BACKGROUND: A critical shortage of donor organs has necessitated an investigation of new strategies to increase the availability of additional organs available for human transplantation. We investigated the amount of apoptosis and expression of GADD45beta in two groups, a GADD45beta-transfected group and untransfected group. MATERIAL AND METHOD: The experimental groups consist of a control group (normal H9C2 cell line) and GADD45beta-transfected group. After injury of the each group, we evaluated the expression of GADD45beta and the level of apoptosis in each group. RESULT: There was a significant increase in the expression of GADD45beta in the GADD45beta-transfected group at 1 hour, 2 hours, and 3 hours after stimuli as compared with the control group. The amount of cardiac myoblast cell line apoptosis was significantly lower in the GADD45beta-transfected group as compared with the control group. The concentration of annexin in the GADD45beta-transfected group was significantly lower than that of the control group after cell injury. CONCLUSION: Transfection of a rat myoblast cell line with the GADD45beta gene results in decreased susceptibility to cell injury of human serum.
Animals ; Apoptosis ; Cell Line ; Humans ; Myoblasts ; Myoblasts, Cardiac ; Rats ; Tissue Donors ; Transfection ; Transplantation, Heterologous ; Transplants

Cellular cardiomyoplasty has recently emerged as a potential new treatment of ischemic heart disease. Combining cellular cardiomyoplasty with gene therapy using myogenic transcription factor might facilitate myocardial regeneration. In this study, we engineered H9c2, L6 using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. Each cell overexpressing MEF2 and Nkx2.5 was generated by gene transfection and clonal selection. CO-culture was performed that each cell line added over cultured cardiomyocyte. H9c2-MEF2c and H9c2-Nkx2.5 became long, spindle shape like cardiomyocyte. Troponin T, cardiac specific marker, was found spot-like pattern in H9c2-Nkx2.5. However, co-culture with cardiomyocyte did not induce differentiation all kinds of cells into cardiomyocyte. Connexin43, which is gap junction marker was increased in H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c and L6-Nkx2.5. Especially, co-culture with cardiomyocyte resulted in elevation of connexin43 levels more than monoculture. Ultrastructurally, formations of gap junction and desmosome were found apparently in L6-Nkx2.5. Long-standing, strong, regular and more frequent contraction were observed in cardiomyocyte co-cultured with H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c, L6-Nkx2.5, respectively. Neverthless, any cell did not have active contraction itself, but passive movement except cardiomyocyte. H9c2-MEF2c, L6-MEF2c and L6-Nkx2.5 had resistance to hypoxia compared with other groups. These results suggested that co-culture and overexpressions of MEF2c and Nkx2.5 induced differentiation into cardiomyocyte and played an important role on intercellular junction formation and hypoxic resistance. This would be a promising source of cellular cardiomyoplasty. Therefore, much more research would be essential for clinical application of cellular cardiomyoplasty and this study would be a basic source for further study of MEF2c and Nkx2.5 in cellular cardiomyoplasty.
Animals ; Anoxia ; Cardiomyoplasty ; Cell Line ; Cell Transplantation ; Coculture Techniques* ; Connexin 43 ; Desmosomes ; Gap Junctions ; Genetic Therapy ; Intercellular Junctions ; Myoblasts ; Myoblasts, Cardiac* ; Myocardial Ischemia ; Myocytes, Cardiac* ; Plasmids ; Rats* ; Regeneration ; Transcription Factors ; Transfection ; Troponin T

BACKGROUND: Nitric oxide(NO) is known to have protective effects on an ischemic heart and to exert triggering effects on ischemic preconditioning. However, the effects of NO during the ischemic period have not been investigated. To investigate the role of exogenous nitric oxide in a model of ischemic heart cell death, we studied the effects of ischemic preconditioning and ischemia in a normal and an ischemic buffer. METHODS: Rat cardiac myoblast cells(H9c2) were cultured in a normal and an ischemic buffered medium. For the ischemic culture of heart cells, the cells were cultured in a dessicator with GasPak for 5 hrs. In ischemic preconditioning, the cells were pretreated with ischemic buffer for 5 min and then perfused with normal medium for 30 min. For the measurement of the cytotoxicity, a MTT(3-4-5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay was performed. A DAPI(4',6-diamidino-2-phenylindole dihydrochloride) staining procedure and a flow cytometry analysis were performed to confirm apoptotic cell death by ischemia. RESULTS: Cell viability, as determined by using a MTT assay, showed that the preconditioned group treated with NO showed more cell death than with the not-preconditioned groups in both normal and ischemic buffers. But, In normal medium and not-preconditioned groups, NO showed protective effect according to the concentrations(100, 1000 microM) . No treatment with NO produced the different results. In normal medium, the protective effect of ischemic preconditioning was demonstrated, but no protective effect of ischemic preconditioning could be seen in the case of the ischemic buffer. The DAPI staining and flow cytometry analysis of heart cells showed characteristic apoptotic features. CONCLUSION: NO added in the ischemic phase had deterious effects on heart cells. Ischemic preconditioning was more harmful than ischemia alone. The toxicity of the cells was characteristic apoptosis.
Animals ; Apoptosis ; Buffers ; Cell Death ; Cell Survival ; Flow Cytometry ; Heart ; Ischemia ; Ischemic Preconditioning ; Myoblasts, Cardiac* ; Nitric Oxide* ; Rats

AIMTo study the effect of thyroid hormone on protein kinase C activity and isoprotein expressions in cardiac myocytes and fibroblasts of rats in vitro.

METHODSCardiac myocytes and fibroblasts were cultured according to the method of Simpson. Cells were pretreated with 1% newborn calf serum (NCS) or Angiotensin II (Ang II) for 24 hours, then Triiodothyronine (T3) was added to the culture medium and the culture was kept for another 48 hours. The protein kinase C activation were measured by PepTaga non-radioactive PKC assay, and the expressions of PKC alpha and PKC epsilon were detected by Western blot method.

RESULTSAt the condition of 1% NCS culture medium, T3 could inhibit PKC activity and PKC epsilon expression in cardiac myocytes significantly, but the expression of PKC alpha in cardiac myocytes was not influenced by T3. In cardiac fibroblasts, neither PKC activity nor PKC alpha and PKC epsilon expressions was influenced by T3. When cells were pretreated with Ang II for 24 hours, PKC activities in cardiac myocytes and fibroblasts were increased significantly, and PKC epsilon expressions in cardiac myocytes were also markedly increased. Following a T3 treatment, PKC activity and PKC epsilon expression in cardiac myocytes were markedly decreased, but PKC activity in cardiac fibroblasts was not changed.

CONCLUSIONWhether at the condition of 1% NCS medium or in a pretreatment with Ang II, thyroid hormone could inhibit the PKC activity and PKC epsilon expression in cardiac myocytes. The influence of thyroid hormone on the PKC signal pathway in cardiac myocyte may be involved in many pathophysiological progress of myocardium.

Animals ; Cells, Cultured ; Myoblasts, Cardiac ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thyroid Hormones ; pharmacology

AIMTo investigate how vasonatrin peptide (VNP) can attenuate the growth-promoting effect of hypoxia in cardiac fibroblasts cultured from neonatal rats.

METHODSThe cultured cardiac fibroblasts were divided randomly into four groups: control group, hypoxia group, hypoxia + VNP group and hypoxia + 8-Bromo-cGMP group. The growth of cardiac myocytes was measured by the means of MTT method. The effect of VNP on the intracellular level of cGMP and PCNA were measured by the means of radioimmunoassay and immunohistochemistry stain respectively.

RESULTSHypoxia (24 h) significantly increased the MTT A490nm value of cardiac fibroblasts (P < 0.05 vs control group). Both VNP (10(-7) mol/L) and 8-Bromo-cGMP (10(-3) mol/L) decreased MTT A490 nm value in cardiac fibroblast (P < 0.05 vs hypoxia group). VNP (10(-7) mol/L) increased the intracellular level of cGMP (P < 0.05 vs control and hypoxia group). Hypoxia (24 h) significantly increased the expression of proliferating cell nuclear antigen (PCNA) in cardiac myocytes (P < 0.05, vs control group), but VNP (10(-7) mol/L) decreased it.

CONCLUSIONVNP can attenuate hypoxia-induced growth-promoting effect in cardiac fibroblasts which is associated with the changes of cGMP and PCNA.

Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cyclic GMP ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMTo investigate whether AcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

METHODSNeonatal rat cardiac fibroblasts were isolated. The cell proliferation was observed by 3H-proline incorporation assay.

RESULTSOn the culture of 0.4% FBS, PDGF stimulated cardiac fibroblasts proliferation and collagen synthesis with a dose-dependent manner at the concentrations from 1 ng/ml to 20 ng/ml, in which 10 ng/ml PDGF reached its peak. AcSDKP at the concentration from 10(-10) mol/L to 10(-8) mol/L could inhibit cardiac fibroblasts proliferation and collagen synthesis mediated by PDGF. 10(-9) mol/L AcSDKP attained its peak on inhibiting cardiac fibroblasts proliferation and collagen synthesis.

CONCLUSIONAcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

Animals ; Cell Proliferation ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Rats, Wistar

AIMTo investigate the effects of pravastatin on endothelin(ET) expression induced by aldosterone in cultured neonatal rat cardiac fibroblasts.

METHODSET concentration in conditioned medium was measured by radioimmunoassay, intracellular ET-1 level was evaluated by flow cytometry, and the expression of preproendothelin-1 (ppET-1) was detected and quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) method.

RESULTSThe cardiac fibroblasts, treated with aldosterone at 107 mol/L, significantly up-regulated ppET-1 mRNA expression, as well as ET-1 synthesis and release. Pravastatin (10(-5), 10(-4), 10(-3) mol/L) dose-dependently blocked these effects. In contrast, pravastatin-induced inhibitory effects were reversed in the presence of mevalonate.

CONCLUSIONPravastatin down-regulated ppET-1 mRNA expression, as well as ET-1 synthesis and release induced by aldosterone in a process specifically related to mevalonate in cardiac fibroblasts.

Aldosterone ; metabolism ; Animals ; Cells, Cultured ; Endothelins ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Myoblasts, Cardiac ; drug effects ; metabolism ; Pravastatin ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe transient receptor potential melastatin 7-like (TRPM7L) expression changes post myocardial infarction (MI) in mouse cardiac fibroblast (CF).

METHODSTRPM7 expression and Ca2+ influx in CF from MI and control mice were quantified by mRNA RT-PCR and whole cell patch clamp technique.

RESULTS(1) TRPM7 expression was significantly upregulated post MI and Ca2+ influx of CF were significantly increased post MI [(7.4 +/- 0.7) pA/pF vs. (16.2 +/- 1.7) pA/pF, P < 0.01] and Ca2+ influx of CF increased 3-fold under lower pH condition; (2) These effects could be blocked by knock-out TRPM7 gene with SiRNA.

CONCLUSIONTRPM7L upregulation post MI and under lower pH condition are responsible for increased Ca2+ influx in CF.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Gene Expression Regulation ; Mice ; Myoblasts, Cardiac ; metabolism ; Myocardial Infarction ; metabolism ; Patch-Clamp Techniques ; RNA, Messenger ; genetics ; TRPM Cation Channels ; metabolism

OBJECTIVERecent studies have shown cardiac protection effects of erythropoietin (EPO). The present experiment was designed to investigate the effects of EPO on TGF-beta1, nitric oxide synthase (NOS), collagen contents induced by angiotensin II (Ang II) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects.

METHODSNeonatal rat CFs was isolated by collagenase and trypsinase digestion methods. PBS, EPO, Ang II in the absence or presence of LY294002, an inhibitor of PI3-K, or L-NAME, an inhibitor of NOS, were added to CFs and cultured for 24 hours. The concentration of collagen I and collagen III in culture medium were quantitated by ELISA. The levels of nitric oxide (NO) and the activities of NOS as well as NOS isoforms were measured by chemical enzymic method. Western blot was applied to detecting the protein expressions of Akt, p-Akt, eNOS, iNOS, and TGF-beta1.

RESULTSThe concentrations of collagen I and collagen III in CFs culture medium were significantly increased while the level of NO was significantly decreased by Ang II and these changes were significantly suppressed by EPO in a dose dependent manner. The effects of EPO on eNOS and NO could be blocked by LY294002. L-NAME could block EPO's effect on NO but not on the eNOS expression. The suppression effects on expressions of TGF-beta1 and collagen by Ang II in CFs were blocked by both LY294002 and L-NAME.

CONCLUSIONEPO suppresses the upregulated expressions of TGF-beta1 and increased production of collagen induced by Ang II through activating the PI3-K/Akt signaling pathway in neonatal rat CFs.

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Collagen ; metabolism ; Erythropoietin ; pharmacology ; Myoblasts, Cardiac ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Transforming Growth Factor beta1 ; metabolism

Aldosterone ; pharmacology ; Animals ; Animals, Newborn ; Cells, Cultured ; Endothelins ; metabolism ; Myoblasts, Cardiac ; drug effects ; secretion ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley