated after myocardial ischemia reperfusion injury.(3) We used hypoxia/reoxygenation to mimic myocardial ischemia-reperfusion injury in H9c2cells can activate Wnt5a/Frizzled-2signaling pathway. And found out that this pathway contribute to calcium overload after hypoxia/reoxygenation. In other words, Wnt5a/Frizzled-2signaling pathway was activated after hypoxia/reoxygenation, may may be one of the mechanisms of calcium overload after myocardial ischemia-reperfusion injury.(3) This study tested the reliability of Stealth RNAi gene silencing, it has high inhibition efficiency;By research on Wnt5a/Frizzled-2signaling pathways in myocardial ischemia reperfusion injury, may contribute to understanding the futher mechanism of reperfusion injury, will provide a new potential therapeutic target for treatment and research. |
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