Jacques Noireaud

Mixed β3-adrenoceptor agonist and α1-adrenoceptor antagonist properties of nebivolol in rat thoracic aorta

Nebivolol, a selective β‐adrenoceptor (β1‐AR) antagonist, induces vasodilatation by an endothelium‐ and NO‐cGMP‐dependent pathway. However, the mechanisms involved in the vascular effect of nebivolol have not been established. Thus, we evaluated the role of α1 and β3‐ARs in nebivolol‐induced vasodilatation. The responses to nebivolol were investigated in vitro in thoracic aortic rings isolated from male Sprague–Dawley rats. Nebivolol (0.1–10 μM) significantly shifted the concentration–response curve to phenylephrine, an α1‐AR agonist, to the right in a concentration‐dependent manner (pA2=6.5). Conversely, the concentration–response curve to endothelin 1 (ET1) was unaffected by nebivolol. In ET1‐precontracted rings, nebivolol induced a concentration‐dependent relaxation, which was unaffected by nadolol (a β1/β2‐AR antagonist) but was significantly reduced by L‐748,337 (a β3‐AR antagonist), endothelium removal or pretreatment with L‐NMMA (an NOS inhibitor). Similar results were obtained with a β3‐AR agonist, SR 58611A. It was concluded that, in rat aorta, nebivolol‐induced relaxation results from both inhibition of α1‐ARs and activation of β3‐ARs. In addition, we confirmed that the endothelium and the NO pathway are involved in the vascular effect of nebivolol. The identification of these vascular targets of nebivolol indicate that it has therapeutic potential for the treatment of pathological conditions associated with an elevation of sympathetic tone, such as heart failure and hypertension.

Recent Insights in the Paracrine Modulation of Cardiomyocyte Contractility by Cardiac Endothelial Cells

The cardiac endothelium is formed by a continuous monolayer of cells that line the cavity of the heart (endocardial endothelial cells (EECs)) and the luminal surface of the myocardial blood vessels (intramyocardial capillary endothelial cells (IMCEs)). EECs and IMCEs can exercise substantial control over the contractility of cardiomyocytes by releasing various factors such as nitric oxide (NO) via a constitutive endothelial NO-synthase (eNOS), endothelin-1, prostaglandins, angiotensin II, peptide growth factors, and neuregulin-1. The purpose of the present paper is actually to shortly review recent new information concerning cardiomyocytes as effectors of endothelium paracrine signaling, focusing particularly on contractile function. The modes of action and the regulatory paracrine role of the main mediators delivered by cardiac endothelial cells upon cardiac contractility identified in cardiomyocytes are complex and not fully described. Thus, careful evaluation of new therapeutic approaches is required targeting important physiological signaling pathways, some of which have been until recently considered as deleterious, like reactive oxygen species. Future works in the field of cardiac endothelial cells and cardiac function will help to better understand the implication of these mediators in cardiac physiopathology.

Increased basal contractility of cardiomyocytes overexpressing protein kinase Cϵ and blunted positive inotropic response to endothelin-1

OBJECTIVE Protein kinase C (PKC) is thought to be involved in the regulation of the mammalian cardiac excitation-contraction coupling process by vasoactive peptides like endothelin-1 (ET-1). However, the demonstration of a causal link between activation of specific PKC isoforms and the increase in contractility mediated by ET-1 is still inferential. METHODS By means of adenovirus-mediated gene transfer, we specifically overexpressed PKC epsilon in cultured adult rabbit ventricular myocytes (Ad-PKC epsilon). Myocyte shortening and [Ca2+]i transients under basal and ET-1-stimulated conditions were measured in Ad-PKC epsilon and Ad-LacZ control transfected cells. RESULTS Infection with Ad-PKC epsilon resulted in a strong, virus dose-dependent increase in PKC epsilon protein levels, whereas protein expression of other PKC isoforms remained unchanged. Using a multiplicity of infection of 100 plaque-forming units/myocyte, basal and cofactor-dependent PKC epsilon kinase activity was increased 28- and 90-fold, respectively, when compared to control. Myocyte basal fractional shortening and [Ca2+]i transient amplitude were both increased by 21% (P < 0.05 each) in Ad-PKC epsilon transfected myocytes when compared to Ad-LacZ transfected control myocytes. The positive inotropic effect of ET-1 in control myocytes was markedly blunted in PKC epsilon-overexpressing myocytes. CONCLUSION Specific overexpression of PKC epsilon in rabbit ventricular myocytes increases basal myocyte contractility and [Ca2+]i transients, and modifies their responsiveness to ET-1.

External calcium dependence of extensor digitorum longus muscle contractility during bupivacaine-induced regeneration.

The effect of the intramuscular injection of bupivacaine hydrochloride on selected morphological characteristics and contractile properties of adult rat extensor digitorum longus muscle was studied. Recovery of normal fiber size was already present 30 days after bupivacaine injection and at 90 days after injection, values of the normalized twitch tension (mN/mg of tissue) and of the fatigue index approached those measured in control muscle, whereas the normalized tetanic tension remained 57% of control. At 7–30 days postinjection, twitch force was decreased by reducing [Ca2+]0 (substituted by Mg2+) or adding Co2+ (5 mmol/L−1). By contrast, potentiation of the twitch was recorded in the presence of Cd2+ (2 mmol/L−1). Glycerol treatment only reduced, but did not eliminate, twitches developed by muscles 7 days after injection. Present results emphasize the importance of the recovery process in the loss of the susceptibility of the contractile responses to extracellular calcium in bupivacaine‐injected muscles. These data may be of interest in the evaluation of functional aspects of muscles in which injections of viral vector or autologous myoblasts have been performed.

Rabbit, a relevant model for the study of cardiac β3‐adrenoceptors

The β3‐adrenoceptors (β3‐ARs) have been identified and characterized in the human heart. Specific β3‐AR stimulation, unlike β1‐AR or β2‐AR stimulation, decreases cardiac contractility, partly via the Gi–NO pathway. However, the precise role of cardiac β3‐ARs is not yet completely understood. Indeed, under normal conditions, the β3‐AR response is present only to a very low degree in rats and mice. Therefore, we evaluated whether β3‐ARs were present and functional in rabbit ventricular cardiomyocytes, and whether the rabbit could serve as a relevant model for the study of cardiac β3‐ARs. We used RT‐PCR and Western blot to measure the β3‐AR transcripts and protein levels in rabbit ventricular cardiomyocytes. We also analysed the effect of β3‐AR stimulation using isoproterenol in combination with nadolol or SR 58611A on cardiomyocyte shortening, Ca2+ transient, L‐type Ca2+ current (ICa,L), delayed rectifier potassium current (IKs) and action potential duration (APD). For the first time, we show that β3‐ARs are expressed in rabbit ventricular cardiomyocytes. The mRNA and protein sequences present a high homology to those of rat and human β3‐ARs. Furthermore, β3‐AR stimulation decreases cardiomyocyte shortening, Ca2+ transient and ICa,L amplitudes, via a Gi–NO pathway. Importantly, β3‐AR stimulation enhances IKs amplitude and shortens the APD. Taken together, our results indicate that the rabbit provides a relevant model, easily used in laboratories, to study the roles of cardiac β3‐ARs in physiological conditions.

Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle

Abstract One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation–contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR α1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507–515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation–contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well.

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.

Pharmacologic evaluation of isometric contraction-relaxation coupling indexes in rabbit ventricular muscle

Investigations of the coupling between contraction and relaxation (contraction-relaxation [CRC] process) in isometric conditions are essential in determining whether pharmacologic interventions or cardiac diseases specifically modify isometric relaxation (intrinsic lusitropic effect) or change it in proportion with the accompanying changes in contractility (or inotropy). For this purpose, the CRC process is quantified by various indexes, derived from differentiation and/or curve fitting the whole or relaxation phase of the isometric twitch, one of the most used being tau, the time constant of the final iso(volu)metric phase of relaxation. Nevertheless, the possible redundancy and validity of such indexes have not been thoroughly investigated. Accordingly, we performed a pharmacologic evaluation of such indexes in isolated rabbit ventricular muscles isometrically contracting in vitro, using modifiers of either intracellular Ca(2)+ handling (nifedipine, ryanodine, 2,5-di-tert-butyl-benzohydroquinone, all negative inotropic compounds, and BAY K 8644, a positive inotropic drug), or myofibrillar Ca(2)+ sensitivity (CGP 48506, a Ca(2)+ sensitizer, and butanedione monoxime, a Ca(2)+ desensitizer, respectively positive and negative inotropic compounds). The isometric twitch in control conditions and in the presence of increasing concentration of each compound was analyzed to determine the classically used CRC and/or lusitropic indexes, derived either from single parameters such as the maximal rate or contraction and relaxation (+dT(max) and -dT(max), respectively), or from curve fitting of the whole, or part, of the twitch. As the rate of isometric relaxation is dependent on myofilament properties, we expected that compounds modifying myofibrillar Ca(2)+ sensitivity in an opposite direction (CGP 48506 vs butanedione monoxime) would be the only drugs exerting an intrinsic lusitropic and opposite effect on a validated CRC index. Results showed that (1) none of the tested compounds affected the slope of the linear relationship between peak twitch tension and dT(max), a previously assumed CRC index, sensitive only to myofibrillar Ca(2)+ sensitivity modifiers; (2) the lusitropic parameter B, derived from mathematical curve fitting of the whole isometric twitch, and the ratio +dT(max)/dT(max), exhibited similar drug- and dose-dependency, but no opposite sensitivity to CGP 48506 and BDM for either index; and (3) negative inotropic compounds dose-dependently slowed relaxation (and conversely for positive inotropes), whether the latter was quantified by the rate constant beta, derived from double exponential curve fitting of the whole relaxation phase, or by the time constants tau(L) and tau(E), derived from the curve fitting (logistic and monoexponential, respectively) of the final phase of relaxation. Nevertheless, the pharmacologicly induced changes in beta were statistically significant at lower concentrations and exhibited less individual variability, compared with the time constants. We demonstrate that intrinsic lusitropic changes can be quantified by the value of the slope of the relationship relating beta to peak isometric tension: the slope value was unchanged by Ca(2)+ handling modifiers, decreased by CGP 48506, and reversed by BDM (indicating number, negative, and positive intrinsic lusitropic effects respectively). Based on these data, we propose that the linear relationship between beta and peak isometric tension could be used a new method to assess whether pharmacologic interventions or cardiac diseases exert intrinsic effects on isometric relaxation.

In situ study of the sarcoplasmic reticulum function in control and mdx mouse diaphragm muscle.

Sarcoplasmic reticulum (SR) calcium handling in diaphragm was compared between mdx mice (7-8 weeks old) and age-matched controls. The total SR Ca2+ load was released from the SR by rapidly cooling muscle bundles from 22 to -1 degree C. The plateau amplitude of the rapid cooling contracture (RCC) was considered as an index of the SR Ca2+ content. The steady-state RCC amplitude was significantly lower by 50% in mdx bundles mainly because of a decreased capacity of the dystrophic diaphragm to generate maximal tension. There was no significant difference between either RCC time to peak or the time to half-relaxation of the transient, spike-like, contractile response induced by muscle rewarming. The recovery process of RCC was studied by using a paired RCC protocol. In both groups, at the shortest interval (10 s) between two RCCs, the amplitude of the second RCC was decreased by 25% compared with the first RCC. Increasing the time interval led to progressive monoexponential recovery of the second RCC with similar time constants in control and mdx diaphragm. These results indicate that the dystrophic process does not significantly alter SR Ca2+ uptake nor Ca2+ redistribution within the muscular cell.

Positive influence of AT1 receptor antagonism upon the impaired celiprolol-induced vasodilatation in aorta from spontaneously hypertensive rats

We evaluated celiprolol-induced vasodilatation in aorta taken from 12-week-old spontaneously hypertensive rats (SHR) and the effect of AT(1) angiotensin II receptor antagonism on the vasodilatory action of celiprolol in Wistar Kyoto (WKY) rats and SHR. In WKY rats, the celiprolol-induced relaxation was greatly decreased in denuded aorta, and completely abolished in intact aorta by N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM). In SHR, celiprolol-induced relaxation was reduced compared to WKY rats (E(max) (value obtained for the highest concentration, 300 microM)=39.1+ or - 3.78%, n=21 vs. 80.4 + or - 3% in WKY rats, n=10; P<0.0001). Endothelium removal or pre-treatment with l-NAME did not alter celiprolol-induced relaxation in SHR. In both strains, relaxation to celiprolol was decreased in the presence of nadolol (a beta(1)/beta(2)-adrenoceptor antagonist, 10 microM). N-[[3-[(2S)-2-hydroxy-3-[[2-[4-[(phenylsulfonyl)amino] phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide (L748337, a beta(3)-adrenoceptor antagonist, 7 microM) had no effect. A 12-day treatment with candesartan cilexetil (an AT(1) receptor antagonist, 0.37 or 1mg/kg/day) reduced systolic blood pressure in both strains, but only improved relaxation to celiprolol in SHR, and only at the highest dose (E(max)=64.2+/-3.9%, n=10, P<0.0001 vs. SHR control). In both strains, local aortic AT(1) receptor antagonism with candesartan CV11974 (100 microM) had no effect. The endothelial beta(1)/beta(2) relaxation induced by celiprolol was therefore impaired in SHR aorta and AT(1) receptor antagonism improved the response to celiprolol, in conjunction with a reduction in blood pressure. This work highlights the need to analyse the potential benefit of a combination of celiprolol/AT(1) receptor antagonist in the treatment of hypertension.