Tor Skomedal

Dual Serotonergic Regulation of Ventricular Contractile Force Through 5-HT2A and 5-HT4 Receptors Induced in the Acute Failing Heart

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.

Effects of dibenzepine and imipramine on the isolated rat heart

Abstract The tricyclic antidepressants, imipramine and dibenzapine and the local anaesthetic drug lidocaine were added to the recirculating perfusate in a rat heart perfusion apparatus. Each drug produced the following effects: (a) A dose-dependent decrease in impulse generation and impulse conduction velocity. (b) A dose-dependent decrease in cardiac contractile force. (c) A dose-dependent decrease in coronary flow preceded by an increase at lower concentrations. (d) A dose-dependent decrease in potassium loss from the heart. (e) Potentiation of the imipramine-induced decrease in contractile force and coronary flow in the presence of lidocaine. Further, in the concentration range in wich the antidepressants produced their cardiac effects, they stabilized eryhrocytes against hypotonic lysis. The antidepressants and the local anaesthetic share this effect and the cardiac effects with a variety of drugs commonly referred to as membrane stabilizers. It is concluded that the membrane stabilizers probably change the properties of biomembranes in a common way, and that the cardiac effects are secondary to these changes.

Natriuretic peptides increase β1-adrenoceptor signalling in failing hearts through phosphodiesterase 3 inhibition

AIMS Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. METHODS AND RESULTS Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. CONCLUSION An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure--a quantitative RT-PCR study and the question of normalisation.

AbstractCongestive heart failure (CHF) induces changes in the neurohumoral system and gene expression in viable myocardium. Several of these genes encode G protein-coupled receptors (GPCRs) involved in mechanisms which compensate for impaired myocardial function. We used real-time quantitative RT-PCR (Q-RT-PCR) to investigate the expression of mRNA encoding 15 different GPCRs possibly involved in CHF, and the effect of normalisation to GAPDH mRNA (GAPDH) or 18S rRNA (18S). CHF was induced in rats by coronary artery ligation, with sham-operated controls (Sham). After 6 weeks, mRNA expression in viable left ventricular myocardium was determined using both 18S and GAPDH as the normalisation standard. An apparent 30% reduction in GAPDH mRNA levels vs. 18S in CHF compared to Sham, although not significant in itself, influenced the interpretation of regulation of other genes.Thus, levels of mRNA encoding receptors for angiotensin II (AT1), endothelin (ETA, ETB) and the muscarinic acetylcholine (mACh) receptor M1 increased significantly in CHF only when normalised to GAPDH. Levels of mRNA encoding the mACh receptors M3 and M4 and the serotonin receptors 5-HT2A and 5-HT4 increased, whereas α1D-adrenoceptor mRNA decreased in CHF irrespective of the normalisation standard. No significant change was detected for M2 and M5 mACh receptors or α1A-, α1B-, β1- or β2-adrenoceptors. Q-RT-PCR is a sensitive and powerful method to monitor changes in GPCR mRNA expression in CHF. However, the normalisation standard used is important for the interpretation of mRNA regulation.

Increase of cyclic AMP in subcellular fractions of rat heart muscle after β-adrenergic stimulation: Prenalterol and isoprenaline caused different distribution of bound cyclic AMP

Although prenalterol is a partial beta-agonist, it has been reported to produce only a marginal, non-significant increase of cAMP in rat hearts. We studied the effect of prenalterol and isoprenaline on free and bound cAMP content in subcellular fractions of perfused rat hearts to see whether prenalterol increased cAMP and whether there were differences in the subcellular distribution of the cAMP contents after stimulation with the drugs. The in situ binding characteristics of cAMP to the protein kinases at different degree of beta-adrenergic stimulation were elucidated. Prenalterol increased significantly both total cAMP content in homogenate, bound cAMP in a 100,000 g particulate fraction and bound, free and total cAMP contents in the resulting supernatant. While prenalterol increased bound cAMP in the particulate fraction and in the supernatant proportionally, isoprenaline caused a relatively greater increase of bound cAMP in the supernatant. At equieffective concentrations regarding inotropic and lusitropic effects, prenalterol and isoprenaline increased bound cAMP in the particulate fraction to the same degree, while the increase in the other fractions was greater after isoprenaline. Thus, the cAMP bound in the particulate fraction seemed to determine the inotropic state of the cell after beta-adrenergic stimulation, supporting a compartmentation of cAMP in myocardium. In controls 31 to 34% of the total binding capacity for cAMP in the particulate fraction was occupied, increasing to 77% after maximal beta-stimulation. The binding capacity in the supernatant was only 15 to 18% saturated in the basal situation and about half saturated after maximal beta-stimulation. cAMP bound in the particulate fraction is of special interest when cAMP and functional effects are studied.

Cyclic AMP formation and morphology of myocardial cells isolated from adult heart: Effect of Ca2+ and Mg2+

Adult rat heart cells were isolated by perfusion with a calcium-free phosphate buffer containing collagenase. Optimal conditions gave a high proportion of elongated cells. Isoprenaline increased cydic AMP content linearly, with ED50 (dose effective in 50% of the population) about 10(-7) M. Ca2+ made the cells spherical, and it nearly abolished cyclic AMP response as did lack of Mg2+.

Substrate Specificities of G Protein-Coupled Receptor Kinase-2 and -3 at Cardiac Myocyte Receptors Provide Basis for Distinct Roles in Regulation of Myocardial Function

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac β-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, α1-adrenergic, and β1-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC50 for GRK3, 5 ± 0.7 pmol/mg of protein; EC50 for GRK3ct, 2 ± 0.2 pmol/mg of protein). The α1-adrenergic receptor was also a preferred substrate of GRK3 (IC50,7 ± 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and α1-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced β1-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at β1-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling β1-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and α1-adrenergic receptors.

Particle-induced cytokine responses in cardiac cell cultures--the effect of particles versus soluble mediators released by particle-exposed lung cells.

Increased levels of particulate matter have been associated with adverse effects in the respiratory as well as the cardiovascular system. The biological mechanisms behind these associations are still unresolved. Among potential mechanisms, particulate matter-associated cardiac effects may be initiated by inhaled small-sized particles, particle components and/or mediators related to inflammation that translocate into the pulmonary circulation. In the present study cytokine responses (interleukin [IL]-6, IL-1beta, and tumor necrosis factor [TNF]-alpha) of primary rat cardiomyocytes and cardiofibroblasts in mono- and cocultures induced by direct exposure to particles, were compared with cytokine responses induced by mediators released by particle-exposed primary rat epithelial lung cells (conditioned media). Cells were exposed to a model ultrafine particle (ultrafine carbon black, Printex 90) and in selected experiments to an urban air particle sample (SRM 1648, St Louis, MO). In lung cell cultures both particle types induced release of IL-6 and IL-1beta, whereas TNF-alpha was only detected upon exposure to St Louis particles. The release of IL-6 by cardiac cells was strongly enhanced upon exposure to conditioned media, and markedly exceeded the response to direct particle exposure. IL-1, but not TNF-alpha, seemed necessary, but not sufficient, for this enhanced IL-6 release. The role of IL-1 was demonstrated by use of an IL-1 receptor antagonist that partially reduced the effect of the conditioned media, and by a stimulating effect on the cardiac cell release of IL-6 by exogenous addition of IL-1alpha and IL-1beta. These in vitro findings lend support to the hypothesis that particle-induced cardiac inflammation and disease may involve lung-derived mediators.

Effects of treatment with a 5-HT4 receptor antagonist in heart failure

Positive inotropic responses (PIR) to 5‐hydroxytryptamine (5‐HT) are induced in the left ventricle (LV) in rats with congestive heart failure (CHF); this is associated with upregulation of the Gs‐coupled 5‐HT4 receptor. We investigated whether chronic 5‐HT4 receptor blockade improved cardiac function in CHF rats.

Cardiac-restricted Expression of the Carboxyl-terminal Fragment of GRK3 Uncovers Distinct Functions of GRK3 in Regulation of Cardiac Contractility and Growth GRK3 CONTROLS CARDIAC α1-ADRENERGIC RECEPTOR RESPONSIVENESS

G protein-coupled receptor kinase-2 and -3 (GRK2 and GRK3) in cardiac myocytes catalyze phosphorylation and desensitization of different G protein-coupled receptors through specificity controlled by their carboxyl-terminal pleckstrin homology domain. Although GRK2 has been extensively investigated, the function of cardiac GRK3 remains unknown. Thus, in this study cardiac function of GRK3 was investigated in transgenic (Tg) mice with cardiac-restricted expression of a competitive inhibitor of GRK3, i.e. the carboxyl-terminal plasma membrane targeting domain of GRK3 (GRK3ct). Cardiac myocytes from Tg-GRK3ct mice displayed significantly enhanced agonist-stimulated α1-adrenergic receptor-mediated activation of ERK1/2 versus cardiac myocytes from nontransgenic littermate control (NLC) mice consistent with inhibition of GRK3. Tg-GRK3ct mice did not display alterations of cardiac mass or left ventricular dimensions compared with NLC mice. Tail-cuff plethysmography of 3- and 9-month-old mice revealed elevated systolic blood pressure in Tg-GRK3ct mice versus control mice (3-month-old mice, 136.8 ± 3.6 versus 118.3 ± 4.7 mm Hg, p < 0.001), an observation confirmed by radiotelemetric recording of blood pressure of conscious, unrestrained mice. Simultaneous recording of left ventricular pressure and volume in vivo by miniaturized conductance micromanometry revealed increased systolic performance with significantly higher stroke volume and stroke work in Tg-GRK3ct mice than in NLC mice. This phenotype was corroborated in electrically paced ex vivo perfused working hearts. However, analysis of left ventricular function ex vivo as a function of increasing filling pressure disclosed significantly reduced (dP/dt)min and prolonged time constant of relaxation (τ) in Tg-GRK3ct hearts at elevated supraphysiological filling pressure compared with control hearts. Thus, inhibition of GRK3 apparently reduces tolerance to elevation of preload. In conclusion, inhibition of cardiac GRK3 causes hypertension because of hyperkinetic myocardium and increased cardiac output relying at least partially on cardiac myocyte α1-adrenergic receptor hyper-responsiveness. The reduced tolerance to elevation of preload may cause impaired ability to withstand pathophysiological mechanisms of heart failure.