Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Expression of alpha1-adrenergic receptor subtypes in heart cell culture.

Three alpha1-AR subtypes have been cloned so far and are designated as alpha1a, alpha1b,, and alpha1d. Organ-specific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of alpha1-AR subtypes on RNA- and protein-level in heart tissue, cultured cardiomyocytes and non-myocytes of the rat. Each alpha1AR-subtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the alpha1a-AR subtype is preferentially expressed in adult cardiomyocytes, the alpha1b-AR subtype was preferentially expressed in the non-myocyte cell fraction. The RT-PCR results were confirmed by Western-blotting (alpha1b) and immunocytochemical studies. Incubation with an alpha1-agonist (phenylephrine) for 72 h led to a significant reduction of the alpha1b-AR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an alpha1-antagonist (prazosin) induced a 1.6 fold upregulation of the alpha1a-AR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the alpha1-AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.

Inducible nitric oxide synthase in the myocard.

Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte--along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

Autoantibodies against the beta- and muscarinic receptors in cardiomyopathy.

The sera of patients with idiopathic dilated cardiomyopathy and the Chagas' disease contain agonist-like autoantibodies directed against the beta 1-adrenoceptor and/or the muscarinic M2-receptor. The anti-beta 1-adrenoceptor antibodies could be directed against amino acid sequences of the first or second extracellular loop. In patients with dilated cardiomyopathy the first as well as the second extracellular loop was identified as an antibody epitope. In Chagas' disease the anti-beta 1-adrenoceptor antibody recognizes only 1 epitope on the second extracellular loop. The anti-beta 1-adrenoceptor antibodies acting like the beta-adrenergic agonist isoprenaline and exert a positive chronotropic effect in cultured rat cardiomyocytes. In contrast to isoprenaline the antibody caused no downregulation of the beta-adrenergic signal transduction cascade within 6 hours. The anti-M2 receptor antibodies recognize in both diseases an epitope on the second extracellular loop. The anti-M2-receptor antibody exert a negative chronotropic response in cultured cardiomyocytes. This antibody induced no downregulation of the muscarinic M2-receptor. The negative chronotropic effect was unabated for 6 hours. Based on these findings it is believed that the agonist-like autoantibodies that act against the beta 1-adrenoceptor and the muscarinic M2-receptor may play a role in the pathogenesis of dilated cardiomyopathy and Chagas' disease.

Agonist-like beta-adrenoceptor antibodies in heart failure.

Anti-beta1-adrenoceptor antibodies may play a harmful role, and the elimination of these antibodies could have beneficial effects for some patients with dilated cardiomyopathy. In vitro experiments showed that the antibody was able to influence the function of cultured cardiomyocytes. In these experiments, the antibody prevented the down-regulation of the beta-adrenoceptor-mediated chronotropic response. This lack of desensitization, which resulted in permanent stimulation, could also influence the Ca2+ homeostasis of cardiomyocytes. However, in longer-term (72 hours)-treated cells, the antibodies were able to decrease subtype-specific expression of the beta1 adrenoceptor. In animal experiments, it was shown that long-term immunization with a peptide corresponding to the second extracellular loop of the beta1 adrenoceptor induced a failing heart similar to that in dilated cardiomyopathy. In humans, we observed a remarkable correlation between disappearance of the antibodies and improvement of heart function. Furthermore, in anti-beta1-adrenoceptor-positive patients with dilated cardiomyopathy treated with the immunoadsorption technique, removal of the antibodies also led to improvement of cardiac function and quality of life. This finding indicates that autoimmune processes may be involved in some patients with dilated cardiomyopathy.

Agonistic anti-beta1-adrenergic receptor autoantibodies from cardiomyopathy patients reduce the beta1-adrenergic receptor expression in neonatal rat cardiomyocytes.

BACKGROUND: Autoantibodies directed against the beta1-adrenergic receptor have been described in patients with dilated cardiomyopathy. These autoantibodies exert an agonistic, chronotropic effect on spontaneously beating cultured neonatal rat cardiomyocytes. We studied the effect of such antibodies on beta1-adrenergic receptor expression. METHODS AND RESULTS: Cardiomyocytes were incubated with either the beta-adrenergic agonist isoproterenol or autoantibodies for 72 hours. beta-Adrenergic receptor expression was studied on the mRNA level with semiquantitative reverse transcription-polymerase chain reaction and on the protein level with immunoblotting. Isoproterenol downregulated both mRNA and beta1- and beta2-adrenergic receptor protein subtypes, whereas the anti-beta1-adrenergic receptor autoantibodies decreased only the beta1-adrenergic receptor mRNA and protein. Long-term incubation of cultured cardiomyocytes with isoproterenol or the anti-beta1-adrenergic receptor autoantibodies reduced the acute stimulatory effect of isoproterenol on the myocytes. These effects were prevented by incubating the cells with isoproterenol in the presence of propranolol or with anti-beta1-adrenergic receptor autoantibodies in the presence of bisoprolol. Bisoprolol also abolished the reduction of the beta1-adrenergic receptor expression caused by longer-term incubation with isoproterenol and the autoantibodies. CONCLUSIONS: We conclude that after longer-term treatment with the anti-beta1-adrenergic receptor autoantibodies, the rat cardiomyocytes showed a beta-adrenergic receptor expression similar to that observed in failing hearts from patients with dilated cardiomyopathy.

Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture.

Recognition of the role of nitric oxide (NO) in cardiovascular regulations raised an acute interest in NO-generating enzymes-nitric oxide synthases (NOS). Nevertheless, the subcellular localization of inducible isoform of NOS (NOS II) and regulation of its expression in the cardiomyocyte still remains to be elucidated. Therefore, we focused this study on the subcellular localization of NOS II in cultured neonatal rat cardiomyocytes using immunocytochemical techniques at the light and electron microscopic level as well as the demonstration of NADPH-diaphorase activity and the Griess assay for NO measurement. Cultivation of neonatal cardiomyocytes during 2 and more days induced a moderate increase in the NOS II immunolabeling in defined cytoplasmic structures and a nuclear NOS II staining in some cells. Exposure of the cell cultures to exogenous cAMP markedly stimulated NO production with a concomitant enhancement of NOS II immunolabeling of cardiomyocytes. cAMP-induced changes were significantly attenuated by dexamethasone. This report provides evidence for the localization of NOS II in the perinuclear space, Golgi complex, mitochondria, plasma membrane and along contractile fibers of cardiomyocytes, as well as for the appearance of NOS II staining of the cell nuclei in the course of cultivation. In non-cardiomyocytes contaminating the cell culture, positive immunoreaction was detected in the Golgi complex and endoplasmic reticulum. Our data point to a notable constitutive expression of NOS II in rat cardiomyocytes apparently dependent on the developmental stage.

Expression of sense and naturally occurring antisense mRNA of myosin heavy chain in rat heart tissue and cultivated cardiomyocytes.

We investigated the expression of sense- as well as naturally occurring antisense-mRNA of myosin heavy chains (MHC) in rat cardiomyocytes during development and cultivation. Relative distribution of the two MHC mRNA isoforms (alpha and beta) was studied by quantitative polymerase chain reaction. Primers were specific for the 3' untranslated region of alpha- and beta-MHC. Sense-alpha-MHC mRNA increased from 0% in the fetal heart to 45 +/- 8% in neonatal and to 75 +/- 5% in adult rat myocardium. In neonatal heart cell culture (10% bovine calf serum), alpha-MHC mRNA levels changed from 49 +/- 3% (day 0 = fresh isolated cells) to 61 +/- 6% (day 5 of cultivation). alpha-MHC mRNA of cultivated adult cardiomyocytes (serum-free) decreased from 90 +/- 7% (day 0) to 30 +/- 5% (day 5). Antisense-mRNA of both alpha- and beta-MHC was detected in rat cardiac tissue and cultivated cardiomyocytes. The expression of antisense-MHC mRNA did not change during development and cultivation. Antisense-alpha-MHC mRNA was the abundant isoform and was half of sense-alpha-MHC mRNA.

The sera of spontaneously hypertensive rats contain agonistic auto-antibodies against the beta 1-adrenoceptor.

OBJECTIVE: To examine auto-antibodies in the sera of spontaneously hypertensive rats (SHR) and investigate the possibility of abnormalities of the immune system in those rats. DESIGN: Blood samples were taken from 18-month-old SHR and age-matched Wistar-Kyoto (WKY) rats. The immunoglobulin fraction was prepared from the rat sera. METHODS: Immunoglobulins were investigated in a sensitive biological test system using spontaneously beating neonatal rat heart myocytes. RESULTS: Immunoglobulins prepared from the sera of the 18-month-old SHR increased the beating frequency of cultured rat heart myocytes. This positive chronotropic effect induced by the auto-antibody-containing immunoglobulin fraction was realized via the beta 1-adrenergic receptor. CONCLUSIONS: The sera of the investigated ageing SHR contain agonistic auto-antibodies directed against the beta 1-adrenoceptor. These anti-beta-adrenoceptor antibodies in the SHR, like those in the sera of cardiomyopathic patients, recognize in most cases the second extracellular loop of the beta 1-adrenoceptor as the site of the antigenic determinant, and act in a similar way to the antibodies observed in the sera of cardiomyopathic patients. The present findings provide the opportunity of using ageing SHR as a model to investigate the development and possible pathogenic role of the agonistic auto-antibodies that recognize the beta 1-adrenoceptor.

Immunocytochemical studies of the Gi protein mediated muscarinic receptor-adenylyl cyclase system.

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that: 1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts. 2. Gia was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. Goa were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures. 3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.

Anti-beta 1-adrenoceptor autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: mapping of epitopes in the first and second extracellular loops.

In a preceding communication (Wallukat et al., 1992, Z Kardiol 81 [Suppl. 4]: 79-83), it was reported that synthetic peptides, corresponding in amino acid sequence to either the first or the second extracellular loop of the human beta 1-adrenoceptor, selectively suppressed the metoprolol- and bisoprolol-sensitive positive chronotropic action exerted in cultures of beating neonatal rat cardiomyocytes by the serum immunoglobulin fraction of patients with myocarditis and idiopathic dilated cardiomyopathy (DCM) and by affinity-purified autoantibodies from that fraction. These observations added to existing evidence that these antibodies were directed against the beta 1-adrenoceptor and might thus contribute to the harmful chronic cardiac adrenergic drive to which patients with DCM are believed to be exposed. Specifically, they pointed to the putative first and second extracellular loops of this receptor (these loops are each identical in man and the rat) as the sites of epitopes recognized by the chronotropically active, beta 1-agonistic autoantibodies. Now we report on the mapping of these epitopes with the help of two series of short synthetic overlap peptides, one series forming part of the first and the other of the second extracellular loop of the beta 1-adrenoceptor. Inhibition of the positive chronotropic response of cultured rat cardiomyocytes to the anti-beta 1-receptor autoantibodies (EC50 = 0.14 +/- 0.01 nM) from the serum immunoglobulin fraction of patients with DCM was taken as reflecting the neutralization of these antibodies by a particular overlap peptide. In this way the sequences S-F-F-C-E-L (residues 129-134) and A-R-R-C-Y-N-D (residues 206-212) emerged as the dominant epitopes in the first and second extracellular loops, respectively, followed with respect to neutralizing ability by the first loop sequence E-Y-G-S-F-F (residues 126-131) and the second loop sequences H-W-W-R-A-E (residues 197-202) and P-K-C-C-D-F (residues 213-218). Synthetic peptides corresponding to the sequences of the third extracellular loop of the beta 1-receptor (residues 346-356) and of the second extracellular loop of the human beta 2-receptor (residues 172-197) failed to neutralize the beta 1-agonistic autoantibodies. Using dithiothreitol as a reducing agent a disulfide bridge between cysteine 132 in the first and cysteine 209 in the second extracellular loop was considered to be essential for the chronotropic action of these autoantibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of the beta-adrenergic response of cardiomyocytes by specific lipoxygenase products involves their incorporation into phosphatidylinositol and activation of protein kinase C.

Arachidonic acid and its 15-lipoxygenase metabolite (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid ((15S)-HETE) modulates the beta-adrenergic response of cultured rat neonatal cardiomyocytes as indicated by an increase in the beating rate following stimulation of the cells with suboptimal isoproterenol concentrations. The effect of 15-HETE was enantioselective for the S-isomer and could be detected at concentrations as low as 10(-12) M. (11S)-HETE and (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetraen oic acid did also exhibit this effect, whereas other mono-, di-, and trihydroxyeicosanoids as well as the 15-lipoxygenase products of 11,14-eicosadienoic acid, of two eicosatrienoic acid isomers and of 5,8,11,14,17-eicosapentaenoic acid were ineffective. Immunohistochemical studies indicated the expression of a 15-lipoxygenase in cardiomyocytes and in resident heart mast cells. Induction of the beta-adrenergic supersensitivity is paralleled by a selective incorporation of (15S)-HETE into the cellular phosphatidylinositol pool. In contrast, (12S)-HETE, which did not induce beta-adrenergic supersensitivity, was incorporated preferentially into phosphatidylcholine and phosphatidylethanolamine. Calphostin C, an inhibitor of protein kinase C, blocked both the induction of supersensitivity by (15S)-HETE and its incorporation into phosphatidylinositol. These data suggest that in cardiomyocytes 15-lipoxygenase metabolites specifically induces a signal transduction cascade leading to a supersensitivity of the cells toward beta-adrenergic agonists, which involves the phosphatidylinositol cycle and a protein kinase C.

Mast cells detected in cultures of neonatal rat heart cells.

Although the presence of mast cells in heart muscle is well documented, their possible presence in heart cell cultures has not yet been considered. In this paper we show for the first time that cultures from neonatal rat hearts contain mast cells in proportions which may exert physiological influences on heart muscle cells. Evidence is based on cytochemistry, immunocytochemistry with a monoclonal antibody (F2) specific for connective tissue mast cells of the rat, and direct estimations of both cellular histamine and histamine released into the culture medium. Since the number of nonmuscle cells immunoreactive with antibody F2 exceeded the number of cells reacting with conventional cytochemical stains for mast cells, a substantial proportion of the former may represent less differentiated (precursor) cells.

[Autoantibodies against the beta 1-adrenergic receptor in myocarditis and dilated cardiomyopathy: localization of two epitopes]. / Autoantikörper gegen den beta 1-adrenergen Rezeptor bei Myokarditis und dilatativer Kardiomyopathie: Lokalisation von zwei Epitopen.

Sera of patients with myocarditis and dilated cardiomyopathy contain stimulatory autoantibodies directed specifically against the beta 1-adrenergic receptor. The binding of the antibodies could be localized to either the first or the second extracellular loop of the beta 1-adrenoceptor. In 73% of the cases investigated the antibodies recognized the second extracellular loop. The agonistic effects of the antibodies were abolished by beta-adrenergic antagonists. Furthermore, the antagonists were able to remove the antibodies from their binding sites.

Arachidonic acid metabolism in cultured adult myocardial cells under short-time hypoxic conditions.

The present study utilized a cultured adult myocardial cell model to examine the arachidonic acid metabolism under different cell-damaging and normoxic conditions. Cell injury was caused by short-time hypoxia, calcium ionophore A 23187-triggered cell-damage under hypoxia and cell disruption by freezing and thawing. The current study demonstrates that under the cell-damaging conditions cultured adult heart myocytes resemble myocardial cells under normoxic conditions in metabolizing arachidonic acid into triacylglycerols and phospholipids as the major route (a), in formation of ETYA-inhibitable indomethacin-resistant lipid metabolites in minor amounts (b) and in being independent of calcium overload in the metabolic pathways of arachidonic acid metabolism (c). The ETYA-inhibitable components were resolved by HPLC. There was no evidence in formation of lipoxygenase products. The results were supported by negative hybridisation experiments of the total mRNA isolated from adult myocardial cells with a cDNA probe of a red-cell-specific lipoxygenase mRNA. We conclude from these observations that cell injury does not result in expression of lipoxygenase activities in heart myocytes.

The cultured myocardial cells as a model in cardiac research.

Cultured myocardial cells have become a standard tool in experimental cardiology. Advantages of this model are pointed out, and some of the results obtained demonstrate the usefulness of this model for investigations of contractile, biochemical, and electrophysiological parameters. The first report on cultivated heart cells was published 75 years ago by BURROWS. He described the independent pulsatory activity of single cells grown out of a tissue explant. This observation confirmed the old view that the cell and not a syncytium acts as the elementary structural and functional unit of heart muscle. More than 40 years later RINALDINI (1954) and CAVANOUGH (1955) succeeded in isolating discrete single cells from embryonic avian heart tissue and HARARY (1963) from postnatal mammalian heart muscle by means of proteolytic enzymes (for review see 1). Today myocardial cells in primary culture can be kept alive and physiologically active for several weeks, but there exist not yet a permanent line of cardiac muscle cells with specific attributes of excitability and contractility. Cultivated myocardial cells have become a standard tool in experimental cardiology during the last 10 to 15 years. They provide a model system to study problems of development, differentiation, adaptation, and regulation of metabolism and function. This model affords unique advantages as compared to intact myocardium for solving some of these problems.