| Background Acute myocardial infarction(AMI)is the single leading cause of death worldwide.Timely reperfusion strategies such as pharmacological thrombolysis and percutaneous coronary intervention(PCI)have contributed to substantial improvement in the prognosis of patients suffering AMI.However,paradoxically,restoration of coronary blood flow causes further tissue damage,referred to as myocardial reperfusion injury.The damage to the myocardium can lead to major clinical consequences such as the no-reflow phenomenon,irreversible and reversible loss of contractile cardiac function,and reperfusion arrhythmias including lethal arrhythmias.Furthermore,in up to half of the patients who are successfully treated with primary PCI,coronary microvascular obstruction occurs and these patients are more likely to have a worse outcome.Thus,there is clearly an unmet need for therapeutic strategies to limit cardiac I/R injury.LncRNAs are a heterogeneous group of ncRNAs that are more than 200 nt long They are involved in many biological processes,such as transcriptional regulation of protein-coding genes,maintenance of genomic integrity,X chromosome inactivation,genomic imprinting,cell differentiation,and development.They are also involved in post-transcriptional regulation,splicing,and translational regulation of various genes.LncRNAs play vital role in various normal physiological and pathological conditions including cell differentiation,metabolism and cancer progression.Emerging evidences suggest that IncRNAs are involved in the regulation of cardiac diseases,such as dilated cardiomyopathy,heart failure,coronary atherosclerosis,myocardial Infarction,and so on.In a recent study,lncRNAs and mRNAs were profiled by microarray at early stage of reperfusion and the expression of some highly-dysregulated lncRNAs was further validated using polymerase chain reaction.The results revealed that 64 lncRNAs were up-regulated and 87 down-regulated.In these differential lncRNAs,Gm2691 attracted our attention due to its high fold change.The aim of the study was to investigate the function and mechanism of lncRNA Gm2691 in ischemic reperfusion injury.Objective 1.To investigate the anti-inflammatory and anti-apoptosis effect of IncRNA Gm2691 and its protective role in ischemic reperfusion injury.2.To confirm the role of PI3K/Akt pathway in the protective effect of lncRNA Gm2691.Methods For vivo study1.Animal model:Male Wistar rats(n=40)were divided into 4 groups(n=10/group):sham,I/R,I/R+Ad-Gm2691 and I/R+Ad-con.I/R+Ad-Gm2691 rats were administered Ad-Gm2691 through intramyocardial injection into the left ventricular myocardium 7 days before I/R operation.The suture was placed around LAD but not tied in Sham rats.2.TTC staining was performed to measure the myocardial infarction size.3.LDH content was measured.4.Echocardiography was performed.5.Western blot(WB)was performed to measure the expression of Caspase 3、Bax、Bcl-2.6.RT-PCR was performed to measure the mRNA level of IL-6,TNF-a and IL-8.For vitro study1.Cultured neonatal rat ventricular cardiomyocytes(NRCMs)were divided into the following five groups:(1)the blank control group(normoxia group):primary myocardial cells were cultured under normoxia condition.(2)the hypoxia group,cardiomyocytes were deoxygenated for 4h after hypoxia for 10h.(3)hypoxia+ad-con group:after transfection of primary myocardial cells with negative control adenovirus,hypoxia was performed for 10h and then reoxygenated for 4h.(4)hypoxia+ad-Gm2691 group:after transfection with overexpressed lncRNA Gm2691 adenovirus,hypoxia for lOh and reoxygenation for 4h.(5)hypoxia+ad-Gm2691+LY294002 group:after transfection with overexpressed lncRNA Gm2691 adenovirus,20μmol/L LY294002 was added,and hypoxia was reduced for lOh and reoxygenated for 4h.2.TUNEL assay was performed to show apoptosis in NRCMs induced by H/R.3.Western blot(WB)was performed to measure the expression of Akt、ERK1/2 and Caspase 3、Bax、Bcl-2.4.RT-PCR was performed to measure the mRNA level of IncRNA Gm2691,IL-6,TNF-a and IL-8.Results1.LncRNA Gm2691 reduced the myocardial infarct size.Compared with I/R alone,I/R+Ad-Gm2691 reduced the infarct size(P=0.000),while rats in I/R+Ad-con group have no significant different infarct size than that of I/R group(P=0.294).2.Changes of heart functional parameters measured by echocardiography.As compared with the sham group,left ventricular internal diameter atend-systole(LVIDs),left ventricular internal diameter at end-diastole(LVIDd),left ventricular end diastolic volume(LVEDV),left ventricular end systolic volume(LVESV)in the I/R group were significantly increased.But left ventricularfraction(LVEF)and fractional shortening(FS)were decreased(all P<0.01).Compared with I/R alone,I/R+Ad-Gm2691 reduced the LVIDs、LVIDd、LVEDV、LVESV,and increased LVEF and LVFS(all P=0.000).The I/R+Ad-con group have no significant difference of the above parameters compared with the I/R group(all P>0.05).3.LncRNA Gm2691 decreased the level of LDH after I/R.The level of LDH in I/R group was higher than that of the sham group(P=0.002).The level of LDH in I/R+Ad-Gm2691 group was lower than that of I/R group(P=0.031).4.LncRNA Gm2691 reduced the expression of protein related to apoptosis in myocardial tissue of I/R rats.Western-blot results showed that compared with sham group,caspase-3 and Bax expression were significantly increased in I/R group,while Bcl-2 expression was significantly decreased,and the corresponding Bax/Bcl-2 ratio was significantly increased(all P<0.01).Compared with I/R group,the caspase-3 and Bax expression in I/R+ad-Gm2691 group were significantly reduced,the Bcl-2 expression was significantly increased,and the Bax/Bcl-2 ratio was significantly reduced(all P<0.05).There was no significant difference between I/R group and I/R+ad-con group(all P>0.05).5.LncRNA Gm2691 effectively reduced the level of myocardial inflammatory cytokines in I/R rats.RT-PCR results showed that,compared with sham group,the expressions of IL-6,TNF-aand IL-8 mRNA in myocardial tissues of I/R group were significantly increased(all P=0.000).Compared with I/R group,the mRNA expression of the above inflammatory factors was significantly reduced in I/R+ad-Gm2691 group(all P<0.05).There was no significant difference between I/R group and I/R+ad-con group(all P>0.05).6.The results suggested that the expression of IncRNA Gm2691 in anoxic induced NRVMs was time-dependent after 6 h pretreatment with IncRNA Gm2691 adenovirus,and the expression of IncRNA Gm2691 was significantly increased 3 h after hypoxia,and reached the peak at 9 h after hypoxia.Hypoxia can reduce the expression of IncRNA Gm2691 in cardiomyocytes,while pre-treatment with IncRNA Gm2691 adenovirus for 6 h before hypoxia can significantly increase the expression of IncRNA Gm2691(all P<0.01).7.LncRNA Gm2691 reduced NRVMs apoptosis.TUNEL staining showed that LncRNA Gm2691 effectively reduced apoptosis of cardiomyocytes caused by hypoxia/reoxygenation(P=0.002).Compared with the control group,the expressions of caspase-3 and Bax and Bcl-2 were significantly increased,and the corresponding Bax/Bcl-2 ratio was significantly increased in the hypoxia group(all P<0.01).Compared with the hypoxia group,the expressions of caspase-3 and Bax,Bcl-2 and Bax/Bcl-2 in the hypoxia+ad-Gm2691 group were significantly reduced(all P<0.05).There was no significant difference between the hypoxia and the hypoxia+ad-con groups(all P>0.05).8.LncRNA Gm2691 effectively reduced the expression of inflammatory cytokines in cardiomyocytes caused by hypoxia/reoxygenation.Rt-PCR results showed that the expressions of IL-6,TNF-a and IL-8 mRNA were significantly increased in the hypoxia group compared with the normal group(all P=0.000).Compared with the hypoxia group,the mRNA expression of these inflammatory factors was significantly reduced in the hypoxia+ad-Gm2691 group(all P<0.05).There was no significant difference between the hypoxia group and the hypoxia+ad-con group(all P>0.05).9.The protective role of lncRNA Gm2691 in myocardial cell injury caused by hypoxia/reoxygenation was through the PI3K/Akt signal transduction pathway.Western-blot results showed that the expression of p-Akt was significantly reduced(P=0.000)and p-ERK1/2 significantly increased in the hypoxia group compared with the normal group(P=0.005).Compared with the hypoxia group,the overexpression of IncRNA Gm2691 significantly increased the expression of p-Akt(P<0.01),while the expression of p-ERK1/2 was not significantly affected(P=0.084).With the addition of PI3K-Akt pathway inhibitor LY294002 at the same time,the effect of IncRNA Gm2691 on inhibiting myocardial cell apoptosis and inflammatory factor expression was significantly reduced(all P<0.01).Conclusion1.LncRNA Gm2691 has anti-inflammatory and anti-apoptotic effects,and it exerts protective effects on myocardial ischemia reperfusion injury2.LncRNAGm2691 plays its protective role through the PI3K/Akt signaling pathway.Background The AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis, which acts to energy homoeostasis whenever cellular energy charge is depleted. Over the last 2 decades, it has become apparent that AMPK regulates several other cellular functions and has specific roles in cardiovascular tissues, acting to regulate cardiac metabolism and contractile function,As well as promoting anticontractile, anti-inflammatory, and antiatherogenic actions in blood vessels. Recent studies have shown that AMPK plays a protective role in myocardial ischemia-reperfusion injury through its above effects.To directly and potently target AMPK,Mirguet et al. developed aThienopyridone- derived compound GRS621 that consistently activated AMPK recombinant heterotrimers in vitro. In cellular assays, GSK621 was more potent than A-769662 at inducing AMPK activation, as measured by the level of ACC phosphorylation. GSK621 markedly increased phosphorylation at AMPKαT172,a marker of AMPK activation, and also stimulated the phosphorylation of ACC(S79) and ULKI(S555). These results suggest that GSK621 is a potent AMPK activator. GSK621 increased nicotinamide adenine dinucleotide phosphate(NADPH)content in osteoblasts to inhibit H2O2-induced reactive oxygen species (ROS) production.Meanwhile, GSK62I activated cytoprotective autophagy in the osteoblasts. However，it has not been reported whether GSK621 can Play a protective role in myocardial ischemia reperfusion injury.This study aims to investigate the protective effect and mechanism of GSK621 on myocardial cell injury induced by oxygen glucose deprivation/ re-oxygenation. To elucidate the protective effect of AMPK and its new agonist GSK621 on cardiac. Muscle cells,so as to provide a new basis for drug development targeting AMPK.Objective 1. To determine whether GSK621 can activate AMPK. In cardiomyocytes.2.To investigates whether GSK621 can inhibit the oxidative damage and apoptosis of cardiomyocytes caused by OGD/R.3. To clarify whether the protective effect of GSK621 on cardomyocytes induced by OGD/R was generated by inducing AMPK activation, and to further explore the role of Nrf2. a downstream signal molecule of AMPK.Methods I. AC1b human myocardial cells and the primary murine myocardiocytes were cultured in DMEM with IDP. fetal bovine serum (FBS) with necessary antibiotics Myocardial cells were seeded into six-well plats at a density of 100, 0co cells per weland placed it an airtight chamber (95% N2 and 5% CO2. The chamber was sealed. nod cells were maintained with OGD for 4 h. Afterwards, cells were re-oxygenated"Mock"cells were placed in regular complete medium with normal oxygen.2.After the OGDR treatment, CCK-8 assay were performed to assess cell viability.3.Cell death was tested through measuring medium lactate dehydrogenase (LDH) content.4. Apoptosis assays : including the caspase-3 activity, the single strand DNA(ssDNA)ELISA, TUNEL staining and Annexin-propidium iodid (PT) FACS.5. Westem blowing(WB) Total proteins were extracted to defense the protein expression of AMPKα1, p-AMPKal, ACC, p-ACC, Cle-caspase-3. Clecaspuse-9, Cle-PARP、Nrf2、 Hol and NQoL6.AMPK activity was tested.7.DCFH-DA assay was perforomed to measure ROS in myocardial ells induced by OGD/R. 8.The thiobarbituric acid reactive subsatances (TBARS) assay was performed to quantitatively analyze the cellular lipid peroxidation contents 9. Changes of mitochondrial membrane potential in myocardial cells was measure by JC-1 assay. 10. Quantitative real-time PCR (qPCR)was performed to measure Nrf-2, HoL NQOI mRNA 11. The lentiviral particles with AMPKαl shRNA were added to ACIecells Following cell viability and apoptosis were detected again. 12. A Lent-CRISPR/Cas9 AMPKα1-knockout (KO)-GFP construct was transfected to AC16 human myocardial cells. Following cell viability and apoptosis was detected again. 13. The lentiviral particles with Nrf2 shRNA were added to AC16cells Following,HO1 expression ,cell viability and apoptosis were detected again.Results 1.GSK621 activates AMPK signaling in myocardial cells. Westen blotting testing AMPK pathway proteins confined that GSK621,in adose-dependent manner, induced phosphorylation of AMPKαl (Thr-172) and itsprimary downstream target acetyl-CoA carboxylase(ACC, at Ser- 79) <(均P＜0. 01)GSK621 dose-dependently increased AMPK activity meanwhile (HA-00002 GSK621 inhibits OGD/R-induced cytotoxicity in myocardial cells.OGDMR prucedure in AClo human myocardial cells induced potent viability?OGD/R-8 OD reduction (P=0. 000),?Imporantly, pretreatment with GSK621, at 2 and I0μM significantly inhibited OGD/R induced viability redaction (P-0.000), OGD/R-induced AC16aell death, evidencd by significantly increased medium LDH?release, was attenuated by GsK621 pretreatment (P-0.000). Further studies?demonstrated that OGD/R induced significant apoptosis activation, evidenced by?mitochondrial depolarization, caspase-3 activity increast.?cleavage ("Cle") of caspase-3 ,caspase-9 and poly (ADP-ribose) polymerase (PARP),single strang DNA (ssDNA) accumulation and TUNEL stating increase (P=0.000). Importantly,GSK621(2 and10μM) pretreatment largely inhibitrd OGD/G-induced apoptosis in AC16 cells (P=0.000). Annexin V-PIFACS assay results, further confirmed that the AMPK activator potently attenuated OGD/R-induced apoptosis (P=0.000).3.?GSK621 inhibits OGD/R -induced oxidative injury in myocardial cells OGD/R treatment induced singnificant ROS production and lipid peroxidation(P=0.000). Such action by OGD/R were largely attenuated by GSK621(均P<0.01).4.?AMPK activation mediates GSK621-induced myocardial cell protrction against OGD/R.?Western blotting confirmed that its expression was significantly downregulated in the stable cells with AMPKαl shRNA ("sh-AMPαl")or AMPKαl KO construct ("ko-AMPαK""). Consequently, GSK621-induced AMPK activation, or AMPKαl-ACC phosphorylation, was almost blocked by AMPKαl ?shRNA or KO. Significantly. in "sh-AMPKαl"and ko-AMPKl" cells, GSK621 was unable to inhibit OGD/R-induced viability reduction and apoptosis (均P< 0.01>).5. GSK621 activates Nrf2 signaling in myocardial cells.PCR assay results in AC16 human myocardial cells confirmed that GSK621 dose-dependently induced mRNA expression of Nrf2-dependent genes, including HOI and NQOl. Although Nrf2 mRNA levels were unchanged, its protein levels were increased dramatically in GSK621-trcated AC16 myocardial cells. HOI and NQoI protein levels were increased as well. PCR results show that GSK621-induced HOI and NQOl expression in AC16 cells was completely blocked by AMPKαl silencing or KO, The lentiviral particles with Nrf2 shRNAs were added to culturedAC16 cells. GSK621-induced HOI and NQOl expression was largely inhibited by the Nrf2 shRNAs in AC16cells. In Nrf2- silenced AC16cells, OGD/R-induced viability and apoptosis were not attenuated by GSK621 pretreatment (P=0.01)Conclusion 1.?GSK621 activates AMPK signaling in myocardial cells2.?GSK621 inhibits OGD/R-induced cytotoxicity and oxidati ?injury in myocardial cell.3.?AMPK activation mediates GSK621-induced myocardial cell protection against OGD/R.4.?GSK621 activates Nrf2 signaling in myocardial cells... |
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