LncRNA ZFAS1 regulates cardiomyocyte differentiation of human embryonic stem cells

Background:Cardiomyocytes derived from human embryonic stem cells(hESCs)are regulated by complex and stringent gene networks during differentiation.Long non-coding RNAs(lncRNAs)exert critical epigenetic regulatory functions in multiple differentiation processes.However,the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated.Here,we identified the key roles of ZFAS1(lncRNA zinc finger antisense 1)in the differentiation of cardiomyocytes from hESCs.Methods:A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method,and the number of beating hESCs-derived cardiomyocytes was calculated.Gene expression was analyzed by quantitative real-time PCR(qRTPCR).Immunofluorescence assays were performed to assess the expression of cardiac troponin T(cTnT)andα-actinin protein in cardiomyocytes.Results:qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells,cardiac progenitor cells,and cardiomyocytes.Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs,which was characterized by reduced expression of the cardiac-specific markers cTnT,α-actinin,myosin heavy chain 6(MYH6),and myosin heavy chain 7(MYH7).In contrast,ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes.In terms of the mechanism,we found that ZFAS1 is an antisense lncRNA at the 5′end of the protein-coding gene ZNFX1.Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1.Furthermore,qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation.Conclusion:Altogether,these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1,which may provide a reference for the treatment of heart disease to a certain extent.

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background:Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period.Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells(hiPS-CM),researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM.In our previous studies,we separately supplemented two polyunsaturated fatty acids(PUFAs),docosahexaenoic acid(DHA)or arachidonic acid(AA),to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation,fatty acid metabolism,and cellular adhesion.The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force.Methods:This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM.In addition to the contractile force and mRNA measurements used in the previous study,we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis,measured glucose and fatty acids metabolites,and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation.Results:It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors,and the supplementations may activate the CD36_ERRγmetabolic pathway.DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters.The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant.AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36.However,finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings.Conclusion:DHA and AA,as having been supplemented in infant formulas,have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually,although they were able to enter the cellular metabolic system.The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM,which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.

Disease modeling of desmosome-related cardiomyopathy using induced pluripotent stem cell-derived cardiomyocytes

Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation.Although optimized medical therapies have been developed for heart failure during the last few decades,some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies.Desmosome,which is a dynamic cell-to-cell junctional component,maintains the structural integrity of heart tissues.Genetic mutations in desmo-somal genes cause arrhythmogenic cardiomyopathy(AC),a rare inheritable disease,and predispose patients to sudden cardiac death and heart failure.Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies.Among desmosomal genes,mutations in PKP2(which encodes PKP2)are most frequently identified in patients with AC.PKP2 deficiency causes various pathological cardiac phenotypes.Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells(iPSCs)in combination with genome editing,which allows the precise arrangement of the targeted genome,are powerful experimental tools for studying disease.This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency.

Changes of c-fos and c-jun mRNA Expression in Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy and Effects of Sodium Tanshinone ⅡA Sulfonate

The changes of proto-oncogene c-fos and c-jun mRNA expression in angiotensin Ⅱ （AngⅡ）-induced hypertrophy and effects of sodium tanshinone ⅡA sulfonate （STS） in the primary culture of neonatal rat cardiomyocytes were investigated. Twelve neonatal clean grade Wistar rats were selected. The cardiomyocytes were isolated, cultured and divided according to different treatments in the medium. The cardiomyocyte size was determined by phase contrast microscope, and the rate of protein synthesis was measured by [3H]-Leucine incorporation. The c-fos and c-jun mRNA expression in cardiomyocytes was detected by reverse transcription polymerase chain reaction （RT-PCR）. It was found after cardiomyocytes were treated with AngⅡ for 30 min, the c-fos and c-jun mRNA expression in cardiomyocytes was increased significantly （P〈0.01）. After treatment with AngⅡ for 24 h, the rate of protein synthesis in AngⅡ group was significantly increased as compared with control group （P〈0.01）. After treatment with AngⅡ for 7 days, the size of cardiomyocytes in AngⅡ group was increased obviously as compared with control group （P〈0.05）. After pretreatment with STS or Valsartan before AngⅡ treatment, both of them could inhibit the above effects of AngⅡ （P〈0.05 or P〈0.01）. It was suggested that STS could ameliorate AngⅡ-induced cardiomyocyte hy- pertrophy by inhibiting c-fos and c-jun mRNA expression and reducing protein synthesis rate of cardiomyocytes.

Expression of Wnt and NCX1 and its correlation with cardiomyocyte apoptosis in mouse with myocardial hypertrophy

Objective:To study the correlation between expression of Wnt and NCXl and cardiomyocyte apoptosis in mouse with myocardial hypertrophy.Methods:C57B/16 male mice were given the subcutaneous injection of 1 mg/kg isoprenaline to build the myocardial hypertrophy model.After 14 d of model building,mice were executed by cervical vertebra luxation.The ratio of heart weight/body weight(HW/BW) and heart weight/tibia length(HW/TL) was observed and proved using HE staining mat detected the size of eaidiomyocytes.40 male C57B/16 mice were randomly divided into the sham group(normal saline) and model group(isoprenaline),with 20 mice in each group.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was applied to detect the cardiomyocyte apoptosis;while Western blot and immunohistochemistry were employed to detect the expression of Wnt and NCX1.Meanwhile,the correlation between these two proteins and cardiomyocyte apoptosis was explored.Results:Compared with the sham group,the ratio of HW/BW and HW/TL was increased in the model group,as well as the bigger and hypertrophied cardiomyocytes,decreased number and increased apoptosis of eaidiomyocytes,and increased positive expression of Wnt3 a,WntSa and NCXl in the cardiac muscle tissue.Besides,there was positive correlation between the expression of Wnt and NCXl and the cardiomyocyte apoptosis.Conclusions:The expression of Wnt3 a,Wnt5a and NCXl in mouse with myocardial hypertrophy is increased and positively correlated with the cardiomyocyte apoptosis.

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.

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.

Current methods for the maturation of induced pluripotent stem cellderived cardiomyocytes

Induced pluripotent stem cells(iPSCs) were first generated by Yamanaka and colleagues over a decade ago. Since then, iPSCs have been successfully differentiated into many distinct cell types, enabling tissue-, disease-, and patientspecific in vitro modelling. Cardiovascular disease is the greatest cause of mortality worldwide but encompasses rarer disorders of conduction and myocardial function for which a cellular model of study is ideal. Although methods to differentiate iPSCs into beating cardiomyocytes(iPSC-CMs) have recently been adequately optimized and commercialized, the resulting cells remain largely immature with regards to their structure and function,demonstrating fetal gene expression, disorganized morphology, reliance on predominantly glycolytic metabolism and contractile characteristics that differ from those of adult cardiomyocytes. As such, disease modelling using iPSC-CMs may be inaccurate and of limited utility. However, this limitation is widely recognized, and numerous groups have made substantial progress in addressing this problem. This review highlights successful methods that have been developed for the maturation of human iPSC-CMs using small molecules,environmental manipulation and 3-dimensional(3 D) growth approaches.

Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes

Objective To detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy. Methods The primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca], endoplasmic reticulum calcium [Ca]ER, and mitochondrial calcium [Ca]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate(SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels. Results The results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca], [Ca]ER, and [Ca]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca], [Ca]ER, and [Ca]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum. Conclusion The calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope(HVJ-E) and tissue maceration. METHODS: Cardiomyocytes(1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets(area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or Na OH maceration: G1: HVJ-E(+), Na OH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaO H(+), Cardiomyocytes(+); G3: HVJ-E(+),Na OH(-), Cardiomyocytes(+); G4: HVJ-E(-), Na OH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas.CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaO H maceration.

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.

Induced Differentiation of Human Cord Blood Mesenchymal Stem/Pro genitor Cells into Cardiomyocyte-like Cells In Vitro

The feasibility of using cord blood mesenchymal stem/progenitor cells (CB-MSPCs) to regenerate cardiomyocytes and the optimal inducing conditions were investigated. The CB mononuclear cells were cultured in low serum DMEM medium to produce an adherent layer. After expansion, the adherent cells were added into cardiomyocyte inducing medium supplemented with 5-azacytidine. Cardiogenic specific contractile protein troponin T staining was performed to identify the cardiomy-ocyte-like cells. The results showed that the frequency of CB-MSPCs clones in CB mononuclear cells was 0. 5×10-6 and about 1. 3×107-fold expansion was achieved within 20 sub-cultivation. After car-diogenic induction, 70 % CB-MSPCs was differentiated into cardiomyocyte-like cells. It was indicated that low serum culture could expand CB-MSPCs extensively and the expanded CB-MSPCs could be induced to differentiate into cardiomyocyte-like cells in high efficiency.

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.

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.

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.

Cytoprotective Effect of Silymarin against Diabetes-Induced Cardiomyocyte Apoptosis in Diabetic Rats

Objective The beneficial effects of silymarin have been extensively studied in the context of inflammation and cancer treatment, yet much less is known about its therapeutic effect on diabetes. The present study was aimed to investigate the cytoprotective activity of silymarin against diabetes-induced cardiomyocyte apoptosis. Methods Rats were randomly divided into： control group, untreated diabetes group and diabetes group treated with silymarin （120 mg/ks·d） for 10 d. Rats were sacrificed, and the cardiac muscle specimens and blood samples were collected. The immunoreactivity of caspase-3 and Bcl-2 in the cardiomyocytes was measured. Total proteins, glucose, insulin, creatinine, AST, ALT, cholesterol, and triglycerides levels were estimated. Results Unlike the treated diabetes group, cardiomyocyte apoptosis increased in the untreated rats, as evidenced by enhanced caspase-3 and declined Bcl-2 activities. The levels of glucose, creatinine, AST, ALT, cholesterol, and triglycerides declined in the treated rats. The declined levels of insulin were enhanced again after treatment of diabetic rats with silymarin, reflecting a restoration of the pancreatic β-cells activity. Conclusion The findings of this study are of great importance, which confirmed for the first time that treatment of diabetic subjects with silymarin may protect cardiomyocytes against apoptosis and promote survival-restoration of the pancreatic β-cells.

Protective role of retinoid X receptor in H9c2 cardiomyocytes from hypoxia/reoxygenation injury in rats

BACKGROUND: Retinoid X receptor(RXR) plays a central role in the regulation of intracellular receptor signaling pathways. The activation of RXR has protective effect on H2O2-induced apoptosis of H9c2 ventricular cells in rats. But the protective effect and mechanism of activating RXR in cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative iniury are still unclear.METHODS: The model of H/R injury was established through hypoxia for 2 hours and reoxygenation for 4 hours in H9c2 cardiomyocytes of rats. 9-cis-retinoic acid(9-cis RA) was obtained as an RXR agonist, and HX531 as an RXR antagonist. Cultured cardiomyocytes were randomly divided into four groups: sham group, H/R group, H/R+9-cis RA-pretreated group(100 nmol/L 9-cis RA), and H/R+9-cis RA+HX531-pretreated group(2.5 μmol/L HX531). The cell viability was measured by MTT, apoptosis rate of cardiomyocytes by flow cytometry analysis, and mitochondrial membrane potential(ΔΨm) by JC-1 fluorescent probe, and protein expressions of Bcl-2, Bax and cleaved caspase-9 with Western blotting. All measurement data were expressed as mean±standard deviation, and analyzed using one-way ANOVA and the Dunnett test. Differences were considered signif icant when P was <0.05.RESULTS: Pretreatment with RXR agonist enhanced cell viability, reduced apoptosis ratio, and stabled ΔΨm. Dot blotting experiments showed that under H/R stress conditions, Bcl-2 protein level decreased, while Bax and cleaved caspase-9 were increased. 9-cis RA administration before H/R stress prevented these effects, but the protective effects of activating RXR on cardiomyocytes against H/R induced oxidative injury were abolished when pretreated with RXR pan-antagonist HX531.CONCLUSION: The activation of RXR has protective effects against H/R injury in H9c2 cardiomyocytes of rats through attenuating signaling pathway of mitochondria apoptosis.

MiR-301a promotes embryonic stem cell differentiation to cardiomyocytes

BACKGROUND Cardiovascular disease is the leading cause of death worldwide.Tissue repair after pathological injury in the heart remains a major challenge due to the limited regenerative ability of cardiomyocytes in adults.Stem cell-derived cardiomyocytes provide a promising source for the cell transplantation-based treatment of injured hearts.AIM To explore the function and mechanisms of miR-301a in regulating cardiomyocyte differentiation of mouse embryonic stem(mES)cells,and provide experimental evidence for applying miR-301a to the cardiomyocyte differentiation induction from stem cells.METHODS mES cells with or without overexpression of miR-301a were applied for all functional assays.The hanging drop technique was applied to form embryoid bodies from mES cells.Cardiac markers including GATA-4,TBX5,MEF2C,andα-actinin were used to determine cardiomyocyte differentiation from mES cells.RESULTS High expression of miR-301a was detected in the heart from late embryonic to neonatal mice.Overexpression of miR-301a in mES cells significantly induced the expression of cardiac transcription factors,thereby promoting cardiomyocyte differentiation and beating cardiomyocyte clone formation.PTEN is a target gene of miR-301a in cardiomyocytes.PTEN-regulated PI3K-AKT-mTOR-Stat3 signaling showed involvement in regulating miR-301a-promoted cardiomyocyte differentiation from mES cells.CONCLUSION MiR-301a is capable of promoting embryonic stem cell differentiation to cardiomyocytes.