The Mechanism About The Inhibition Of Klotho Protein On Stress-induced Myocardial Injury

Background：Cardiac remodeling is the common pathological alteration in heart responsing tostimuli, which is charactered by the change of the structure, volume and function of heart.Chronic cardiac remodeling finally induced the decline of cardiac diastolic and systolicfunction. Since the global aging population increasing, the incidence and mortality of heartfailure increase year by year, which is a major disease affecting the life quality and survivalof the older. Cardiac remodeling after myocardial infarction is one of the most main cause.Although the development of clinical medical level has saved the dying myocardium at agreater degree, such measures still can’t completely reverse the development of ventricularremodeling and heart failure. Researchers show that neurohumoral factors play animportant role in the pathological process of ventricular remodeling. At early stage, theactivation of β-adrenergic receptor can enhance contraction of myocardial responsing to thechange of blood flow. However, long-term activation of β-adrenergic receptor inducespathological myocardial hypertrophy, apoptosis and interstitial fibrosis, and finally resultsin cardiac dysfunction. Isoproterenol, as β-adrenergic agonists, is commonly used as anideal inducer for cardiac injury, which includes myocyte hypertrophy, apoptosis andinterstitial fibrosis. To explore the key factor reversing the above pathological changes is ofgreat significance in the reduction of heart failure and sudden death.Klotho protein is a newly discovered anti-aging factor, which plays biological effectsthrough the membrane-bound type and secreted type. Based on the literature and previousresearches, we speculated that Klotho protein can play a cardiovascular protection role.Firstly, Klotho protein can resist to oxidation stress, which is widely involved in thedevelopment of many disease, including ventricular remodeling. Secondly, Klotho proteincan increase the biological activity of nitric oxide (NO), or regulate the calcium ionchannels on vascular endothelial cells to maintain endothelial integrity and endothelialfunction. Thirdly, Klotho protein control hypertensive, and the increased pressure load is the main cause of chronic hear failure. In addition, Klotho protein inhibits endothelial cellapoptosis and inflammation. Clinical studies show that low level of plasma Klotho proteinmay be related to cardiovascular disease induced by chronic kidney disease, our previouswork also found the single nucleotide polymorphisms in Klotho gene are closely associatedwith cardiovascular diseases such as arteriosclerosis, so it is reasonable to speculate thatKlotho protein can inhibit ventricular remodeling. Recent study found that ISO can result ina significant reduction of the cardiac systolic function in mice, and the effect can beenhancedin Klotho deficient mice, suggesting that Klotho protein plays a protective role oncardiac systolic function.Myocardial cell apoptosis is an important pathological change of ventricularremodeling, which is the key factors that lead to cardiac dysfunction and even sudden death.In addition to mitochondria, endoplasmic reticulum is another important organ involved inapoptosis, whose main functions include regulating calcium level, protein folding andmature. When the balance of the endoplasmic reticulum was disrupted (such as damagedcalcium level, excessive generation of protein or protein misfolding), it would induce theexpression of endoplasmic reticulum chaperone such as glucose regulatory protein78andheat shock protein47，and this process is known as endoplasmic reticulum stress. At theinitial stage, endoplasmic reticulum stress is the body’s adaptive response to externalstimuli, which will help to maintain the balance of internal environment. Chronicendoplasmic reticulum stress will lead to the activation of apoptosis signaling pathways,including the induction of pro-apoptosis transcription factors CHOP. CHOP signalingpathway is one of the key apoptotic signaling pathways mediated by endoplasmic reticulumstress. Endoplasmic reticulum stress mediated apoptosis is the hot studying field. A largenumber of experiments showed that excessive or chronic ER stress can lead to the apoptosisof myocardial cells in vivo and in vitro. Studies reported that ISO could induce apoptosis ofcultured myocardial cell accompanied by the increased expression of the endoplasmicreticulum chaperone and pro-apoptosis transcription factors CHOP. And the inhibition ofendoplasmic reticulum stress can prevent cardiomyocytes apoptosis. And whether theprotection of cardiac systolic function by Klotho protein is related to CHOP-mediatedapoptosis signal induced by ER stress has not been reported.Cells respond to external stimuli mainly through intracellular signaling pathways. Mitogen-activated protein kinases is an ubiquitous intracellular signaling pathways,including extracellular signal regulated kinase1/2, p38and c-Jun NH2-terminal kinase.Mitogen-activated protein kinases play an important role in cells’ proliferation,differentiation, and it also involves in ER stress-mediated apoptosis. Whether Klothoprotein regulates ER stress mediated apoptosis through these signal pathways has not beenfully elucidated and need further research.Objective：This experiment made anti-apoptosis effect of Klotho protein as the breakthroughpoint to explorer the effect of Klotho protein on ventricular remodeling especiallymyocardial cell apoptosis based on the construction of ventricular remodeling mouse model.And further study was performed to define whether the anti-apoptotic mechanism of Klothoprotein was related with ER stress mediated apoptosis signal. Finally, it intended todetermine whether Klotho inhibited ER stress mediated apoptosis through regulation ofMAPK signal pathway through signal study.The main experimental methods and results:1. PART ONE:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into control group and ISO group. CON group: subcutaneous injection of100ulsaline solution, total9days; ISO group: subcutaneous injection of5mg/kg ISO, dissolved in100ul saline solution, total9days. At the9thof ISO injection, mice were weight on theelectronic balance, and then execute them and remove their hearts gently. Finally wecalculate the ratio of HW/BW. Cut each cardiac tissue into6pieces,3piece of whichpacked with tinfoil, and then put them into the frozen storage tube, preserved in liquidnitrogen tank to performe quantitative PCR test. The rest of the myocardial tissue soak in4%paraformaldehyde solution for24hours, and then embed with paraffin.4um paraffinsection was cut on slicer. Serial4um paraffin sections were used to performe HE staining,Masson staining, Tunel staining for morphological analysis, quantitative analysis ofcollagen and apoptosis. Results indicated that subcutaneous injection of ISO could causecardiomyocytes hypertrophy and the disorders of muscle fibers. By measuring the ratio ofHW/BW, we also found that ISO could increase the ratio of HW/BW statistically, whichwas closely related to cardiomyocytes hypertrophy. HE staining technology found thatcontinuous subcutaneous injection ISO daily (9days) could cause myocardial cell hypertrophy and make muscle fibers arranged in disorder. In addition to the pathologicalchanges of myocardial cells, we also found that fibroblast infiltration and proliferationsignificantly increased in ISO group, suggesting the subcutaneous injection of ISO inducedmyocardial interstitial fibrosis. And Masson staining found that the collagen volume ofmyocardial interstitium in ISO group wss significantly higher than the control group, whichindicated that ISO could induced myocardial fibrosis successfully. Real time PCR wasperformed to detect college mRNA levels in the control group and the ISO group. Resultfound that ISO could increase the college mRNA level. Subcutaneous injection of ISO canincrease the rate of myocardial cell apoptosis. Tunel staining found that the apoptosis rateof myocardial cells in ISO group was higher than in the CON group. Therefore ISO, as abeta-adrenergic agonist, can be used to construct the ventricular remodeling mouse modelsuccessfully.2. PART TWO:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into the following groups. CON group: subcutaneous injection of saline solution(100ul, qd, a total of9days), and intraperitoneal injection of saline solution (100ul, qod, atotal of4days). ISO group: subcutaneous injection of ISO (at the concentration of5mg/kg/d, qd a total of9days), and intraperitoneal injection of saline solution100ul, qod, atotal of4days). ISO+KL group: subcutaneous injection of ISO (at the concentration of5mg/kg, a total of9days), and intraperitoneal injections of Klotho protein (at theconcentration of0.01mg/kg, qod, a total of4days). Mice were decapitated at2,5,9day afterISO injection. We determine the serum Klotho protein levels in mice after intraperitonealinjection of Klotho protein by ELISA technology. And HE staining, Massonn staining andTUNEL staining were performed to determine the pathological changes of heart tissue.ROS detection technology was performed to detect the ROS production in heart.Immunohistochemical staining, Western blot and Real-time PCR was used to determine theprotein level and mRNA level of ER stress related protein GRP78, HSP47andpro-apoptosis transcription factors CHOP. At the same time, H9c2cells were cultured toobserve the effect of Klotho protein on ISO induced cardiacmyocytes injury. Resultsshowed that serum Klotho protein level was increased significantly after the injection ofKlotho protein (P <0.05). We observed that Klotho protein inhibit the pathologic changesin heart tissue induced by ISO. In the control group, myocardial fibers were normally arranged, however cardiac tissue showed irregular myocardial fibrosis and fibroblastshyperplasia after ISO injection, charactered by increased nucleus density and mononuclearcell infiltrates. These suggested serious pathological changes in the ISO group. However,Klotho can relieve the above change, suggesting cardiac protective effect of Klotho protein.Masson stain experiments showed the collagen of interstitial and perivascular issignificantly higher from5days after the injection of ISO in ISO group than in CON group.However Klotho can reduce myocardial fibrosis, showed that Klotho proteins inhibit ISOcaused cardiac structural changes. TUNEL staining experiments showed that Klotho proteincould reduce ISO-induce apoptosis of myocardial cells. At9th day after ISO stimulation,ISO significantly increases the percentage of TUNEL positive cells. The Klotho proteinscan significantly reduced the percentage of TUNEL positive cells, with statistical difference(P <0.05). In accordance with the result, the generation of ROS in ISO group increasedsignificantly, and it was significantly inhibitte in ISO+KL group. Further research showedthat anti-apoptotic effect of Klotho protein was related to the inhibition of ER stress-relatedapoptotic signals in heart tissue. Immunohistochemistry, Western blot and Real-time PCRtechniques had confirmed that Klotho proteins could significantly reduce the protein leveland mRNA level of ER chaperonin GRP78, HSP47and CHOP. In vitro study had shownthat10um ISO induced a significant increase in myocardial cell apoptosis, and Klothoprotein significantly inhibit H9c2cell apoptosis induced by ISO. The study also found thatISO promote the generation of ROS, and Klotho inhibited the generation of ROS. And1ug/ml Klotho proteins can significantly suppress the ER stress protein markers GRP78HSP47and CHOP expression induced by the ISO statistically. So Klotho can improve thepathological change of heart through inhibiting ER stress-mediated apoptosis.3. PART THREE：Cultured H9c2cells were passaged into6-well plates when cells reachedto80-90%confluence. In order to determine the role of MAPK signaling pathways in theinhibition of endoplasmic reticulum stress mediated apoptosis by Klotho, different MAPKinhibitors was used to stimulate H9c2cells. Then they were randomly divided into differentgroups: ISO group (2ml DMEM), ISO+KL group (2ml DMEM containing0.1ug/mlKlotho protein), ISO+SB20380(2ul SB203580+2ml DMEM), ISO+PD98059(10ulPD98058+2ml DMEM), ISO+SP600125(10ul SP600125+2ml DMEM). They wereincubated in an incubator for1h, and then10uM ISO were added to each group. Flow cytometry and Western blot technique were performed to detect cell apoptosis and theexpression of ER stress-related proteins. At the same time, in order to detect the directimpact of Klotho protein of MAPK signal pathway, the cells were exposed to ISO and/ordifferent concentrations of Klotho protein (0.01ug/ml,0.1ug/ml,1ug/ml,10ug/ml).Western blot technique was applied to detect the phosphorylation of P38MAPK, JNK andERK1/2. The results showed that SB203580and SP600125reduced H9c2cell apoptosislike Klotho protein, but PD98059did not affect the apoptosis significantly. In addition, theexpression of GRP78, HSP47and CHOP can be inhibited by Klotho, SB203580orSP600125statistically, but not significantly inhibited by PD98059.These results indicatedthat Klotho may inhibit cell apoptosis through P38MAPK and JNK.10uM ISO can activatethe MAPK signaling pathway in H9c2cells, and different concentrations of Klotho(0.01-10ug/ml) does not significantly affect the level of phosphorylated ERK1/2, but0.1ug/ml and1ug/ml of Klotho significantly stimulated the phosphorylation of JNK andP38MAPK. The results showed that the anti-apoptosis effect of Klotho was related to thedownregulation of P38and JNK pathway.Conclusion:1. Subcutaneous injection of ISO can induce myocardial hypertrophy, apoptosis,muscle fiber disorder and interstitial myocardial fibrosis, indicating that subcutaneousinjection of ISO can successfully construct ventricular remodeling mouse model whichcan effectively simulate the pathological process of chronic heart failure；2. Klotho protein inhibited the development of chronic heart failure in ISO-inducedBalb/C mouse model. Importantly this study confirmed that Klotho protein can reduce theISO-induced apoptosis and the expression of endoplasmic reticulum stress-related proteinGRP78, HSP47and pro-apoptosis transcription factor CHOP in heart tissue and culturedcardiomyocytes, indicating the anti-apoptosis role of Klotho protein was related with theinhibition of ER stress-related apoptosis signal pathway;3. P38inhibitor, SB203580and JNK inhibitor, SP600125have the same anti-apoptosiseffect with Klotho protein in ISO-stimulated H9c2cells, however ERK1/2inhibitor,PD98059did not affect the apoptosis significantly. Klotho protein (0.01-10ug/ml)significantly inhibited the phosphorylation of JNK and P38, but did not significantly affectthe phosphorylation of ERK1/2, suggesting that anti-apoptosis effect of Klotho protein was partially dependent on the inhibition of JNK and P38MAPK signaling pathway.