To corroborate alterations in the functional responses to β-adrenergic receptor (β-AR) stimulation with changes in β-AR signaling in failing cardiomyocytes, contractile and L-type Ca 2+ current responses to isoproterenol along with stimulated cAMP generation were compared among cardiomyocytes isolated from canines with tachycardia-induced heart failure or healthy hearts. The magnitude of shortening of failing cardiomyocytes was significantly depressed (by 22 ± 4.4%) under basal conditions, and the maximal response to isoproterenol was significantly reduced (by 45 ± 18%). Similar results were obtained when the responses in the rate of contraction and rate of relaxation to isoproterenol were considered. The L-type Ca 2+ current amplitude measured in failing cardiomyocytes under basal conditions was unchanged, but the responses to isoproterenol were significantly reduced compared with healthy cells. Isoproterenol-stimulated cAMP generation was similar in sarcolemmal membranes derived from the homogenates of failing (45 ± 6.8) and healthy cardiomyocytes (52 ± 8.5 pmol cAMP · mg protein −1 · min −1 ). However, stimulated cAMP generation was found to be significantly reduced when the membranes were derived from the homogenates of whole tissue (failing: 67 ± 8.1 vs. healthy: 140 ± 27.8 pmol cAMP · mg protein −1 · min −1 ). Total β-AR density was not reduced in membranes derived from either whole tissue or isolated cardiomyocyte homogenates, but the β 1 /β 2 ratio was significantly reduced in the former (failing: 45/55 vs. healthy: 72/28) without being altered in the latter (failing: 72/28, healthy: 77/23). We thus conclude that, in tachycardia-induced heart failure, reduction in the functional responses of isolated cardiomyocytes to β-AR stimulation may be attributed to alterations in the excitation-contraction machinery rather than to limitation of cAMP generation.
Introduction: Cough and airway irritation are adverse effects (AEs) associated with inhaled treprostinil (TRE). The site of action causing these AEs is unknown. Aim: Nebulized TRE, at a concentration that causes cough in guinea pigs, was administered to the isolated laryngeal airway of rats and guinea pigs to determine if the actions of TRE involved activation of laryngeal reflexes. Methods: The laryngeal airway was isolated in anesthetized male Sprague Dawley rats and Dunkin Hartley guinea pigs for administration (20 sec) of either nebulized TRE (30 µg/mL), citric acid (CA), hypertonic saline (HS) or phosphate buffered saline (PBS). Pulmonary airflow and expiratory duration (TE) were measured with a pneumotachograph connected to a tracheal catheter. The apneic index (TE post-challenge/TE pre-challenge), mean arterial blood pressure (MAP), heart rate (HR), arterial oxygen saturation (SaO2) and the number of swallows were measured before and after drugs. Results: In rats, nebulized CA (0, 0.01, 0.1 and 1 M) increased the apneic index (1, 32, 90, 101, respectively), MAP and swallowing and decreased SaO2 and HR. Similar effects occurred with nebulized HS (1.5, 3, 5 and 7 %). However, nebulized TRE (30 µg/mL), like nebulized PBS had no effects. In guinea pigs, nebulized CA (0, 0.1 and 1 M) increased the apneic index (2, 3 and 15, respectively) and produced cough and swallowing. Nebulized TRE (30 µg/mL) had no effects. Conclusions: These results demonstrate that nebulization of TRE to the laryngeal airway of rats and guinea pigs does not produce cough and airway irritation and suggest that sites more distal in the tracheobronchial tree and lung are involved.
Pulmonary arterial hypertension (PAH), a condition that is defined by pulmonary vasculature constriction and remodeling, involves dysfunctional signaling of the serotonin (5-HT) receptors, 5-HT2A/2B/7. In a rat model of monocrotaline (MCT)-induced PAH, the effectiveness of RP5063 (RP), a dopamine and 5-HT receptor modulator, was evaluated as monotherapy and as an adjunct to standard PAH treatments. After a single 60 mg/kg dose of MCT, rats received vehicle (MCT+Veh; gavage twice-daily [b.i.d.]), RP (10 mg/kg; gavage b.i.d.), bosentan (B; 100 mg/kg; gavage BID), sildenafil (S; 50 mg/kg; gavage, BID), treprostinil (T; 100 ng/kg/min over 24 h intravenous), RP+B, RP+S, and RP+T for 28 days. Single-agent RP limited the functional and structural effects of PAH seen in the MCT+Veh group, with significant improvements in pulmonary hemodynamics, right ventricular (RV) hypertrophy, SO2, and pulmonary blood vessel structural changes. These effects appeared comparable with those associated with B, S, and T. Adjunctive RP treatment resulted in significantly lower mean pulmonary arterial pressures, RV systolic pressure. It also improved SO2 measurements, as compared with MCT+Veh (P < 0.05), and diastolic pulmonary artery pressure (P < 0.05), as compared with single-agent B and S therapy (Bonferroni method adjusting for multiplicity). RP+S appeared to show the most consistent and extensive effects on pulmonary hemodynamics, respiratory parameters, and histopathologic changes. These results corroborate earlier preclinical findings supporting the efficacy of single-agent RP in PAH. RP, as mono and adjunctive therapy compared with induced-control, mitigated the functional and structural effects of MCT-induced PAH.
To investigate the capacity of efferent sympathetic neurons to modulate the failing heart, stellate ganglion stimulation was performed in dogs with biventricular heart failure induced by rapid ventricular pacing (240 beats/min) for 4-6 weeks. Less noradrenaline was released from cardiac myoneural junctions into coronary sinus blood in response to left stellate ganglion stimulation in anesthetized failing heart preparations (582 pg/mL, lower and upper 95% confidence intervals of 288 and 1174 pg/mL, n = 19) compared with healthy heart preparations (6391 pg/mL, 95% confidence intervals of 4180 and 9770 pg/mL, n = 14; p < 0.001). There was substantial adrenaline extraction by failing hearts (49 +/- 6%), although it was slightly lower than in healthy heart preparations (65 +/- 9%, p = 0.055). In contrast with healthy heart preparations, no net release of adrenaline occurred during stellate ganglion stimulation in any of the failing heart preparations, and ventricular tissue levels of adrenaline fell below the sensitivity limit of the HPLC technique. In failing heart preparations, maximal electrical stimulation of right or left stellate ganglia resulted in minimal augmentation of left ventricular intramyocardial (17%) and chamber (12%) systolic pressures. These indices were augmented by 145 and 97%, respectively, following exogenous noradrenaline administration. Thus, the cardiac efferent sympathetic neurons' reduced capacity to release noradrenaline and modify cardiac function can contribute to reduction of sympathetic support to the failing heart.
ABSTRACT Pulmonary arterial hypertension (PAH) is a rare chronic life-threatening disorder, characterized by the elevation of the mean pulmonary arterial pressure above 20 mmHg at rest. Histologically, PAH induces lung vascular remodeling, with the thickening of vessel wall. The conventional histological analysis commonly used in non-clinical models to assess lung vascular remodeling relies on manual measurements of representative lung vessels and is time-consuming. We have developed a fully automated reader-independent software (MorphoQuant-Lung) to both specifically detect vessels and measure vascular wall components from a-SMA rat lung sections. Analysis was performed on monocrotaline-and Sugen/hypoxia-induced PH rat models, treated or not with Sildenafil. The software requires 3-5 minutes to detect up to 1500 vessels per section, classify them per size, quantify intima, media and wall thicknesses, and calculate their level of occlusion. A comparison of our digital analysis results with those of the pathologist’s conventional visual analysis was performed for wall thickness and lumen radius showing a strong correlation between the two techniques (r: 0.80 and r: 0.88) regardless of the rat model. In addition, the occlusion estimated by automated analysis also strongly correlated with the mean pulmonary arterial pressure and the pulmonary vascular resistance (r ranging from 0.71 to 0.83) in both rat models. The added value of the present digital analysis paves the way for a more in-depth understanding of the PAH physiopathology in preclinical research and provides a robust and reliable tool for efficient therapeutic drug development.
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