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PubMed2000

Coupling function of endogenous alpha(1)- and beta-adrenergic receptors in mouse cardiomyocytes.

Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. beta(1)-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective beta(2)-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. beta(2)-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. alpha(1)-ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G(q) alpha-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via beta-ARs in mouse cardiomyocytes, but beta-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in alpha(1)-AR signaling and a defect in beta(2)-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

Animals↗
PubMed2002

Generation of cardiomyocytes from embryonic stem cells experimental studies.

BACKGROUND: Cardiomyopathy is characterized by the loss of functional cardiomyocytes resulting in heart failure. More recently, there is increasing evidence from animal studies that transplantation of cardiomyocytes may represent a valuable approach for the treatment of severe heart failure. DEVELOPMENT OF CARDIAC CELLS: Treatment of cardiovascular diseases using cardiomyocytes derived from embryonic stem cells prerequisites establishment of pure lineages of early embryonic cardiomyocytes from human embryonic stem cells. The development of cardiac cells from embryonic stem cells is regulated by several growth factors such as TGF-beta, IGF FGF and erythropoietin. After binding to their receptor, growth factors induce expression of a number of cardiac specific transcription factors such as the zinc finger GATA proteins and Nkx-2.5, a coactivator of the GATA-4 protein. The GATA-4 transcription factor and Nkx-2.5 are essential for heart development. In parallel to adult cardiomyocytes, embryonic stem cell-derived cardiomyocytes developmentally express cardiac specific proteins and ion channels. GENERATION FROM EMBRYONIC STEM CELLS: Recently, it has been shown that pure cardiomyocytes can be generated from genetically manipulated embryonic stem cells. In order to achieve the selective cardiac differentiation of embryonic stem cells different culture conditions are currently tested to examine in the future the influence of different growth factors. However, although significant progress has been made in generating pure cardiomyocytes, further efforts are required to avoid possible immunological rejection of the engrafted cardiomyocytes. Thus, a main challenge in the future will be the establishment of pure lineages of cardiomyocytes derived from human embryonic stem cells.

Adult↗
PubMed2004

Molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia.

To explore the molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia, burn serum was collected from Wistar rats inflicted with 30% third-degree burn of the total body surface area. The cardiomyocytes of neonatal Wistar rats were cultured and then treated with burn serum and hypoxia (a gas mixture containing 1% O2). The constructed c-jun antisense gene recombinant was transfected into the cardiomyocytes of neonatal Wistar rats. TdT-mediated d-utp nick end labeling (TUNEL) was adopted to examine cardiomyocyte apoptosis. Morphological changes of cardiomyocytes were observed under an optic-microscope and an electron-microscope. Expression of troponin T and beta-tubulin protein, c-jun protein, protein kinase Ca (PKCa), and c-jun N-terminal kinase (JNK) were assayed with Western blot in the transfected and non-transfected groups. The morphology of cardiomyocytes in the non-transfected group changed explicitly, but the change was not so obvious in the transfected cardiomyocytes. The expression of beta-tubulin and troponin increased significantly in the transfected group as compared with the non-transfected group. In the non-transfected group, numbers of apoptotic cardiomyocytes were significantly higher than in the transfected group. The c-jun protein, PKCa, and JNK were significantly expressed in the non-transfected group, and they reached a maximum at the 24th hour after cardiomyocytes were treated with burn serum and hypoxia. In the transfected group, however, expressions of c-jun protein, PKCa, and JNK decreased significantly compared with the non-transfected group. The c-jun antisense gene recombinant transfection alleviates injury to cardiomyocytes treated with burn serum and hypoxia, probably through low expression of PKCa and JNK.

Animals↗
PubMed2005

Suppression of physiological cardiomyocyte proliferation in the rat pup after neonatal glucocorticosteroid treatment.

BACKGROUND: Glucocorticosteroids (mostly dexamethasone) are widely used to prevent chronic lung disease in premature infants. Neonatal rats treated with dexamethasone have been shown to have reduced cardiac mass and cardiomyocyte hypertrophy, suggesting a lower number of cardiomyocytes at adult age, and a severely reduced life expectancy. In the present study we tested the hypothesis that a lower number of cardiomyocytes in later life is caused by a reduced cardiomyocyte proliferation and/or by early cell death (apoptosis). METHODS AND RESULTS: Rat pups received dexamethasone or saline control on day 1, 2 and 3 and were sacrificed at day 0, 2, 4, 7 and 21. The cardiomyocytes of dexamethasone treated pups showed a reduced proliferation as indicated by a lower mitotic index and reduced number of Ki-67 positive cardiomyocytes on day 2 and 4 as compared to day 0 and day 7 and also as compared to the age-matched saline pups. On day 7 and day 21 the mitotic index was not different between groups. From day 2 onward up to day 21 dexamethasone treated pups showed a lower number of cardiomyocytes. The cardiomyocytes showed no signs (<<1%) of apoptosis (Caspase-3 and cleaved-PARP) in any group. CONCLUSION: The temporary suppression of cardiomyocyte hyperplasia found in dexamethasone treated pups eventually leads to a reduced number and hypertrophy of cardiomyocytes during adult life.

Animals↗
PubMed2002

Electrophysiology of single cardiomyocytes isolated from rabbit pulmonary veins: implication in initiation of focal atrial fibrillation.

Pulmonary veins (PVs) are important foci in initiation of paroxysmal atrial fibrillation. However, the mechanisms of the high arrhythmogenic activity of PVs are unclear. This study aimed to isolate single cardiomyocytes from PVs and evaluate their electrophysiological characteristics and arrhythmogenic potential. Cardiomyocytes of rabbit PVs were isolated by retrograde perfusion with digestive enzymes from aorta via left ventricle and left atrium. The action potentials and ionic currents were investigated in isolated single PV cardiomyocytes using the whole-cell clamp technique. Dissociation of PVs yielded single pacemaker cardiomyocytes (76%) and non-pacemaker cardiomyocytes with a fast response action potential. Both the pacemaker and non-pacemaker cardiomyocytes had similar inward Ca2+ currents and transient outward K+ currents. However, the pacemaker cardiomyocytes had a smaller inward rectifier K+ current (1.50 +/- 0.22 versus 4.21 +/- 1.15 pA/pF, P < 0.005) and a larger delayed rectifier K+ current (0.60 +/- 0.05 versus 0.24 +/- 0.05 pA/pF, P < 0.005) than non-pacemaker cardiomyocytes. Acetylcholine induced hyperpolarization and inhibited the spontaneous action potential. Isoproterenol (10 nM) accelerated the spontaneous activity and induced early or delayed afterdepolarization, which could be suppressed by nifedipine. The PV cardiomyocytes with early afterdepolarization have a greater prolongation of action potential duration (deltaAPD, +67 +/- 17 versus -109 +/- 20 ms, P < 0.0001) and a greater increase of inward Ca2+ current (0.90 +/- 0.23 versus 0.38 +/- 0.08 pA/pF, P < 0.05) after isoproterenol than those with out early afterdepolarization. These findings suggest that PV cardiomyocytes have distinct action potentials and ionic current profiles, which may be responsible for the high arrhythmogenic activity of the PVs.

Acetylcholine↗
PubMed2006

Enrichment of cardiomyocytes derived from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cell-derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. METHODS: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. RESULTS: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including alpha-MHC, beta-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell-derived cardiac cell clusters. Moreover, the mES cell-derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. CONCLUSIONS: mES cell-derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering.

Animals↗
PubMed2005

Human adult bone marrow mesenchymal stem cells repair experimental conduction block in rat cardiomyocyte cultures.

OBJECTIVES: We evaluated whether human adult bone marrow-derived mesenchymal stem cells (hMSCs) could repair an experimentally induced conduction block in cardiomyocyte cultures. BACKGROUND: Autologous stem cell therapy is a novel treatment option for patients with heart disease. However, detailed electrophysiological characterization of hMSCs is still lacking. METHODS: Neonatal rat cardiomyocytes were seeded on multi-electrode arrays. After 48 h, abrasion of a 200- to 450-microm-wide channel caused conduction block. Next, we applied adult hMSCs (hMSC group, n = 8), human skeletal myoblasts (myoblast group, n = 7), rat cardiac fibroblasts (fibroblast group, n = 7), or no cells (control group, n = 7) in a channel-crossing pattern. Cross-channel electrical conduction was analyzed after 24 and 48 h. Intracellular action potentials of hMSCs and cardiomyocytes were recorded. Immunostaining for connexins and intercellular dye transfer (calcein) assessed the presence of functional gap junctions. RESULTS: After creation of conduction block, two asynchronously beating fields of cardiomyocytes were present. Application of hMSCs restored synchronization between the two fields in five of eight cultures after 24 h. Conduction velocity across hMSCs (0.9 +/- 0.4 cm/s) was approximately 11-fold slower than across cardiomyocytes (10.4 +/- 5.8 cm/s). No resynchronization occurred in the myoblast, fibroblast, or control group. Intracellular action potential recordings indicated that conduction across the channel presumably occurred by electrotonic impulse propagation. Connexin-43 was present along regions of hMSC-to-cardiomyocyte contact, but not along regions of cardiomyocyte-to-myoblast or cardiomyocyte-to-fibroblast contact. Calcein transfer from cardiomyocytes to hMSCs was observed within 24 h after co-culture initiation. CONCLUSIONS: Human mesenchymal stem cells are able to repair conduction block in cardiomyocyte cultures, probably through connexin-mediated coupling.

Animals↗
PubMed2005

The effect of extracellular matrix on embryonic stem cell-derived cardiomyocytes.

Extracellular matrix (ECM) components significantly influence the growth characteristics of cardiomyocytes, development of spontaneous contractile activity and morphologic differentiation. The present study involves characterization of ES cell-derived cardiomyocytes cultured on different ECMs. We hypothesized that cardiogel, which is a naturally occurring ECM containing a complex mixture of laminin, fibronectin may better support ES cell-derived cardiomyocytes maturation in terms of chronotropic characteristics and subcellular structure development. Spontaneously differentiated cardiomyocytes from 7-day embryoid bodies (EBs) derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a fibroblast-derived ECM (cardiogel), and control (without ECM) for 7 + 21 days. The growth characteristics of cardiomyocytes were assessed by immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR), positive and negative chronotropic drugs and transmission electron microscopy (TEM). The spontaneously beating cells expressed markers characteristic of cardiomyocytes including alpha-actinin, desmin, troponin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin-43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF) and myosin light chain isoform-2V (MLC-2V). In addition, spontaneous beating of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel, by carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine and/or Bay-K. Ultrastructure analysis showed myofibrillar bundle organization and exhibited intercalated discs, T-tubules, Z-, A-, I-, H- and M-bands and the showed earlier maturation of the cardiomyocytes on cardiogel. We conclude that substructurally matured cardiomyocytes can be produced from ES cells and the cardiac fibroblast-derived ECM (cardiogel) supported earlier maturation of ES-derived cardiomyocytes, in terms of chronotropic characteristics and subcellular structure development.

Animals↗
PubMed2006

Cyclosporine A attenuates mitochondrial permeability transition and improves mitochondrial respiratory function in cardiomyocytes isolated from dogs with heart failure.

We used isolated cardiomyocytes to investigate a possible role of mitochondrial permeability transition pore in mitochondrial abnormalities associated with heart failure. Cardiomyocytes were isolated from LV myocardium of normal control dogs and dogs with heart failure produced by intracoronary microembolizations. Mitochondrial permeability transition was measured in isolated cardiomyocytes with intact sarcolemma with and without 0.2 microM cyclosporin A using calcein AM and the fluorometer. State-3 mitochondrial respiration was also measured with the Clark electrode. Mitochondrial membrane potential was measured with JC-1 probe using the fluorometer. Propidium iodide was used to ensure sarcolemma integrity. 200 min after loading with calcein AM, mitochondria of failing cardiomyocytes showed only 50% of maximal level of calcein fluorescence while it remained unchanged in normal cells. The mitochondrial membrane potential in failing cardiomyocytes was significantly decreased by 38% compared to normal cardiomyocytes. Cyclosporine A significantly slowed the exit of calcein from mitochondria of failing cardiomyocytes and increased mitochondrial membrane potential by 29%. State-3 respiration was not affected with cyclosporine A in normal cardiomyocytes while it was significantly increased in failing cardiomyocytes by 20%. Exit of calcein (m.w. 1.0 kDa) from mitochondria of viable failing cardiomyocytes with intact sarcolemma suggests an existence of a reversible transitory permeability transition opening in high conductance mode. Attenuation of calcein exit, DeltaPsi(m) and improvement of state-3 respiration achieved with CsA (0.2 microM) show that permeability transition opening could be a cause of mitochondrial dysfunction described in the failing heart.

Animals↗
PubMed2001

Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro.

OBJECTIVE: Adrenomedullin is a potent hypotensive, natriuretic and diuretic peptide that is coexpressed in the heart with its receptor, suggesting that it may have localized actions as a modulator of cardiac function. Although expression of adrenomedullin is upregulated in the pathological heart, its cardiac function has not been clearly elucidated and it is not known whether this represents a common feature of cardiac hypertrophy, nor whether this is restricted to cardiac myocytes. We have determined the direct effects of hypertrophic agents on cardiomyocyte adrenomedullin gene expression and peptide secretion and have examined the effects of adrenomedullin on biochemical markers of cardiomyocyte hypertrophy. METHODS: Regulation of adrenomedullin expression and its effects on the hypertrophic response were studied in cultured rat neonatal ventricular cardiomyocytes. RESULTS: Incubation with phenylephrine or endothelin for 48 h led to a hypertrophic response with an associated fivefold stimulation of ANP gene expression. In contrast, adrenomedullin mRNA was inhibited by 30-50% in response to phenylephrine or endothelin-mediated hypertrophy, and this was associated with a 35-45% reduction in secretion of immunoreactive adrenomedullin. Phorbol ester mediated activation of protein kinase C and increasing intracellular Ca(2+) with ionomycin led to significant downregulation of adrenomedullin gene expression in cardiomyocytes. Co-incubation with 100 nM adrenomedullin for 48 h inhibited phenylephrine-induced cardiomyocyte hypertrophy as determined by protein:DNA ratio. Adrenomedullin partially blocked phenylephrine-mediated transcriptional activation of ANP and MLC-2 reporter gene expression in cardiomyocytes and this effect was mimicked by 2 microM forskolin, suggesting that this response was mediated via the activation of adenylate cyclase. CONCLUSION: These data demonstrate that the cardiomyocyte adrenomedullin gene is repressed by phenylephrine or endothelin-mediated hypertrophy. The inhibitory effects of adrenomedullin on the cardiomyocyte hypertrophic response suggests that this peptide acts as a regulated autocrine or paracrine modulator of cardiomyocyte function and that downregulation of adrenomedullin expression may play a role in induction and maintenance of cardiomyocyte hypertrophy.

Adenylyl Cyclases↗
PubMed1999

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes.

OBJECTIVES: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. METHODS: Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. RESULTS: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. CONCLUSIONS: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

Animals↗
PubMed2003

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner.

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.

Adenoviridae↗
PubMed2000

Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts.

Growth factors and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac fibroblasts, we tested the involvement of growth factors and hormones in this process. We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 +/- 3%. Moreover, CM derived from 2 h stretched fibroblasts increased the rate of protein synthesis in static fibroblasts as well as in static cardiomyocytes by 8 +/- 2 and 6 +/- 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and fibroblasts released at least three factors with MW < or = 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGFbeta1) being increased by 45 +/- 17 and 21 +/- 4% upon 4 h of stretch, respectively. Fibroblasts released TGFbeta1 and very low quantity of endothelin-1 (ET-1). The release of TGFbeta1 was significantly increased by 18 +/- 4% after 24 h of stretch in fibroblasts. Both cell types released no detectable amount of angiotensin II (Ang II). In conclusion, upon cyclic stretch cardiomyocytes and fibroblasts secrete growth factors and hormones which induce growth responses in cardiomyocytes and fibroblasts in an autocrine/paracrine way. TGFbeta secreted by cardiomyocytes and fibroblasts, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.

Animals↗
PubMed2002

Stretch-induced paracrine hypertrophic stimuli increase TGF-beta1 expression in cardiomyocytes.

Cardiac hypertrophy refers to the abnormal growth of cardiomyocytes, and is often caused by valvular heart disease and hypertension. It involves the activation of growth, including increased protein synthesis and changes in gene expression. Transforming growth factor-beta1 (TGF-beta1) may play a central role in protecting the heart during the hypertrophic response by helping to restore normal functions of the affected myocardium. We tested the hypothesis that cardiomyocytes respond to stretch-induced paracrine hypertrophic stimuli with increased expression of TGF-beta1. To that purpose, we investigated whether angiotensin II (All), endothelin- I (ET-1) and TGF-beta, secreted by stretched cardiac and vascular cells, are involved in the paracrine mechanisms of stretch-induced changes of TGF-beta1 mRNA expression in stationary (i.e. non-stretched) cardiomyocytes. Our results indicated that TGF-beta1 mRNA expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 30-60 min. Furthermore, it is likely that ET-1 and TGF-beta were released by stretched cardiac fibroblasts and endothelial cells to induce TGF-beta1 mRNA expression in stationary cardiomyocytes. Stretched vascular smooth muscle cells did not influence TGF-beta1 mRNA expression in stationary cardiomyocytes. These results indicate that AII, ET-I and TGF-beta, released by cardiac cell types, act as paracrine mediators of TGF-beta1 mRNA expression in cardiomyocytes. Therefore, we conclude that in stretched myocardium the cardiomyocytes, cardiac fibroblasts and endothelial cells take part in intercellular interactions contributing to cardiomyocyte hypertrophy.

Angiotensin II↗
PubMed2001

Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.

1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

Animals↗
PubMed2006

c-Jun N-terminal kinase-mediated stabilization of microsomal prostaglandin E2 synthase-1 mRNA regulates delayed microsomal prostaglandin E2 synthase-1 expression and prostaglandin E2 biosynthesis by cardiomyocytes.

Microsomal prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE(2), a key proinflammatory mediator. The purpose of this study was to elucidate the regulation of mPGES-1 mRNA expression in cardiomyocytes, define the role of JNK enzymes in this process, and characterize the role of mPGES-1 in cardiomyocyte PGE(2) biosynthesis. In neonatal cardiomyocytes, interleukin-1beta and lipopolysaccharide (LPS) both stimulated mPGES-1 mRNA expression and increased mPGES-1 mRNA stability and protein synthesis but failed to increase mPGES-1 mRNA transcription. Treatment with the JNK1/2 inhibitor, SP600125, abrogated the increases in mPGES-1 mRNA stability, mPGES-1 protein synthesis, and PGE(2) release induced by interleukin-1beta or LPS. mPGES-1 protein synthesis was observed in LPS-stimulated neonatal cardiomyocytes from jnk1(-/-) or jnk2(-/-) mice. In contrast, infection of jnk1(-/-) cardiomyocytes with an adenovirus encoding phosphorylation-resistant JNK2 (ad-JNK2-DN), or of jnk2(-/-) cardiomyocytes with ad-JNK1-DN, significantly decreased LPS-stimulated mPGES-1 protein synthesis. Similarly, co-infection with ad-JNK1-DN and ad-JNK2-DN attenuated LPS-stimulated mPGES-1 protein synthesis in cardiomyocytes from wild type mice. Targeted deletion of the gene encoding mPGES-1 led to a 3.2-fold decrease in LPS-stimulated PGE(2) release by cardiomyocytes in comparison with wild type cells but had no effect on COX-1, COX-2, mPGES-2, or cytosolic PGES mRNA levels. These studies provide direct evidence that mPGES-1 mRNA levels in cardiomyocytes are augmented by stabilization of mPGES-1 mRNA, that JNK1 or JNK2 can participate in the regulation of mPGES-1 protein synthesis in these cells, and that mPGES-1 catalyzes the majority of LPS-induced PGE(2) biosynthesis by cardiomyocytes.

Animals↗
PubMed2003

Cardiac beta-adrenoceptor changes in monocrotaline-treated rats: differences between membrane preparations from whole ventricles and isolated ventricular cardiomyocytes.

In monocrotaline (MCT)-treated rats the beta-adrenoceptor-G-protein-adenylyl cyclase system-determined in crude membrane preparations from whole ventricular tissue-was desensitized not only in right (RV) but also in left ventricles (LV). This study aimed to assess the specific contribution of cardiomyocytes to these beta-adrenoceptor changes. Six-week-old male Wistar rats were treated with 60 mg/kg body weight MCT intraperitoneally; within 4-6 weeks, rats developed marked RV hypertrophy. Cardiomyocytes were isolated from RVs and LVs. In RV cardiomyocytes of MCT-treated rats, beta-adrenoceptor density was significantly reduced whereas it was unaltered in LV cardiomyocytes. Reduction of RV cardiomyocyte beta-adrenoceptors was due to a selective beta(1)-adrenoceptor reduction. Isoprenaline (100 microM)-induced cAMP increase was significantly reduced in RV but not in LV cardiomyocytes of MCT-treated rats. G protein-coupled receptor kinase activity was increased in RV but not in LV cardiomyocytes. alpha(1)-Adrenoceptor density and noradrenaline-induced increase in inositol phosphate formation were significantly reduced only in RV but not in LV cardiomyocytes from MCT-treated rats. It is concluded that in cardiomyocytes of MCT-treated rats, cardiac beta-adrenoceptors and alpha -adrenoceptors are chamber-specifically desensitized only in the RV. Thus, changes in cardiac beta-adrenoceptors determined in membrane preparations from whole tissue homogenates do not correctly reflect changes occurring in cardiomyocytes.

Animals↗
PubMed2004

Different roles of alpha1-adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats.

1. Three different alpha1-adrenoceptor subtypes, designated alpha1A, alpha1B and alpha1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that alpha1-adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of alpha1-adrenoceptors is involved in this response. In the present study, we investigated the different role of each alpha1-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 alpha1-adrenoceptor subtype in mediating cardiomyocyte protein synthesis was investigated by the effect of specific alpha1-adrenoceptor subtype-selective antagonists on noradrenaline-induced [3H]-leucine incorporation. In addition, pKB values for alpha1-adrenoceptor subtype-selective antagonists were calculated and compared with the corresponding pKi values to further identify their effects. 4. Activation of alpha1-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 alpha1-adrenoceptor subtype-selective antagonists and pKi values obtained from cloned alpha1A-, alpha1B- or alpha1D-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 alpha1-adrenoceptor is dominantly responsible for adrenergic hypertrophy of cultured cardiomyocytes in neonatal rats. The efficiency in mediating cardiomyocyte protein synthesis is alpha1A > alpha1B >> alpha1D.

Adrenergic alpha-Agonists↗