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Rat Cardiomyocytes (rat + cardiomyocyte)
Kinds of Rat Cardiomyocytes Selected AbstractsBehavior of Cardiomyocytes and Skeletal Muscle Cells on Different Extracellular Matrix Components,Relevance for Cardiac Tissue EngineeringARTIFICIAL ORGANS, Issue 1 2007Karin Macfelda Abstract:, Myocardial cell transplantation in patients with heart failure is emerging as a potential therapeutic option to augment the function of remaining myocytes. Nevertheless, further investigations on basic issues such as ideal cell type continue to be evaluated. Therefore, the aim of our studies was to compare the performance of skeletal muscle cells and cardiomyocytes with respect to their proliferation rate and viability on different extracellular matrix components (EMCs). Rat cardiomyocytes (RCM) and rat skeletal muscle cells (RSMC) were cultured on EMCs such as collagen type I, type IV, laminin, and fibronectin. The components were used as "single coating" as well as "double coating." Proliferation rates were determined by proliferation assays on days 1, 2, 4, and 8 after inoculation of the cells. The most essential result is that collagen type I enhances the proliferation rate of RSMC but decreases the proliferation of RCM significantly. This effect is independent of the second EMC used for the double-coating studies. Other EMCs also influence cellular behavior, whereas the sequence of the EMCs is essential. Results obtained in our studies reveal the significant different proliferation behavior of RCM and RSMC under identical conditions. As skeletal muscle cells are also used in heart tissue engineering models, these results are essential and should be investigated in further studies to prove the applicability of skeletal muscle cells for heart tissue engineering purposes. [source] Reduced calcium tolerance in rat cardiomyocytes after myocardial infarctionACTA PHYSIOLOGICA, Issue 4 2002I. Sjaastad ABSTRACT During ischaemia and reperfusion the intracellular Na+ concentration is elevated in the cardiomyocytes and the cells are depolarized, both favouring reverse mode Na,Ca-exchange loading of the cell with Ca2+. We examined whether cardiomyocytes from rats with congestive heart failure (CHF) and younger rats (HINCX) which both have a high expression of the Na,Ca-exchanger protein (NCX) showed reduced tolerance to extracellular Ca2+. The CHF was induced in Isofluran anaesthetized rats by left coronary artery ligation. Isolated cardiomyocytes were loaded with Fura-2AM and 140 mm Na+ and exposed to 0.05 mm Ca2+. Expression of the Na,Ca-exchanger protein was analysed. Fura-2 340/380 ratio rose more rapidly in HINCX and CHF than in SHAM, and the rise was abolished by Ni2+. Hypercontracture developed more frequently in HINCX and CHF than in SHAM cells. The amount of NCX was 54% higher in HINCX and 76% higher in CHF compared with SHAM. Na+ -loaded cardiomyocytes from CHF and HINCX rats are more susceptible to Ca2+ overload than SHAM cells because of the increased capacity for Na,Ca-exchange. [source] miR133a regulates cardiomyocyte hypertrophy in diabetesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 1 2010Biao Feng Abstract Background Diabetic cardiomyopathy, characterized by cardiac hypertrophy and contractile dysfunction, eventually leads to heart failure. We have previously shown that alterations of a number of key molecules are involved in producing cardiomyocyte hypertrophy in diabetes. The aim of the present study was to determine whether microRNAs (miRNA) play a role in mediating altered gene expression and structural/functional deficits in the heart in diabetes. Methods STZ-induced diabetic mice were haemodynamically investigated after 2 months of diabetes to establish the development of cardiomyopathy. The tissues were then examined for gene expression and microRNA analysis. We further investigated neonatal rat cardiomyocytes to identify the mechanisms of glucose-induced hypertrophy and the potential role of miR133a. Results Diabetic mice showed myocardial contractile dysfunction and augmented mRNA expression of atrial and brain natriuretic peptides (ANP, BNP), MEF2A and MEF2C, SGK1 and IGF1R compared to age- and sex-matched controls. Cardiac tissues from these mice showed alteration of multiple miRNAs by array analysis including miR133a, which was confirmed by RT-PCR. In vitro exposure of cardiomyocytes to high levels of glucose produced hypertrophic changes and reduced expression of miRNA133a. Finally, transfection of miR133a mimics prevented altered gene expression and hypertrophic changes. Conclusion Data from these studies demonstrate a novel glucose-induced mechanism regulating gene expression and cardiomyocyte hypertrophy in diabetes which is mediated through miR133a. Copyright © 2009 John Wiley & Sons, Ltd. [source] Leptin and endothelin-1 mediated increased extracellular matrix protein production and cardiomyocyte hypertrophy in diabetic heart diseaseDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2009Pijush Majumdar Abstract Background We investigated the role of leptin and its interaction with endothelin 1 (ET-1) in fibronectin (FN) synthesis and cardiomyocyte hypertrophy, two characteristic features of diabetic cardiomyopathy. Methods Endothelial cells [human umbilical vein endothelial cells (HUVECs)] were examined for FN production and neonatal rat cardiomyocytes for hypertrophy, following incubation with glucose, ET-1, leptin and specific blockers. FN, ET-1, leptin and leptin receptors mRNA expression and FN protein were measured. Myocytes were also morphometrically examined. Furthermore, hearts from streptozotocin-diabetic rats were analysed. Results Glucose caused increased FN mRNA and protein expression in HUVECs and cardiomyocytes hypertrophy along with upregulation of ET-1 mRNA, leptin mRNA and protein. Glucosemimetic effects were seen with leptin and ET-1. Leptin receptor antagonist (leptin quadruple mutant) and dual endothelin A endothelin B (ETA/ETB) receptor blocker bosentan normalized such abnormalities. Hearts from the diabetic animals showed hypertrophy and similar mRNA changes. Conclusion These data indicate that in diabetes increased FN production and cardiomyocyte hypertrophy may be mediated through leptin with its interaction with ET-1. Copyright © 2009 John Wiley & Sons, Ltd. [source] Measurement of electrophoretic mobility of cardiomyocytesELECTROPHORESIS, Issue 21 2009Ying Zhou Abstract The electrophoretic mobility (EPM) of rat cardiomyocytes with or without the treatment of neuraminidase was studied by cell electrophoresis. The EPM was found to change over a range from 0 to 8.67,,m,s,1/V,cm,1, depending on ionic strength, transmembrane potential, pH value, and/or surface charges. It is interesting that zero EPM was observed but reverse of the mobility was not. These results suggested that the negative charges carried on the cardiomyocyte surface might comprehensively consist of surface sialic acid, plasmalemma proteins, phospholipids, and transmembrane potential. The aberrant electrical double layer formed between the carried negative charges and adions had a big adsorption layer and a diffusion layer whose sizes changed circularly, making only negative charges be carried on the surface of living cardiomyocytes. The special structures on the surface of cardiomyocytes probably play a considerable role in the process of cardiac electrical activity. [source] Protective Effect of HOE642, a Selective Blocker of Na+ -H+ Exchange, Against the Development of Rigor Contracture in Rat Ventricular MyocytesEXPERIMENTAL PHYSIOLOGY, Issue 1 2000Marisol Ruiz-Meana The objective of this study was to investigate the effect of Na+ -H+ exchange (NHE) and HCO3, -Na+ symport inhibition on the development of rigor contracture. Freshly isolated adult rat cardiomyocytes were subjected to 60 min metabolic inhibition (MI) and 5 min re-energization (Rx). The effects of perfusion of HCO3, or HCO3, -free buffer with or without the NHE inhibitor HOE642 (7 ,M) were investigated during MI and Rx. In HCO3, -free conditions, HOE642 reduced the percentage of cells developing rigor during MI from 79 ± 1% to 40 ± 4% (P < 0.001) without modifying the time at which rigor appeared. This resulted in a 30% reduction of hypercontracture during Rx (P < 0.01). The presence of HCO3, abolished the protective effect of HOE642 against rigor. Cells that had developed rigor underwent hypercontracture during Rx independently of treatment allocation. Ratiofluorescence measurement demonstrated that the rise in cytosolic Ca2+ (fura-2) occurred only after the onset of rigor, and was not influenced by HOE642. NHE inhibition did not modify Na+ rise (SBFI) during MI, but exaggerated the initial fall of intracellular pH (BCEFC). In conclusion, HOE642 has a protective effect against rigor during energy deprivation, but only when HCO3, -dependent transporters are inhibited. This effect is independent of changes in cytosolic Na+ or Ca2+ concentrations. [source] ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Yi-Qiang Zhang Abstract Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2. J. Cell. Biochem. 99: 575,588, 2006. © 2006 Wiley-Liss, Inc. [source] Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell-penetrating abilityJOURNAL OF PEPTIDE SCIENCE, Issue 1 2010Yvonne Turner Abstract A 12-mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell-penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell-penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA-peptide conjugates to be not primarily related to the cell-penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA-peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes.PHYTOTHERAPY RESEARCH, Issue 7 2004Part III. Abstract Flavonoids are found universally in plants and act as free radical scavenging and chelating agents with antiin,ammatory, antiischemic, vasodilating and chemoprotective properties. In this study, the antilipoperoxidative and cytoprotective effects of apigenin, baicalein, kaempferol, luteolin and quercetin against doxorubicin-induced oxidative stress were investigated in isolated rat heart cardiac myocytes, mitochondria and microsomes. After preincubation of cardiomyocytes with the test compounds for 1 h the cardiomyocytes were treated with the toxic agent, doxorubicin (100 µM for 8 h). Cardiomyocyte protection was assessed by extracellular LDH and cellular ADP and ATP production. Cytoprotection was concentration dependent for baicalein > luteolin , apigenin > quercetin > kaempferol. All test compounds had signi,cantly better protective effects than dexrazoxan, an agent currently used for adjuvant therapy during anthracycline antibiotic therapy. In microsomes/mitochondria the IC50 values of lipid peroxidation inhibition for quercetin, baicalein, kaempferol, luteolin, and apigenin were 3.1 ± 0.2/8.2 ± 0.6, 3.3 ± 0.3/9.6 ± 0.5, 3.9 ± 0.3/10.1 ± 0.8, 22.9 ± 1.7/18.2 ± 0.7, and 338.8 ± 23.1/73.1 ± 6.4 mM, respectively. The antilipoperoxidative activity of apigenin differed from its cytoprotective effects, but correlated with the free radical scavenging of 2,2-diphenyl-1-picrylhydrazyl radical and half peak oxidation potential (Ep/2). Apigenin was the least effective of the ,avonoids studied in all models except the cardiomyocyte model where its cardiomyocyte cytoprotective effect was comparable to other compounds. Copyright © 2004 John Wiley & Sons, Ltd. [source] Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes.PHYTOTHERAPY RESEARCH, Issue 2 2004Part I. Silymarin, its flavonolignans Abstract Silymarin, an extract of ,avonolignans from the dried fruits of milk thistle (Silybum marianum L. Gaertneri) and its constituents silibinin, dehydrosilibinin, silychristin and silydianin were tested for protective effects on rat cardiomyocytes exposed to doxorubicin. Silymarin and individual ,avonolignans did not exert cytotoxicity in the range 25,100 µm (incubation 9 h). Dehydrosilibinin was tested only at 25 µm concentration due to its low solubility. All substances increased the cell ATP level. Silymarin and ,avonolignans displayed a dose-dependent cytoprotection against doxorubicin (100 µm, incubation 8 h). The protective effects of silymarin, silibinin, dehydrosilibinin and silychristin were comparable to that of dexrasoxane, while silydianin exerted the best protective effect. The ability of silymarin complex and its components to protect cardiomyocytes against doxorubicin-induced oxidative stress is due mainly to their cell membrane stabilization effect, radical scavenging and iron chelating potency. Copyright © 2004 John Wiley & Sons, Ltd. [source] Proteomic profiling reveals comprehensive insights into adrenergic receptor-mediated hypertrophy in neonatal rat cardiomyocytesPROTEOMICS - CLINICAL APPLICATIONS, Issue 12 2009Zijian Li Abstract Myocardial adrenergic receptors (ARs) play important roles in cardiac hypertrophy. However, the detailed molecular mechanism of AR-mediated cardiac hypertrophy remains elusive to date. To gain full insight into how ARs are involved in the regulation of cardiac hypertrophy, protein expression profiling was performed with comparative proteomics approach on neonatal rat cardiomyocytes. Forty-six proteins were identified as differentially expressed in hypertrophic cardiomyocytes induced by AR stimulation. To better understand the biological significance of the obtained proteomic data, we utilized the ingenuity pathway analysis tool to construct biological networks and analyze function and pathways that might associate with AR-mediated cardiac hypertrophy. Pathway analysis strongly suggested that ROS may be involved in the development of AR-mediated cardiac hypertrophy, which was then confirmed by further experimentation. The results showed that a marked increase in ROS production was detected in AR-mediated cardiac hypertrophy and blocking of ROS production significantly inhibited AR-mediated cardiac hypertrophy. We further proved that the ROS production was through NADPH oxidase or the mitochondrial electron transport chain and this ROS accumulation resulted in activation of extracellular signal-regulated kinase 1/2 leading to AR-mediated cardiac hypertrophy. These experimental results support the hypothesis, from the ingenuity pathway analysis, that AR-mediated cardiac hypertrophy is associated with the dysregulation of a complicated oxidative stress-regulatory network. In conclusion, our results provide a basis for understanding the detailed molecular mechanisms of AR-mediated cardiac hypertrophy. [source] Divergent regulation of cardiac KCND3 potassium channel expression by the thyroid hormone receptors ,1 and ,1THE JOURNAL OF PHYSIOLOGY, Issue 6 2009Natig Gassanov The cardiac transient outward current Ito is regulated by thyroid hormone (T3). However, it remains unclear whether T3 directly modulates underlying gene transcription and which thyroid receptor (TR) isoform might be responsible for gene transactivation. To clarify this situation, we analysed the role of T3 and its receptors ,1 (TR,1) and ,1 (TR,1) in regulation of KCNA4, KCND2, KCND3 and KCNIP2 transcription in rat cardiomyocytes. Initial results demonstrated a T3-mediated increase of Ito current density. T3 stimulation enhanced KCND2 and KCND3 expression and decreased KCNA4 transcription, while KCNIP2 remained unaffected. To dissect the role of TR,1 and TR,1 in T3-dependent Ito modulation, TR,1 and TR,1 were overexpressed in cardiomyocytes by adenovirus-mediated gene transfer. TR,1 increased Ito, while TR,1 significantly reduced Ito in size, which was associated with TR,1-mediated increase and TR,1-mediated reduction of KCND2/3 transcription. To further evaluate a possible direct interaction of TR,1 and TR,1 with the KCND3 promoter, TR expression vectors were cotransfected with a construct containing 2335 bp of the KCND3 5,-flanking sequence linked to a luciferase reporter into ventricular myocytes. While the TR,1 aporeceptor enhanced KCND3 transcription, the TR,1 aporeceptor suppressed KCND3 expression, with both effects exhibiting ligand-dependent amplification upon T3 stimulation. Deletion of the KCND3 5,-flanking region localized the suppressible promoter sequence for TR,1 to within ,293 bp and the activating promoter sequence for TR,1 to within ,2335 to ,1654 bp of the transcription start site. Disruption of putative TR binding sites by mutagenesis abolished the TR,1- (G-1651T) and TR,1- (G-73T) mediated effects, indicating that TR,1 and TR,1 response elements map to different regions of the KCND3 promoter. Thus, Ito is modulated by diverse T3-dependent regulation of underlying gene transcription. TR,1 and TR,1 exhibit distinct effects on KCND3 transactivation with TR,1 enhancing and TR,1 suppressing KCND3 transcription. [source] Effect of intracellular lipid droplets on cytosolic Ca2+ and cell death during ischaemia,reperfusion injury in cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 6 2009Ignasi Barba Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia,reperfusion injury. The addition of oleate,bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and 1H-nuclear magnetic resonance spectroscopy. Simulated ischaemia,reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7 ± 2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P= 0.02). The magnitude of LD accumulation was inversely correlated (r2= 0.68, P= 0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca2+ -sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca2+ overload than control cells. These results suggest that LD exert a protective effect during ischaemia,reperfusion by sequestering free fatty acids and Ca2+. [source] A New Method for Manufacturing Cardiac Cell Sheets Using Fibrin-Coated Dishes and Its Electrophysiological Studies by Optical MappingARTIFICIAL ORGANS, Issue 2 2005Yuji Itabashi Abstract:, We developed a novel simple method for making functional myocardial cell sheets that may be used as transplants. Polymerized human fibrin-coated dishes were prepared with fibrinogen monomers mixed with thrombin. Neonatal rat cardiomyocytes cultured on these dishes formed myocardial cell sheets within 4 days. These cell sheets were easily dissociated intact from the polymerized fibrin layer, because the fibrin had been digested by intrinsic protease. Two overlaid myocardial cell sheets exhibited synchronized spontaneous beating and captured artificial pacing. Optical mapping confirmed that the conduction of the action potential between two partially overlaid myocardial cell sheets was established, and the action potential propagated across the junction without any delay. Transplanted three-layered myocardial cell sheets exhibited strong spontaneous beating and showed well-differentiated striations and an increase in cell size. This simple method of cell sheet engineering may also be applicable for various other cell types. [source] Delayed Protection of Tetramethylpyrazine on Neonatal Rat Cardiomyocytes Subjected to Anoxia-Reoxygenation InjuryBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 6 2007He-Ping Chen Cultured neonatal rat cardiomyocytes were preconditioned using TMP at different concentrations (100, 200 and 500 µM). Cell viability, lactate dehydrogenase release, malondialdehyde formation, superoxide dismutase activity and glutathione peroxidase activity were measured to determine the protective effects against anoxia-reoxygenation injury. The expression of heat shock protein 70 (Hsp70) was measured 24 hr after TMP preconditioning by Western blot analysis. The results showed that TMP decreased lactate dehydrogenase release, increased cell viability, suppressed malondialdehyde formation and augmented activities of superoxide dismutase and glutathione peroxidase in a concentration-dependent manner. Moreover, the delayed protection was abolished by pre-treating with either protein kinase C inhibitor chelerythrine chloride or PD98059, a selective inhibitor of extracellular signal-regulated protein kinase 1/2, respectively, and the expression of Hsp70 was significantly increased in 24 hr after TMP preconditioning that was also suppressed by chelerythrine chloride or PD98059. These results suggest that TMP can induce delayed cardioprotective effects by activation of protein kinase C and extracellular signal-regulated protein kinase 1/2 signalling pathways and subsequent increased expression of Hsp70 in rat neonatal cardiomyocytes. [source] ,-Adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKABRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2010Jorid T Stuenęs Background and purpose:, Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and ,-adrenergic stimulation in regulation of PKB phosphorylation. Experimental approach:, Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies. Key results:, Isoprenaline increased insulin-stimulated PKB Ser473 and Thr308 phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3, Ser9 phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (,1 -agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (,2 -agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser16 phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N6 -benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2,-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N6 -benzoyl-cAMP, but not 007, increased phospholamban Ser16 phosphorylation. Conclusions and implications:, ,-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during ,-adrenergic receptors stimulation. [source] The signal transduction cascade regulating the expression of the gap junction protein connexin43 by ,-adrenoceptorsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2009A Salameh Background and purpose:, In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the ,-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which ,-adrenoceptor stimulation may control Cx43 expression. Experimental approach:, Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH2 -terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM). Key results:, Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC50 of 7.92 ± 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes. Conclusion and implication:, ,-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution. [source] Diazoxide acts more as a PKC- , activator, and indirectly activates the mitochondrial KATP channel conferring cardioprotection against hypoxic injuryBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2006M-Y Kim Background and purpose: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoKATP) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoKATP channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoKATP channel in diazoxide-induced cardioprotection. Experimental approach: In H9c2 cells and neonatal rat cardiomyocytes, PKC-, activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca2+ and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. Key results: Diazoxide (100 ,M) induced translocation of PKC-, from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-, by either ,V1-2 or dominant negative mutant PKC-, (PKC-, KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either ,V1-2 or PKC-, KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-, induced by diazoxide. Transfection with wild type PKC-, mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by ,V1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by ,V1-2 or 5-HD. Conclusions and Implications: Diazoxide induced isoform-specific translocation of PKC-, as an upstream signaling molecule for the mitoKATP channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway. British Journal of Pharmacology (2006) 149, 1059,1070. doi:10.1038/sj.bjp.0706922 [source] Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2001Cyril Goudet Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 ,M), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels. British Journal of Pharmacology (2001) 134, 1195,1206; doi:10.1038/sj.bjp.0704361 [source] The Transcriptional Coactivator p300 Plays a Critical Role in the Hypertrophic and Protective Pathways Induced by Phenylephrine in Cardiac Cells but Is Specific to the Hypertrophic Effect of UrocortinCHEMBIOCHEM, Issue 1 2005Sean M. Davidson Dr. Abstract Anacardic acid is an alkylsalicylic acid obtained from cashew-nut-shell liquid, and is a potent inhibitor of p300 histone acetyl-transferase (HAT) activity. We have used anacardic acid to prevent the induction of hypertrophy in isolated neonatal rat cardiomyocytes. Hypertrophy was detected as an increase in cell size, the rearrangement of sarcomeres into a striated pattern, and the induction of embryonic genes ,-MHC and ANF. p300 inhibition was equally effective at preventing hypertrophy whether it was induced by treatment with the ,1-adrenergic agonist, phenylephrine, or by treatment with urocortin, a member of the corticotrophin-releasing-factor family, which stimulates specific G protein-coupled receptors. Spiruchostatin A is a natural-product inhibitor of histone deacetylases (HDAC) similar to the depsipeptide FK228 molecule. We have recently synthesized spiruchostatin A and now show that, although HDACs act in opposition to HATs, spiruchostatin A has the same effect as anacardic acid, that is, it prevents the induction of hypertrophy in response to phenylephrine or urocortin. Pretreatment with either phenylephrine or urocortin reduced the extent of death observed after the exposure of isolated cardiomyocytes to simulated ischaemia and reoxygenation. Inhibition of p300 or HDAC activity eliminated the protection conferred by phenylephrine; however, it did not affect the protection conferred by urocortin. Therefore, it might eventually be possible to use chemical inhibitors such as these in a therapeutic setting to dissociate the protective effect and hypertrophic effect of urocortin, enhancing the survival of cardiomyocytes exposed to transient ischemia, while inhibiting the hypertrophic pathway that would otherwise be induced concurrently. [source] The phosphatidylinositol 3-kinase,Akt pathway protects cardiomyocytes from ischaemic and hypoxic apoptosis via mitochondrial functionCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2010Hua-Pei Song Summary 1.,After a severe burn, a marked decrease in myocardial blood flow results in ischaemic and hypoxic injury, which subsequently leads to apoptosis or necrosis. Phosphatidylinositol 3-kinase (PI3-K)/Akt is an important intracellular signal transduction molecule that regulates cell proliferation, differentiation, glucose metabolism and migration. However, the function and mechanisms of the PI3-K,Akt pathway in cardiomyocyte apoptosis after a burn remain unclear. 2.,In the present study, an in vivo rat model of burn injury and an in vitro hypoxic model using rat cardiomyocytes were established. In burned rats, the expression of PI3-K and phosphorylated (p-) Akt expression increased, as did myocardial apoptosis. Inhibition of the PI3-K,Akt pathway with 1.4 mg/kg LY294002 caused a significant increase in the myocardial apoptotic index compared with hypoxia alone in the in vivo model. 3.,Cardiomyocytes cultured under hypoxic conditions exhibited increased apoptosis, decreased cell viability, enhanced caspase 3 activity, a decreased mitochondrial membrane potential, increased cytoplasmic calcium transients and increased p53 and Bax mRNA expression. Pretreatment with 50 ,mol/L LY294002 significantly enhanced all these negative indicators compared with hypoxia alone. In contrast, pretreatment of cells with 200 ng/mL insulin-like growth factor-1, an activator of PI3-K,Akt, significantly ameliorated the effects of hypoxia, although control levels were not reached. 4.,These findings indicate that activation of the PI3-K,Akt pathway induced by ischaemia and hypoxia after a severe burn can protect cardiomyocytes from apoptosis. This anti-apoptotic effect is most likely mediated via the mitochondria and changes in p53 and Bax gene expression, intracellular [Ca2+] and caspase 3 activity. [source] ADENOVIRUS-MEDIATED FKBP12.6 OVEREXPRESSION INDUCES HYPERTROPHY AND APOPTOSIS IN CULTURED NEONATAL CARDIOMYOCYTESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 2 2009Jian Zhong SUMMARY 1Cardiac ryanodine RyR2 receptors regulate Ca2+ release from the sarcoplasmic reticulum (SR). FK506 binding protein (FKBP) 12.6 prevents aberrant SR Ca2+ leakage during diastole, thereby maintaining the integrity of RyR2 function. Previous studies have focused mainly on FKBP12.6 deficiency and so the pathophysiological consequences of FKBP12.6 overexpression remain unclear. Herein, we investigate the effect of FKBP12.6 overexpression on cardiac hypertrophic and apoptotic signalling. 2Human FKBP12.6 cDNA was cloned into pAdTrack-CMV and the resulting plasmid, along with a control empty plasmid, were transfected into bacteria. The resulting virus, namely Ad-FKBP12.6 containing green fluorescent protein, was propagated and purified. Neonatal rat cardiomyocytes were infected with this virus. Protein and DNA synthesis were measured by [3H]-leucine and [3H]-thymidine incorporation, respectively. Expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated extracellular signal-regulated kinase 1 or 2 (p-ERK1/2) and Bax were examined by western blotting. 3Compared with control cells, cardiomyocytes that overexpressed FKBP12.6 became hypertrophic and hyperplastic, with increased levels of both p38 MAPK and p-ERK1/2. At the same time, overexpression of FKBP12.6 induced apoptosis of cardiomyocytes, as determined by both Bax protein expression and DNA fragmentation. Rapamycin treatment downregulated the expression of p-ERK1/2, p38 MAPK and Bax in stimulated cardiomyocytes, with or without FKBP12.6 overexpression, and enhanced protein synthesis, but had no effect on DNA synthesis in cardiomyocytes. 4In conclusion, FKBP12.6 overexpression may participate in pathophysiological processes through both hypertrophic and apoptotic signalling pathways, leading to cardiomyocyte damage and death. [source] CREATINE KINASE INHIBITOR IODOACETAMIDE ANTAGONIZES CALCIUM-STIMULATED INOTROPY IN CARDIOMYOCYTESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 2 2009Jun Ren SUMMARY 1Inhibition of creatine kinase is known to suppress cardiac contractile reserve in intact hearts, although the underlying mechanism has not been elucidated. 2The present study was designed to examine whether cardiac depression induced by creatine kinase inhibition was due to action at the level of the essential contractile element, namely cardiomyocytes. Adult rat cardiomyocytes were perfused with the creatine kinase inhibitor iodoacetamide (90 µmol/L) for 90 min. Mechanical and intracellular Ca2+ properties were evaluated using edge-detection and fluorescence microscopy, respectively. Myocytes were superfused with normal (1.3 mmol/L) or high (3.3 mmol/L) extracellular Ca2+ contractile buffer. Mechanical function was examined, including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time to 90% PS (TPS90), time to 90% relengthening (TR90) and integration of shortening/relengthening (normalized to PS). Intracellular Ca2+ transients were evaluated using the following indices: resting and rise of fura-2 fluorescence intensity (,FFI) and intracellular Ca2+ decay time constant. 3The results indicate that elevated extracellular Ca2+ stimulated cardiomyocyte positive inotrope, manifested as increased PS, ±dL/dt, area of shortening, resting FFI and ,FFI associated with a shortened TR90 and intracellular Ca2+ decay time constant. High extracellular Ca2+ did not affect TPS90 and area of relengthening. Iodoacetamide ablated high Ca2+ -induced increases in PS, ±dL/dt, area of shortening, resting FFI, ,FFI and shortened TR90 and intracellular Ca2+ decay time constant. Iodoacetamide itself significantly enhanced the area of relengthening and TR90 without affecting other indices. 4Collectively, these data demonstrate that inhibition of creatine kinase blunts high extracellular Ca2+ -induced increases in cardiomyocyte contractile response (i.e. cardiac contractile reserve). [source] Different roles of ,1 -adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal ratsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 9 2004Yongzhen Zhang Summary 1.,Three different ,1 -adrenoceptor subtypes, designated ,1A, ,1B and ,1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that ,1 -adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of ,1 -adrenoceptors is involved in this response. In the present study, we investigated the different role of each ,1 -adrenoceptor subtype in mediating cardiomyocyte protein synthesis, which is a most important characteristic of cardiac hypertrophy in cultured neonatal rat cardiomyocytes. 2.,Cardiomyocyte hypertrophy was monitored by the following characteristic phenotypic changes: (i) an increase in protein synthesis; (ii) an increase in total protein content; and (iii) an increase in cardiomyocyte size. 3.,The role of each ,1 -adrenoceptor subtype in mediating cardiomyocyte protein synthesis was investigated by the effect of specific ,1 -adrenoceptor subtype-selective antagonists on noradrenaline-induced [3H]-leucine incorporation. In addition, pKB values for ,1 -adrenoceptor subtype-selective antagonists were calculated and compared with the corresponding pKi values to further identify their effects. 4.,Activation of ,1 -adrenoceptors by phenylephrine or noradrenaline in the presence of propranolol significantly increased [3H]-leucine incorporation, protein content and cell size. 5.,Pre-incubating cardiomyocytes with 5-methyl-urapidil, RS 17053 or WB 4101 significantly inhibited noradrenaline-induced [3H]-leucine incorporation. However, there was no effect when cardiomyocytes were pre-incubated with BMY 7378. The correlation coefficients between pKB values for ,1 -adrenoceptor subtype-selective antagonists and pKi values obtained from cloned ,1A -, ,1B - or ,1D -adrenoceptors were 0.92 (P < 0.01), 0.66 (P > 0.05) and 0.24 (P > 0.05), respectively. 6.,Our results suggest that the ,1 -adrenoceptor is dominantly responsible for adrenergic hypertrophy of cultured cardiomyocytes in neonatal rats. The efficiency in mediating cardiomyocyte protein synthesis is ,1A > ,1B , ,1D. [source] | ||||||||||||||||