| Background:Acute myocardial infarction is now recognized as the most major cause to result in death in cardiovascular diseases. During the past two decades, the AMI reperfusion therapy experienced from drug thrombolytic therapy to percutaneous coronary intervention (Percutaneous Coronary Intervention, PCI) and coronary artery bypass graft surgery (Coronary Artery Bypass Grafting, CABG),got a rapid development and highly improve the patient’s life quality in a great extent. But at the same time, it also brings a potential threat we can not be ignored:subsequent myocardial ischemia-reperfusion (I/R) injury. IRI definition was first proposed in1960by Jennings: when ischemic myocardium regain perfusion or oxygen supply, would be exacerbated the original ischemic tissue damage. The major clinical manifestations are reperfusion arrhythmia; myocardial stunning, microvascular stunning and myocardial structural changes, ultimately resulting in myocardial irreversible damage necrosis, recent researches now indicate that there are various factors be involved in the pathophysiological process of IRI, for example, free radical injury, the effects of leucocytes,calcium overload and microvascular injury. Large amount of the oxygen free radicals (oxygen free radical, OFR) is the most injury factor involved in I/R.OFR act directly on the nucleus DNA, cause to gene mutations and chromosomal aberrations; decrease myocardial cell membrane fluidity; make the extracellular matrix degradate and; damage intracellular protein, ultimately leading to cell apoptosis and necrosis. Therefore, seek a positive and effective way to resist oxidative stress will be the new trend in clinical treatment during I/R injury.Klotho is a gene that encodes a novel protein regulating multiple functions, first discovered in1997by Kuro-o and his colleagues. There are a high degree of homology between the rat, mouse and human, it resides on chromosome13q12with a size of over50kb. Klotho is mainly expressed in the renal tubules and brain, but it also exists as circulating soluble form detectable in the blood, with systemic effects. Klotho gene plays a critical role in regulating aging and the development of age-related diseases in mammals.lt function as a multi-functional humoral factor that influences multiple biological processes.(ⅰ)Klotho inhibits insulin and IGF-1signaling,increases the resistance to oxidative stress,(ⅱ) Klotho may protect the cardiovascular system by increasing NO production and inhibiting oxidative stress.(iii)Klotho involves in organ protection and influences several intracellular signaling pathways. In a recent research suggested that klotho could reduce apoptosis in experimental ischaemic acute renal failure and has therapeutic potential in managing ischaemic renal damage.Then, may it also have some beneficial function in ischaemic cardiac injury? Therefore, in this paper we aim to construct recombinant adenovirus-klotho and to explore its role on myocardial IRI in rats.Methods:Part one:Neonatal ventricular myocytes were prepared from the hearts of1-3day SD rats were randomly distributed in different experimental groups as follows:1) control group:cardiomyocyte were seeded in6-well plates with aerobic Tyrode’s solution during whole experiment period.2) H/R group:cardiomyocyte were seeded in6-well plates incubated with anaerobic Tyrode’s solution for3h hypoxia followed by2h reoxygenation.3) klotho group:cardiomyocytes were incubated with0.1-0.4ug/ml Klotho protein for16h then subjected to3h hypoxia/2h reoxygenation as described above. Cell counting kit (CCK)-8assays were used for the cell viability assay;In order to evaluate cardiomyocyte damage, cell viability, lactate dehydrogenase (LDH) release levels were measured using an activity assay; Cell apoptosis was measured by flow cytometry; The2’,7’-dichlorofluorescein diacetate (DCFH-DA) reagent was used to estimate the intracellular generation of ROS; immunofluorescence analysis were used to test whether the Klotho-induced decrease in FOXO phosphorylation associated with its nuclear translocation; immunoblot analysis performed for the detection of FOXO1, p-FOXOl; Akt, p-Akt and SOD2protein expression, respectively.Part two:AgeI digestion GV135plasmid vectors by aims bands. PCR and restriction enzyme digestion were use followed the method of recombinant plasmid was identified GV135-Klotho. Then, use automatic DNA sequencing analyzer to confirm that the fragment connected correctly. Then the competent cells were prepared for producing large scale of plasmid DNA. Choose the293cells in a growth state and plasmids were transfected into the cells to produce recombinant adenovirus Ad-kl. Calculate the virus titer after amplification and purification the Ad-k1. The final virus titeris3×1011PFU. Part three:Adult male SD rats were randomly divided into4groups: sham-operated rats treated with sham operation (sham group,), I/R+NS group n=15), I/R+Ad-GFP virus (Ad-GFP group, n=15) and I/R+Ad-klotho (Ad-klotho group, n=15). Process of I/R is:operated rats were subjected to30min ischemia followed by24h reperfusion. Either ad-kl or adenovirus was intravenously administered at a dose of1.6x1010pfu to rats3days before the IRI procedure.The rats of SH groups recepted only clamping in ramus descendens anterior arteriae coronariae sinistrae. Transfection effeciency was investigated by fluorecent microscope. The left artrium samples were used to determine the infarction area by Evans blue/TTC staining; the blood of cardial chambers were collected for LDH.. CK-MB examination; cellular apoptosis was evaluated histological TUNEL staining. The mRNA and protein level of Klotho were detected by RT-PCR and Western blot.Results:Part one:In vitro experiments:, Cell viability was significantly decreased in H/R group, the level of LDH in the culture medium of cardiomyocytes were significantly increased and cell apoptosis rate were enhanced compared with control group. While compared with H/R group, cellular survival rate of H/R+klotho group was significantly higher than that of HR group and Ad group (P<0.01); treatment with klotho protein showed a significant resistance in apoptosis in cardiomyocytes and the release of LDH decreased. Cardiomyocyte ROS in the H/R group was significantly elevated above control group levels (P<0.05). After processing with klotho (0.1,0.2or0.4ug/ml), endocellular ROS significantly deceased compared with those in the H/R group (P<0.05) and were similar to those in control group. From immunoblotting analysis, we confirmed that klotho protein increased FOXO1levels in the nuclear fraction and decreased FOXO1levels in the cytoplasm. Furthermore, immunofluorescence confirmed that the addition of exogenous Klotho protein promoted translocation of FOXO1from the cytoplasm to the nucleus. In addition, the administration of Klotho protein suppressed the basal level of phosphorylation FOXO1and Akt. Phosphorylated levels of FOXO1and Akt were significantly down-regulated and Klotho prominently increased the expression levels of SOD2protein.Part two:Recombinant Ad-Klotho was constructed successfully. Klotho gene was proliferation of new recombinant Ad-klotho, was amplified and by HEK293cells and purified. The virus titer was measured3×1011pfu/ml. Klotho mRNA and protein were detected3days after transfection indicating that Klotho gene overexpressed stably in SD Rat.Part three:In vivo:By fluorecent microscope, Frozen section samples of myocardialm, liver and kidney tissues in I/R+Ad and I/R+Ad-klotho group were detected GFP positive with transfection rate of Ad53.1±2.3%and56.7±4.0%. After IRI procedure, the expression of klotho mRNA and protein in I/R+Ad-klotho group were significantly higher than those of NS group and Ad group. Compared with sham group, in I/R+NS、I/R+Ad and I/R+Ad-klotho group:the levels of LDH and CK-MB in blood serum were significantly increased; infarct size and the index of apoptosis were markedly increased. All these change indicated the success of model construction.Tissue samples of I/R+Ad-klotho groups showed less pathological changes, less ISW and ISW/AARW (P<0.01). The release of LDH、CK-MB and index of apoptosis decreased.Klotho gene overexpressed suppress the activation of Akt; induced a markedly inhibition of NF-kB protein levels.Conclusions:1. The present study successfully constructed the in vitro myocardial cells hypoxia and re-oxygen model. Treatment with klotho protein have beneficial effect on cardiomyocytes undergoing H/R injury, the mechanism of this effect might be through suppressin of apoptosis,inhibit Akt phosphorylation and prevent Foxo1nuclear transfer.2. Reconstructed adenovirus vector Ad-Klotho was successfully constructed.3. Klotho gene was effectively transfected into IRI model rats mediated by adenovirus injection. Klotho gene transfection had significant protective effects on Ischemia Reperfusion Injury of myocardium in rats.4. The present studies potentialized the role and mechanism of Klotho gene on IRI. Reveal a potentially novel mechanism for age-related cardiovascular disease. |
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