Study On The Roles And Mechanisms Of Melatonin In Facilitating Stem Cells-Mediated Repair Of Myocardial Ischemic Injury

Backgrounds and aimsMyocardial infarction（MI）and post-infarction chronic heart failure（CHF）remain the major health burden worldwide.In the absence of reperfusion,sustained myocardial ischemia leads to progressive necrosis of cardiomyocytes,which reduces the contractile function of the heart and eventually leads to CHF.Current drug treatments can only delay or at best halt the progression of CHF,but do not restore the function of cardiac tissue completely.Therefore,the clinical demand for the treatment of heart failure after irreversible ischemic injury call for new therapeutics to be developed.One possible option for new treatments is cellular therapy,which is based on injecting live autologous or allogeneic stem cells into the injured heart.At present,the main types of stem cells used in the treatment of myocardial infarction includes but are not limited to:embryonic stem cells（ESCs）,hematopoietic CD34⁺stem cells（HSCs）,induced pluripotent stem cells（i PSCs）and mesenchymal stem cells（MSCs）.Preclinical studies have shown that ESC-derived exosomes can enhance the function of infarcted myocardium.However,the clinical use of ESCs is still discouraged due to limitations such as increased risk of tumor formation,high rejection rates,and ethical issues.Hematopoietic stem cells and mesenchymal stem cells are easily accessible stem cells types mainly derived from bone marrow,peripheral blood and umbilical cord blood with negligible ethical issues.Mesenchymal cells are pluripotent stem cells that can differentiate into a variety of cell types,such as mesodermal cells（adipocytes,osteoblasts,chondrocytes）and myogenic cells.This property of mesenchymal stem cells makes them an attractive therapeutic agent,however,the benefits of these treatments are not stable in the long term,and the end result is highly heterogeneous among patients.The main reason for these limitations is assumed as the poor implantable nature of the transplanted cells.Researchers have developed separate strategies to improve mesenchymal stem cell transplantation:one is designed to increase the survival of transplanted MSCs in recipient tissues,making them more resistant to harmful microenvironments（cell preconditioning）;Another strategy is to make damaged tissue more receptive to transplanted cells and promote their interaction（tissue preconditioning）.We believe that the combined use of these different treatment strategies can accelerate the process of transforming experimental evidence from preclinical research into routine clinical practice.Melatonin,chemically known as N-acetyl-5-methoxytryptamine,is a substance found in mammals,plants,fungi and bacteria.In mammals,it is generally regarded as a hormone secreted mainly by the pineal gland controlling day-night rhythm.Recent studies have suggested that melatonin may have a variety of functions,including anti-inflammatory,antioxidant,anti-apoptosis,autophagy regulation,and anti-tumor effects.Melatonin has been shown to provide cellular protection against ischemic damage to the heart,brain,liver and kidneys.In particular,melatonin can induce the expression of catalase and superoxide dismutase-1 to increase the resistance of MSCs to peroxy-dependent apoptosis.Since improving the ischemic microenvironment by regulating excessive inflammation and oxidative stress has been shown to improve the efficacy of mesenchymal stem cells（MSCs）for ischemic injury,this study was designed to evaluate the effect of melatonin on adipose derived mesenchymal stem cells（AD-MSCs）-based treatment of myocardial infarction.Our aim is to provide a simple,safe,reliable and effective adjuvant therapy for mesenchymal stem cell therapy,so as to accelerate the clinical translation of mesenchymal stem cell therapy.In addition,in the area of myocardial ischemia-reperfusion injury（MI/R）,a large number of preclinical and clinical studies studies have provided evidence that melatonin is beneficial in reducing myocardial ischemia-reperfusion injury（MI/R）.These studies further confirmed that the cardioprotective effect of melatonin is primarily due to the activation of the melatonin membrane receptor,since the cardiovascular protective effect of melatonin can be neutralized by the non-specific melatonin receptor antagonist luzindole.However,there are two kinds of melatonin receptors in mammals including membrane melatonin receptor 1 and 2（MT1 and MT2）,and it is not clear which specific melatonin membrane receptor mediated the cardioprotective effect of melatonin.Therefore,we used in vivo gene silencing and overexpression methods to determine which specific melatonin membrane receptor mediates the cardiac protective effect of melatonin so as to further deepen the understanding of the cardioprotective effects of melatonin.MethodsPart i:Melatonin facilitated-stem cell therapy for ischemic heart disease.A.Melatonin alleviates hypoxia/serum deprivation（H/SD）injury of AD-MSCs,which was associated with alleviated inflammation and oxidative stress.AD-MSCs were isolated from firefly luciferase-enhanced green fluorescent protein double reporter mice（fluc-egfp double trasngenic mice,C57BL/6 background）by a mature method established by our group.The H/SD injury model was used to simulate the microenvironment of myocardial ischemia injury.in vitro bioluminescence imaging and Annexin v-PI dual-channel flow cytometry were used to detect the survival of AD-MSCs.The Caspase 3 activity kit further validated the apoptosis data.The expression of gp91^phox（heme binding subunit of NADPH oxidase that produces superoxide anions）detected by Western blot,NADPH oxidase activity kit were employed to evaluate the oxidative stress level of AD-MSCs.The levels of inflammatory factors（IL-10,TNF-α,IL-6）and paracrine factors（VEGF,b FGF）in the supernatant were detected by ELISA.B.Melatonin promoted the survival and efficacy of AD-MSCs cells in ischemic myocardium,which was associated with alleviated inflammation and oxidative stress.As described above,AD-MSCs with dual reporter genes were isolated and C57BL/6 mice with the syngeneic background were divided into the following 5 groups:Sham group,MI+PBS group（MI）,MI+AD-MSCs（AD-MSCs）,MI+melatonin group（Mel）,MI+AD-MSCs+melatonin pretreatment（AD-MSCs Mel pre）.Living imaging bioluminescence image was used to trace the survival of the transplanted cells.Tissue frozen section-based observation of GFP positive was used to observe cell engraftment.Masson trichrome staining was used to interrogate tissue fibrosis and scar area.TUNEL method was performed to detect myocardial tissue apoptosis level.Caspase 3 activity kit further validated tissue apoptosis level.Dynamic echocardiography was performed on post-operational day 1,7,14 and 28 to evaluate heart pump function.DHE staining,ELSIA-based detection of MDA level were executed to reflect myocardial oxidative stress level.ELISA method were also employed to detect myocardial inflammatory cytokines（IL-10,TNF-α,and IL-6）and paracrine factors（VEGF and bFGF）.C.The possible downstream mechanism of melatonin,whether the activation of silence information regulator 1（SIRT1）mediates the effect of melatonin in vivo and in vitro.RNA interference（RNAi）method was used to silence SIRT1 mRNA and protein levels in AD-MSCs.Loss-of-function studies were conducted to observe whether the protective effect of melatonin was weakened in vitro and in vivo,detailed methods were described above.Part ii:Melatonin receptor mediated protection against myocardial ischemia/reperfusion injury.A.To examine the effects of melatonin receptor silencing on melatonin-elicited cardioprotective effects.The expression levels of MT1 and MT2 in normal condition and myocardial ischemia-reperfusion injury at different time points of expression were examined by Western blot and immunofluorescence.Melatonin receptor silencing in heart was achieved by minicircle vectors.Evans blue-TTC double staining was conducted to examine myocardial infarction area.The changes of serum myocardial enzymes,tissue apoptosis were also tested to reflect myocardial injury.Myocardial functional assays including non-invasive echocardiography and invasive hemodynamics were employed to evaluate cardiac function.In addition,the key enzyme of three kinds of apoptotic pathways were examined by corresponding enzymes kit.Cardiac oxidative and nitrosative stress were evaluated by corresponding markers detection.B.Melatonin receptor overexpression protected against myocardial ischemia-reperfusion injury.Melatonin receptor overexpression in heart was achieved by AAV9 vectors.Evans blue-TTC double staining was conducted to examine myocardial infarction area.The changes of serum myocardial enzymes,tissue apoptosis were also tested to reflect myocardial injury.Myocardial functional assays including non-invasive echocardiography and invasive hemodynamics were employed to evaluate cardiac function.In addition,the key enzyme of three kinds of apoptotic pathways were examined by corresponding enzymes kit.Cardiac oxidative and nitrosative stress were evaluated by corresponding markers detection.C.The downstream signalling of MT2 in hypoxia/reoxygenation（H/R）-injured cardiomyocytes.Cell vitality and LDH release were detected upon melatonin receptors silence.Cell vitality and LDH release were detected upon melatonin receptor 2 silence and overexpression by adenovirus vectors.Cell vitality and LDH release were detected upontreatment with MT2 selective agonist IIK7 at different doses.Immunoco-precipitation was used to detect the interaction between melatonin receptors and Notch1.The expression of Notch1/Hes1/PTEN/Akt signal and ROR upon MT2silence or overexpression were also detected by Western blot.Results1.In vitro bioluminescence imaging（BLI）and Annexin V-PI dual-channel flow cytometry results showed that melatonin significantly improved survival of AD-MSCs under hypoxia/serum deprivation（H/SD）injury.The results of Caspase 3 activity kit showed that melatonin significantly inhibited the increase of Caspase 3 activity induced by H/SD.The expression of gp91^phoxwas significantly inhibited by melatonin.Both NADPH oxidase activity and DCFH-DA fluorescence intensity assay demonstrated that oxidative stress in AD-MSCs induced by H/SD could be inhibited by melatonin.The release of inflammatory cytokines IL-10,TNF-α,and IL-6 in the supernatant of AD-MSCs induced by H/SD was inhibited by melatonin.The level of paracrine factora（VEGF,b FGF）in the AD-MSCs supernatant triggered by H/SD was further augmented by melatonin.2.The survival of transplanted cells in the microenvironment of ischemic and hypoxic myocardium could be significantly improved by melatonin as revealed by in vivo BLI.The GFP staining results of frozen slices on POD 7 further confirmed the results of bioluminescence imaging.Masson trichrome staining showed that both melatonin and AD-MSCs alone could significantly reduce the scar area and interstitial fibrosis caused by myocardial infarction.This effect was more pronounced in the melatonin post-treatment and AD-MSCs combined treatment group,but not in the melatonin pre-treatment group.TUNEL staining results showed that either melatonin or AD-MSCs alone could significantly reduce apoptosis of myocardial cells induced by myocardial infarction.This effect was strengthened in melatonin post-treatment and AD-MSCs combined group,and the activity of Caspase 3 further verified the TUNEL staining results.Dynamic echocardiography showed that both melatonin alone and AD-MSCs alone could significantly reverse the deterioration of cardiac function caused by myocardial infarction.This effect was amplified in melatonin post-treatment and AD-MSCs combined treatment group,but not in melatonin pre-treatment group.DHE staining and ELSIA detection of MDA levels indicated that both melatonin administration alone and AD-MSCs treatment alone could significantly reduce the level of myocardial oxidative stress induced by myocardial infarction,which was more significant in melatonin post-treatment and AD-MSCs combined treatment group.The levels of inflammatory factors（IL-10,TNF-α,and IL-6）in myocardial tissue extracts were significantly reduced when treated with melatonin alone or AD-MSCs alone,which was more pronounced in combined treatment group.ELISA detected that the secretion factor（VEGF,b FGF）level in the boarding zone of infarcted heart triggered by myocardial infarction could be further increased by combined treatment of Mel-AD-MSCs than melatonin or AD-MSCs alone.3.Melatonin treatment significantly up-regulated the expression of SIRT1 and anti-apoptotic protein Bcl2,and effectively reduced the acetylation level of P53,NF-κB,and FOXO1.RNA interference（RNAi）method can effectively knockdown the AD-MSCs SIRT1 mRNA and protein level.The in vitro effect of melatonin-mediated anti-inflammatory and antioxidant stress,and pro-paracrine effect can be significantly inhibit by SIRT1 silencing.Similarly,melatonin-mediated in vivo effects including the scar area reduction,tissue apoptosis alleviation,anti-inflammatory and antioxidant stress actions,pro-paracrine effect can be significantly abolished by SIRT1 silencing.These pieces of evidence suggests that melatonin’s effect on promoting stem cell therapy may be achieved by activating SIRT1.4.Melatonin receptor 2 silence abolished the effects of melatonin on myocardial ischemia-reperfusion injury.Western Blot and immunofluorescence showed that both MT1 and MT2 expressed in murine myocardium,but MT2 is selectivity up-regulated during reperfusion.Minicircle vectors can realize highly efficient silencing of melatonin receptor genes in myocardium.Melatonin-mediated myocardial protection is weakened in the MT2-silenced mice.In addition,melatonin attenuated endoplasmic reticulum stress and mitochondria mediated apoptosis effect is MT2 dependent.Melatonin-mediated alleviation of oxidative/nitrosative stress effects also depends on the activation of MT2.5.Overexpression of MT2 but not MT1 attenuated MI/R-induced myocardial injury and dysfuncction.MT2 rather than MT1 overexpression inhibited ER stress-and mitochondrial-mediated apoptosis and suppressed oxidative/nitrative stress.6.MT2 but not MT1 protected against hypoxia/reoxygenation injury in neonatal cardiomyocytes via the Notch1/Hes1/RORαsignaling.Endogenous MT2 protein level significantly increased after reoxygenation at 2 hrs,4 hrs and 6 hrs,whereas MT1expression remained mostly unaffected.Silencing MT2 with Mc-Sh-MT2 minicircle exacerbated H/R injury.H/R-driven cell injury was accentuated and attenuated by MT2silencing and MT2 overexpression,respectively.MT2 selective agonist IIK7 treatment during reoxygenation was revealed to preserve cell viability,reduce caspase3 activity and LDH release in a dose-depend manner.Co-immunoprecipitation assay showed that the MT2 receptor but not MT1 interacted with Notch1 and activated Notch1 signaling in murine neonatal cardiomyocytes.MT2 overexpression induced up-regulation of Notch1,Hes1,P-Akt and RORα,as well as down-regulation of PTEN.Conclusions1.Melatonin can be used as an important adjunct of MSC for the treatment ofischemic heart disease.2.Melatonin receptor 2 is an important endogenous myocardial protective receptor.