Effect of emulsified isoflurane on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytes

Objective:To explore the effect of emulsified isoflurane(EI)on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytea and relevant protein expression.Methods:Cardiac muscle anoxiareoxygenation damage model was established with culture in vitro neonatal rat cardiomyocytes.The cardiomyocytes were divided into control group,model group,fat emulsion group and EI group.The cardiomyocytes apoptosis rates and lactic dehydrogenase(LDH),superoxide dismutase(SOD)and malondialdehyde(MDA)index standardization were detected after relevant treatment The expression of apoptosis-related proteins Bel-2,Bax and Caspase-3 were detected with Western blot approach.Results:After hypoxia/reoxygenation(H/R)model was treated by EI,the cells apoptosis rate decreased and was dramatically below the fat emulsion group(P<0.05),Cardiomyocytes biochemical index detection presented that,compared with the control group that the LDH activity and MDA content dramatically increased(P<0.05),while the SOD activity notably decreased(P<0.05);compared with the H/R group,the SOD activity of the fat emulsion group and EI group increased(P<0.05);while the LDH activity and MDA content decreased(P<0.05).And the change of the EI group was more remarkable than the fat emulsion group(P<0.05).The Western blot analysis presented that,compared with the control group,the Bcl-2 protein expression of the other groups significantly decreased(P<0.05),the expressions of Bax protein and Caspase-3protein increased significantly(P<0.05);compared with H/R group,cardiomyocytes Bc1-2protein expression of EI group increased significantly(P<0.05),the expressions of Bax protein and Caspase-3 protein decreased significantly(P<0.05),and the change of EI group was more remarkable than the fat emulsion group(P<0.05).Conclusions:EI can inhabit the apoptosis of anoxia-reoxygenation damage model cardiomyocytes,and may he related to the up-regulation of expression of Bcl-2 and down-regulation of expression of Caspase-3 protein.

Effectof Ginsenoside Re on Cardiomyocyte Apoptosis and Expression of Bcl-2/Bax Gene after Ischemia and Reperfusion in Rats

To observe the effectof ginsenoside Re on cardiomyocyte apoptosis and Bcl- 2 / Bax gene expression after ischemia (30 m in) and reperfusion (6 h) in rats and to elucidate the possible m echanism s of ginsenoside Re on inhibition of cardiom yocyte apoptosis,the ischem ia/ reperfusion heart m odel was established by ligating the left anterior descending branch of coronary artery in Wistar rats.The apoptotic cardiom yocytes were confirmed by transm ission electron m icroscopy and counted by in situ nick end labeling(TU NEL) method and lightm icroscopy.The m RNA and protein expression of Bcl- 2 and Bax genes were studied by in situ hybridization and im munohis- tochemical staining.Mean optical density (OD) value of the positive fields of m RNA and protein expression was quantitatively exam ined by im age analysis system.The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischem ia/ reperfusion group and weren't observed in the sham- operation group by transmission electron microscopy;(2 ) The num bers of the apoptotic cells were134.4 5± 4 5 .6 1/ field in the ischemia/ reperfusion group,and 90 .6 6± 19.2 2 / field in the ginsenoside Re- treated group.The differences was significant between two groups(P<0 .0 1) ;(3) Gene expression of Bcl- 2 and Bax were increased significantly in the is- chemia/ reperfusion group and ginsenoside Re- treated group when compared with the sham - opera- tion group.There was no significant difference in the gene expression of Bcl- 2 between the gin- senoside Re- treated group and ischemia/ reperfusion group(P>0 .0 5 ) ,but gene expression of Bax was decreased significantly in the ginsenoside Re- treated group as compared with the ischem ia/ reperfusion group(P<0 .0 1) .The ratio of Bcl- 2 / Bax was increased significantly in the ginseno- side Re- treated group when com pared with the ischem ia/ reperfusion group and sham- operation group.These findings suggest that m yocardial ischem ia- reperfusion can induce cardiom yocyte apoptosis,and ginsenoside Re can significantly inhibit cardiom yocyte apoptosis induced by ischemi- a- reperfusion in rats.It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by in- hibiting expression of pro- apoptotic Bax gene and raising the ratio of Bcl- 2 / Bax.

Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress

Background Endoplasmic reticulum （ER） stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin （PQS） is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured eardiomyocytes were stimulated with thapsigargin （TG）, which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG （1 μmol/L） treatment for 24 h, following PQS pre-treatment （160 μg/mL） for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase （Si-PERK） for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 （GRP78）, calreticulin, PERK, eukaryotic translation initiation factor 2α （elF2c0, activating transcription factor 4 （ATF4）, and C/EBP homologous protein （CHOP） were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.

Effect of microRNA-208a on mitochondrial apoptosis of cardiomyocytes of neonatal rats

Objective: To explore the effect and mechanism of microRNA-208a(mi R-208a) in the mitochondrial apoptosis of cardiomyocytes of neonatal rats. Methods: The primary cultured cardiomyocytes of neonatal rats were added into the hypoxia incubator for the hypoxia induction. The overexpression system for mi R-208 a of cardiomyocytes of neonatal rats was built. The l ow cytometry assay was employed to detect the incidence of apoptosis in the overexpressed mi R-208 a. The mitochondrial staining technique was used to detect the change in the mitochondrial morphology of over-expressed mi R-208 a. The bioinformatic analysis was chosen to analyze and predict the target gene of mi R-208 a. Results: Firstly, the primary culture system of cardiomyocytes of neonatal rats was successfully built. The mi R-208 a was over-expressed in cardiomyocytes of neonatal rats by mi R-208 a Mimics. Results of flow cytometry assay showed that the over-expressed mi R-208 a could signii cantly reduce the incidence of apoptosis; while results of mitochondrial staining indicated the change in the mitochondrial morphology of over-expressed mi R-208 a and the mitochondrialfission process was inhibited. In conclusion, it was supposed that mi R-208 a could inhibit the activation of mitochondrialfission process to keep the cardiomyocytes from apoptosis. Conclusions: The over-expressed mi R-208 a can reduce the incidence of apoptosis in the cardiomyocytes of neonatal rats, signii cantly change the mitochondrial morphology and inhibit the mitochondrial fission process.

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G_(2)/M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.

Overexpression of angiopoietin-1 reduces doxorubicin-induced apoptosis in cardiomyocytes

Doxorubicin （Dox） is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 （Ad5-Ang-1） 24 h before the cells were chal- lenged with Dox at a concentration of 2 ~tmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced in- creases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpres- sion of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase （PI3K）/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB （NF-~：B） activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.

Salubrinal protects against tunicamycin and hypoxia induced cardiomyocyte apoptosis via the PERK-eIF2a signaling pathway

Objectives This study examined the protective effect of salubrinal and the mechanism underlying this protection against tunicamycin （TM）- and hypoxia-induced apoptosis in rat cardiomyocytes. Methods Neonatal rat cardiomyocytes were cultured from the ventricles of l-day-old Wistar rats. Cells were exposed to different concentrations of salubrinal （10, 20, and 40 gmol/L） for 30 min followed by TM treatment or hypoxia for 36 h. Apoptosis was measured by a multiparameter HCS （high content screening） apoptosis assay, TUNEL assay and flow cytometry. The phosphorylation of eukaryotic translation initiation factor 2 subunit alpha （eIF2c0 and the expression of cleaved caspase-12 were determined by Western blotting. C/EBP homologous protein （CHOP） was detected by immunocytochemistry. Results HCS, TUNEL assays and flow cytometry showed that salubrinal protected cardiomyocytes against apoptosis induced by TM or hypoxia. Western blotting showed that salubrinal protected cardiomyocytes against apoptosis by inducing eIF2ct phosphorylation and down-regulating the expression of the endoplasmic reticulum stress-mediated apoptotic proteins, CHOP and cleaved caspase-12. Conclusions Our study suggests that salubrinal protects rat cardiomyocytes against TM- or hypoxia-associated apoptosis via a mechanism involving the inhibition of ER stress-mediated apoptosis.

Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway

Background Myocardial injury caused by microvascular obstruction(MVO)is characterized by persistent ischemia/hypoxia(IH)of cardiomyocytes after microembolization.Autophagy and Egr-1 were closely associated with various cardiovascular diseases,including MVO.Bim and Beclin-1 are the important genes for autophagy and apoptosis.We aimed to explore whether the Egr-1/Bim/Beclin-1 pathway is involved in regulating autophagy and apoptosis in IH-exposed cardiomyocytes.Methods Neonatal rat cardiomyocytes exposed to the IH environment in vitro were transfected with lentivirus expressing Egr-1 or Egr-1 sh RNA,or further treated with 3-methyladenine(3-MA).The expressions of autophagy and apoptosis-associated genes were evaluated using RT-q PCR and Western blots assays.Autophagic vacuoles and autophagic flux were detected by transmission electron microscopy(TEM)and confocal microscope,respectively.Cell injury was assessed by lactate dehydrogenase(LDH)leakage,and apoptosis was determined by flow cytometry.Results IH exposure elevated Egr-1 and Bim expressions,and decreased Beclin-1 expression in rat cardiomyocytes.Egr-1 overexpression in IH-exposed cardiomyocytes significantly up-regulated the levels of Egr-1 and Bim,and down-regulated the level of Beclin-1.Egr-1 knockdown resulted in down-regulated expressions of Egr-1 and Bim,as well as up-regulated expression of Beclin-1.In addition,Egr-1 knockdown induced autophagy was suppressed by 3-MA treatments.TEM and autophagic flux experiments also confirmed that Egr-1 inhibited autophagy progression in IH-exposed cardiomyocytes.Egr-1 suppression protected cardiomyocytes from IH-induced injury,as evidenced by the positive correlations between Egr-1 expression and LDH leakage or apoptosis index in IH-exposed cardiomyocytes.Conclusions IH-induced cardiomyocyte autophagy and apoptosis are regulated by the Egr-1/Bim/Beclin-1 pathway,which is a potential target for treating cardiomyocyte injury caused by MVO in the IH environment.

Activation of calcium-sensing receptors is associated with apoptosis in a model of simulated cardiomyocytes ischemia/reperfusion

Objective： Calcium-sensing receptors （CaSRs） are G-protein coupled receptors which maintain systemic calcium homeostasis and participate in hormone secretion, activation of ion channels, cell apoptosis, proliferation, and differentiation. Previous studies have shown that CaSRs induce apoptosis in isolated adult rat heart and in normal neonatal rat cardiomyocytes by G-protein-PLC-IP3 signaling transduction. However, little knowledge is presently available concerning the role of CaSRs in the apoptosis induced by ischemia and reperfusion in neonatal cardiomyocytes. Methods： Primary neonatal rat ventricular cardiomyocytes were incubated in ischemiamimetic solution for 2 h, and then re-incubated in normal culture medium for 24 h to establish a model of simu- lated ischemia/reperfusion （I/R）. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling （TUNEL）. The expression of CaSRs mRNA was detected by real-time reverse transcription polymerase chain reaction （RT-PCR）. In addition, the expressions of caspase-3 and Bcl-2 were analyzed by western blot. Results： The simulated I/R enhanced the expression of CaSRs and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSRs, further increased the expression of CaSRs and cardiomyocyte apoptosis, along with up-regulation of caspase-3 and down-regulation of Bcl-2. Conclusion： CaSRs are associated with UR injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting caspase-3 expression.

Effects of Hypoxia on Oxidative Stress, Autophagy and Apoptosis in Cardiomyocytes

Coronary heart disease (CHD) is a hypoxia related disease. However, the relationship of the hypoxia-induced oxidative stress, autophagy and apoptosis in cardiomyocyte remains unclear. In this study, we used CoCl2 to mimic hypoxic conditions in H9c2 cardiomyocytes and study the effects of CoCl2-induced hypoxia on oxidative stress, apoptosis and autophagy, as well as the relationships among these processes. Cell viability and levels of ROS, LC3-II, p62, caspase-3 and PARP were assessed. The viability and morphology of cardiomyocytes were affected by hypoxia, and hypoxia enhanced levels of ROS and the levels of the LC3-II, p62, caspase-3 and PARP proteins in H9c2 cells in a dose-dependent manner. ROS levels rise gradually in the presence of hypoxia;however, it shrinks when hypoxia reaches a certain level. Caspase-3 and PARP levels were raised with the increasing of hypoxia level. Enhanced level of LC3 and decreased levels of p62 in hypoxic cells indicate that autophagy levels are in accord with hypoxia. Based on these results, hypoxia induces oxidative stress, apoptosis and autophagy in cardiomyocytes. Autophagy is a double-edged sword. At a low level, autophagy can resist oxidative stress and protect cardiomyocytes from oxidative stress, while high level autophagy can promote apoptosis of cardiomyocytes.

Panax notogiseng saponin inhibits ischemia-induced apoptosis by activating PI3K/Akt signal pathway in cardiomyocytes

The panax notoginseng saponin(PNS) had been clinically used for the treatment of cardiovascular diseases and stroke in China.It had been demonstrated that PNS could protect cardiomyocytes from injury induced by ischemi- a,but the underlying molecular mechanisms of this protective effect were still unclear.This study was aimed to investigate the protective effect and molecular mechanisms of PNS on apoptosis in H9c2 cells in vitro and rat myocardial ischemia injury model in vivo.Annexin-V/PI assay shew that PNS could protect H9c2 cells from apoptosis induced by serum, glucose and oxygen deprivation(SGOD) in a dose-dependent manner.However,the anti-apoptotic effect of PNS was reversed by LY294002,a specific PI3K inhibitor.This antiapoptotic effect of PNS was confirmed by JC-1,a specific probe of mitochondrial membrane potential staining.PNS could significantly increase phos-Akt in H9c2 cells by Western blot assays and its effect could be inhibited by LY294002.Furthermore,PNS could improve ischemic-induced left ventricular function as reflected by EF,LVDd and LVDs.PNS could also inhibited cellular apoptosis in myocardial tissues in ischemic rats by TUNEL assay.PNS administration also increased the expression of phos-Akt in rat ischemic myocardial tissues.These results suggested that PNS could protect myocardial cells from apoptosis induced by ischemia in vitro model and in vivo model through activating-PI3K/Akt signal pathway which may be meaningful for further understanding the molecular mechanisms of cardiac protection of PNS.And the results might be useful in treatment of myocardial ischemia in future.

Nonhematopoietic erythropoietin derivative protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis

Objective：Carbamylated EPO（CEPO） is a derivative of erythropoietin（EPO） by subjecting it to carbamylation. It does not stimulate erythropoiesis, but effectively protects tissue from injury. The present study was to investigate the effect of CEPO treatment using in vitro models of hypoxia/reoxygenation（H/R）. Methods：Cardiomyocytes were exposed to hypoxia（95% N2 and 5% CO2） for 1 hour followed by 4 hours of reoxygenation（95% O2 and 5% CO2）. CEPO was administered after hypoxia, just before reoxygenation. The apoptotic cardiomyocytes were determined by flow cytometry. The level of protein was assessed by western blot analysis. Results： CEPO treatment significantly decreased the apoptotic cardiomyocytes by 54.20% compared with H/R group. Western blot analysis showed that CEPO administration increased the level of Bcl-2（an antiapoptotic protein） by 62.22% compared with H/R group. Conclusion： Acute administration of CEPO protected cardiomyocytes from H/R-induced apoptosis. CEPO protected cardiomyocytes with a concomitant upregulation of Bcl-2 after H/R injury.

Influence of Silencing Soluble Epoxide Hydrolase with RNA Interference on Cardiomyocytes Apoptosis Induced By Doxorubicin

In order to investigate the influence of silencing soluble epoxide hydrolase（sEH） with double-stranded small interfering RNA（siRNA） on cardiomyocytes apoptosis induced by doxorubicin（DOX）,two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence（PCN） served as the negative control.Cardiomyocytes were divided into four groups：control group,DOX group,PCN＋DOX group,and EH-R＋DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R＋DOX group were significantly decreased as compared with other groups（P0.01）.As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased（P0.01）.However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R＋DOX group when compared with those in the DOX group and the PCN＋DOX group（P0.01 for each）.It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Study on the Effects of Losartan on Cardiomyocyte Apoptosis and Gene Expression After Ischemia and Reperfusion in vivo in Rats

Summary: In order to study the effects of losartan on cardiomyocyte apoptosis following ischemia (0. 5 h) and reperfusion (48 h) in vivo and bcl-2 and bax gene expression, TUNEL staining method, immunohistochemistry and in situ hybridization histochemistry (ISHH) were used to monitor the apoptotic cells, mRNA and protein of gene expression, respectively. Image processing system was used to quantitively dispose the positive metric substance of both immunohistochemistry and ISHH through the average optical density (OD) value. The number of the apop- totic cells were 38±9 (control group), 0-1 (sham operation group) and 9±4 (losartan-treated group) in each visual field respectively with the difference among the groups being significant (P< 0. 001 ). OD values of bcl-2 (ISHH) were 0. 07425± 0. 02029 (control group ), 0. 05961± 0. 009932 (sham operation group) and 0. 07619±0. 01445 (losartan-treated group ) respectively, while OD values of bcl-2 (immunohistochemistry) were 0. 1374±0. 01367 (control group ), 0. 08510±0. 01862 (sham operation group) and 0. 1252±0. 02064 (losartan-treated group). hcl-2 gene expression was increased significantly in the control group and losartan-treated group as com- pared with sham operation group (P < 0. 05 ). OD value of bax (immunohistochemistry) was 09727±0. 02230 (control group), 0. 06182±0. 01430 (sham operation group) and 0. 06213± 0. 01420 (losartan-treated group). bax gene expression was decreased very significantly in losartan-treated group and sham operation group as compared with control group (P<0. 001 ). Bcl-2/ bax ratio was 1. 413 (control group), 1. 376 (sham operation group) and 2. 016 (losartan-treated group) respectively. The results indicated that losartan might inhibit cardiomyocyte apoptosis following ischemia and reperfusion. The mechanism might be that bax gene expression was inhibited to increase bcl-2/bax ratio.

Effects of Cornus Officinal Is Total Glycoside on Cardiomyocyte Apoptosis and Calcium Overload after Hypoxia/Reoxygenation Injury in Rats

Objective: To investigate the mechanism of Cornus officinalis Total Glycosides (COTG) on myocardial protection, by studying effects of COTG on cardiomyocyte apoptosis induced by hypoxia/reoxygenation and calcium concentration in rats. Methods: The myocardial cells of born 1-3d SD rats were isolated by enzyme digestion, cultured for 3 days. Cells were divided into five groups: Control group, H/R group, Cornus officinalis Total Glycosides low-dose group (LDG), Cornus officinalis Total Glycosides middle-dose group (MDG) and Cornus officinalis Total Glycosides high-dose group (HDG). Three drug groups were pretreated with different doses of Cornus officinalis Total Glycosides before hypoxia/reoxygenation treatment. The apoptotic rate was determined by flow cytometry assay, the intracellular free calcium concentration was examined by flow cytometry, and the ultrastructure of myocardial cells was observed under transmission electron microscope. Results: The results revealed that Cornus officinalis Total Glycosides pretreatment decreased apoptosis rate, but the effect of lower dosage is not significant. Furthermore, Cornus officinalis Total Glycosides can attenuate mitochondrial calcium overload, improve mitochondrial morphology and inhibit cardiomyocyte apoptosis caused by H/R. Conclusion: Cornus officinalis Total Glycosides pretreatment can inhibit cardiomyocyte apoptosis and calcium overload during H/R injury. However, the underlying mechanisms require us to further study.

β3-adrenoceptor Impacts Apoptosis in Cultured Cardiomyocytes via Activation of PI3K/Akt and p38MAPK

β3-adrenoceptor（β3-AR） has been shown to promote myocardial apoptosis. However, the exact physiological role and importance of this receptor in the human myocardium, and its underlying mode of action, have not been fully elucidated. The present study aimed to determine the effects of β3-AR on the promotion of myocardial apoptosis and on norepinephrine（NE） injury. We analyzed NE-induced cardiomyocyte（CM） apoptosis by using a TUNEL and an annexin V/propidium iodide apoptosis assay. Furthermore, we investigated the NE-induced expression of the apoptosis marker genes Akt and p38 MAPK, their phosphorylated counterparts p-Akt and p-p38 MAPK, caspase-3, Bcl-2, and Bax. In addition, we determined the effect of a 48-h treatment with a β3-AR agonist and antagonist on expression of these marker genes. β3-AR overexpression was found to increase CM apoptosis, accompanied by an increased expression of caspase-3, bax/bcl-2, and p-p38 MAPK. In contrast, the β3-blocker reduced apoptosis of CMs and the associated elevated Akt expression. We identified a novel and potent anti-apoptosis mechanism via the PI3K/Akt pathway and a pro-apoptosis pathway mediated by p38 MAPK.

APEX1 protects against oxidative damage-induced cardiomyocyte apoptosis

Apurine/pyrimidine-free endonuclease 1(APEX1)is a multifunctional enzyme that contributes to oxidization-mediated DNA-cleaved base excision repair and redox activation of transcription factors.However,the role of APEX1 during cardiomyocyte oxidative stress injury is not completely understood.In the present study,whether APEX1 protects oxidative damage-induced cardiomyocytes was investigated.mRNA and protein expression levels of APEX1 were downregulated in the mouse model of cardiac ischemia-reperfusion injury.Furthermore,the expression of APEX1 in hydrogen peroxide(H 2 O 2)-treated neonatal mice cardiomyocytes was also decreased.APEX1 knockdown aggravated H 2 O 2-treated cardiomyocyte apoptosis indexes.By contrast,APEX1 overexpression reversed H 2 O 2-induced oxidative damage,as demonstrated by decreased caspase 3 and Bax expression levels.Moreover,homeobox A5 upregulated APEX1.The results of the present study indicated that APEX1 displayed protective effects against oxidative damage,suggesting that APEX1 may serve as a unique protective strategy for cardiac ischemia-reperfusion injury.

Selenium nanoparticles reduce oxidative stress-induced cardiomyocyte apoptosis in ascites syndrome in broiler chickens via the ATF6-DR5 signaling pathway

Broiler ascites syndrome(AS)is one of the main diseases threatening the health of broilers.It is well documented that myocardial hypertrophy and failure is one of the key mechanisms of broiler ascites syndrome.Therefore,prevention of cardiac hypertrophy and failure would be one goal to reduce broiler ascites syndrome incidence.Myocardial hyper-trophy and failure are closely related to endoplasmic reticulum stress(ERS)in cardiac myocytes,and the endoplasmic reticulum stress signaling system(ATF6-DR5)is one of the important pathways of myocardial apoptosis.Excessive hyper-trophy will affect the heart muscle's normal contraction and diastole function,and the heart will turn from compen-sated to decompensate thus causing myocardial injury.Myocardial apoptosis is a core component of the pathological changes of this myocardial injury.Nano-selenium is a kind of red elemental selenium nanoparticle.Due to its excellent physical,chemical and biological properties,it has attracted extensive academic attention in recent years.It has been proven to have excellent antioxidant,antibacterial,antitumor,antihypertrophic,and antiapoptotic abilties.Herein,nano-selenium(1μmol/L)can inhibit hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in broiler primary cardiomyocytes,and at the same time reduce cardiomyocyte apoptosis.In vivo,nano-selenium can reduce broiler myocardial injury-related enzyme indicators(AST,CK and LDH),and alleviate myocardial injury.It can also activate the antioxidant enzyme system(SOD,GSH-Px and CAT)and reduce MDA,and make the recovery ofT-AOC ability in the organization.Meanwhile,nano-selenium can down-regulate the genes and proteins expression of ATF-6,GRP-78,CHOP and caspase 12 in the ERS-related signaling pathway,and inhibit that of downstream-related caspase 3,Bax and caspase 9,and increase that of the downstream anti-apoptotic Bcl-2,thereby maintaining the homeostasis of the endoplasmic reticulum and alleviating cardiomyocyte apoptosis.It can be seen that nano-selenium can protect the damaged myocardium in the broiler ascites caused by high-salt drinking by regulating the ATF6-DR5 signaling pathway.This study was performed in chickens and cardiomyocyte cells and attempted to demonstrate that selenium nanoparticles can protect the damaged myocar-dium in broiler ascites.This paper provides a new idea for preventing and treating broiler ascites syndrome.

miR-133 contributes to high glucose-induced cardiomyocyte apoptosis via IGF1 receptor

MicroRNAs(miRNAs) are endogenous 20 -23 -nucleotide (nt) -containing small non-coding RNAs that negatively regulate gene expression in diverse biological and pathological processes,including cell differentiation,proliferation, apoptosis,heart disease and human cancers.We investigated miR-133 expression and its potential role in a high glucose-induced myocardium in Streptozotocin(STZ)-induced C57bl6 mouse model of diabetes.miR-133 expression was significantly increased in myocardium in a time-dependent manner after STZ treatment.IGF1 receptor(IGF1R) protein was dramatically decreased without obvious up-regulation of its mRNA level post hyperglycemia.IGF1R protein level was decreaed with increase of its transcript level in neonatal mouse ventricular cardiomyocytes induced by high D-glucose concentration. Dual luciferase assay revealed that miR133 could interact with specific sites in the 3’UTR of IGF1R gene.p-ERK and p-Akt levels were reduced in neonatal mouse cardiomyocytes over-expressed with miR133 after IGF treatment.Introduction of functional miR-133,IGF1R siRNA into neonatal mouse cardiomyocytes could enhance cardiomyocyte apoptosis.These results implicate that miR-133 is involved in contributing to high glucose-induced cardiomyocyte apoptosis via regulating IGF1R expression post-transcriptionally.