Developing a Clinically Relevant Hemorrhagic Shock Model in Rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p < 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p < 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p < 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

Overexpression of endothelial ß3 -adrenergic receptor induces diastolic dysfunction in rats.

AIMS: Diastolic dysfunction is common in cardiovascular diseases, particularly in the case of heart failure with preserved ejection fraction. The challenge is to develop adequate animal models to envision human therapies in the future. It has been hypothesized that this diastolic dysfunction is linked to alterations in the nitric oxide (• NO) pathway. To investigate this issue further, we investigated the cardiac functions of a transgenic rat model (Tgß3 ) that overexpresses the human ß3 -adrenoceptor (hß3 -AR) in the endothelium with the underlying rationale that the • NO pathway should be stimulated in the endothelium. METHODS AND RESULTS: Transgenic rats (Tgß3 ) that express hß3 -AR under the control of intercellular adhesion molecule 2 promoter were developed for a specific expression in endothelial cells. Transcriptomic analyses were performed on left ventricular tissue from 45-week-old rats. Among all altered genes, we focus on • NO synthase expression and endothelial function with arterial reactivity and evaluation of • NO and O2 •- production. Cardiac function was characterized by echocardiography, invasive haemodynamic studies, and working heart studies. Transcriptome analyses illustrate that several key genes are regulated by the hß3 -AR overexpression. Overexpression of hß3 -AR leads to a reduction of Nos3 mRNA expression (-72%; P < 0.05) associated with a decrease in protein expression (-19%; P < 0.05). Concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgß3 aorta (-10%; P < 0.05), while • NO and O2 •- production was increased. In the same time, Tgß3 rats display progressively increasing diastolic dysfunction with age, as shown by an increase in the E/A filing ratio [1.15 ± 0.01 (wild type, WT) vs. 1.33 ± 0.04 (Tgß3 ); P < 0.05] and in left ventricular end-diastolic pressure [5.57 ± 1.23 mmHg (WT) vs. 11.68 ± 1.11 mmHg (Tgß3 ); P < 0.05]. In isolated working hearts, diastolic stress using increasing preload levels led to a 20% decrease in aortic flow [55.4 ± 1.9 mL/min (WT) vs. 45.8 ± 2.5 mL/min (Tgß3 ); P < 0.05]. CONCLUSIONS: The Tgß3 rat model displays the expected increase in • NO production upon ageing and develops diastolic dysfunction. These findings provide a further link between endothelial and cardiac dysfunction. This rat model should be valuable for future preclinical evaluation of candidate drugs aimed at correcting diastolic dysfunction.

Maurocalcin and its analog MCaE12A facilitate Ca2+ mobilization in cardiomyocytes.

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells.

ß1-Adrenergic cardiac contractility is increased during early endotoxemic shock: Involvement of cyclooxygenases.

AIMS: Endothelial dysfunction is one of the earliest symptoms in septic patients and plays an important role in the cardiovascular alterations. However, the endothelial mechanisms involved in the impaired sympathetic regulation of the cardiovascular system are not clear. This study aimed to determine the role of the endocardial endothelium (EE) in the cardiac ß-adrenergic (ß-AR) remodeling at the early phase of endotoxemic shock. MAIN METHODS: Rats received either lipopolysaccharide (LPS) or saline (control) intravenously. Three hours later, ß-AR cardiac contractility was evaluated on papillary muscles with or without a functional EE. KEY FINDINGS: Isoproterenol-induced contractility was strongly increased in papillary muscles from LPS rats. A similar increase was observed with a ß1-AR stimulation, whereas ß2-AR and ß3-AR produced similar contractility in control and LPS treatments. The removal of the EE did not modify ß1-AR-induced contractility in controls, whereas it abolished the increased ß1-AR response in LPS-treated muscles. In LPS-treated papillary muscle, the increased ß1-AR-induced contractility was not modified by pretreatment with a NOS inhibitor or an endothelin receptor antagonist. Conversely, the increased ß1-AR-induced contractility was abolished by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, as well as by selective inhibitors of COX1 and COX2. An early treatment with indomethacin improved the survival of LPS rat. SIGNIFICANCE: Our results suggest that the EE is involved in the increased cardiac ß1-AR contractility in the early phase of endotoxemic shock. This effect is mediated through the activation of COX1 and COX2 and suggests these may be novel putative therapeutic targets during endotoxemic shock.

Nebivolol, a vasodilating selective beta(1)-blocker, is a beta(3)-adrenoceptor agonist in the nonfailing transplanted human heart.

OBJECTIVES: The present study was to assess whether nebivolol could activate beta(3)-adrenergic receptors (ARs) in the human heart. BACKGROUND: Nebivolol is a third-generation beta-blocker used in the treatment of heart failure. It associates selective beta(1)-adrenergic antagonist properties with endothelial and nitric oxide (NO)-dependent vasodilation. Several studies reported that this vasodilation could result from an activation of beta(3)-ARs, but no data are available in the heart. METHODS: The effect of nebivolol (0.1 nmol/l to 10 micromol/l) upon the developed peak tension was tested in endomyocardial biopsies from human nonrejecting transplanted hearts. Tension was recorded at steady state using a mechanoelectric force transducer. RESULTS: Nebivolol induced a concentration-dependent decrease in peak tension (maximum effect obtained at 10 micromol/l: -55 +/- 4%, n = 6), which was similar to that obtained with a preferential beta(3)-AR agonist, BRL 37344 (maximum effect obtained at 1 micromol/l: -45 +/- 2%, n = 12). The nebivolol effect was not modified by 10 micromol/l nadolol, a beta(1,2)-AR antagonist, but was significantly reduced in the presence of 1 micromol/l L-748,337, a selective beta(3)-AR antagonist, and after pre-treatment with 100 micromol/l N(G)-monomethyl-L-arginine, an NOS inhibitor. CONCLUSIONS: Our study demonstrated that nebivolol activated beta(3)-AR in the human ventricle. The NO-dependent negative inotropic effect of nebivolol associated with its vasodilating properties previously described in human microcoronary arteries could improve the energetic balance in heart. Those effects could explain the improvement of hemodynamic parameters obtained in patients with heart failure after nebivolol administration as previously described in clinical trials.

beta3-Adrenergic stimulation produces a decrease of cardiac contractility ex vivo in mice overexpressing the human beta3-adrenergic receptor.

OBJECTIVES: The regulation of cardiac function by catecholamines involves three populations of beta-adrenoceptor (beta-AR). beta(1)- and beta(2)-AR stimulations produce an increase in contractility and beta(3)-AR stimulation mediates a negative inotropic effect in human ventricular muscle. Because of the lack of suitable animal models, we have generated transgenic mice with cardiac-specific expression of the human beta(3)-AR (TG beta(3) mice). METHODS: TG beta(3) mice were produced by microinjection of the human beta(3)-AR under the control of the alpha myosin heavy chain promoter. Phenotypic analyses comprised beta(3)-AR mRNA and protein determinations, histological studies, electrocardiogram, contractility and cyclic nucleotide measurements. RESULTS: TG beta(3) mice presented no histological evidence of myocyte hypertrophy or fibrogenesis. In basal conditions, TG beta(3) mice were characterized by an increase in heart rate and an acceleration of twitch parameters without modification of its amplitude. beta(3)-AR agonists (CL 316243, SR 58611A) decreased contractility at low concentrations (1-100 nM). At high concentrations, the negative inotropic effect was abolished. Pretreatment with nadolol, a beta(1)/beta(2)-AR blocker, blunted the rebound in peak tension elicited by beta(3)-AR agonists suggesting a non-specific action of these compounds on beta(1)- and beta(2)-AR. The involvement of beta(3)-AR in the negative inotropic effect was confirmed by the pretreatment with bupranolol, a non-selective beta-AR antagonist, which fully abolished the effects of SR 58611A. The negative inotropic effect was associated with an increase in intracellular cGMP level. CONCLUSIONS: We conclude that cardiac overexpression of beta(3)-AR in mice reproduces ex vivo the negative inotropic effects obtained with beta(3)-AR stimulation in human ventricular tissues.