Search

International audience; Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that β-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the β-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P < 0.001]. However, no differences in MDA were observed between groups. These data suggest that myocardial dysfunction observed after PSE was not due to β-adrenergic receptor desensitization but could be due to a signaling oxidative stress from the Nox enzyme.

Background— β1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. β3-AR expression is enhanced in diseased human myocardium, but its impact on remodeling is unknown. Methods and Results— Mice with cardiac myocyte-specific expression of human β3-AR (β3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). β3-TG and WT had similar morphometric and hemodynamic parameters at baseline. β3-AR colocalized with caveolin-3, endothelial nitric oxide synthase (NOS) and neuronal NOS in adult transgenic myocytes, which constitutively produced more cyclic GMP, detected with a new transgenic FRET sensor. Iso and Ang II produced hypertrophy and fibrosis in WT mice, but not in β3-TG mice, which also had less re-expression of fetal genes and transforming growth factor β1. Protection from Iso-induced hypertrophy was reversed by nonspecific NOS inhibition at low dose Iso, and by preferential neuronal NOS inhibition at high-dose Iso. Adenoviral overexpression of β3-AR in isolated cardiac myocytes also increased NO production and attenuated hypertrophy to Iso and phenylephrine. Hypertrophy was restored on NOS or protein kinase G inhibition. Mechanistically, β3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. Conclusions— Cardiac-specific overexpression of β3-AR does not affect cardiac morphology at baseline but inhibits the hypertrophic response to neurohormonal stimulation in vivo and in vitro, through a NOS-mediated mechanism. Activation of the cardiac β3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

Adenylyl cyclase (AC) type 5 (AC5) and AC type 6 (AC6) are the two major AC isoforms in the heart. Cardiac overexpression of AC6 has been shown to be protective in response to several interventions. In this investigation, we examined the effects of chronic pressure overload in AC6 transgenic (TG) mice. In the absence of any stress, AC6 TG mice exhibited enhanced contractile function compared with their wild-type (WT) littermates, i.e., increased ( P < 0.05) left ventricular (LV) ejection fraction (EF) (75 ± 0.9 vs. 71 ± 0.5%) and LV dP/d t (7,850 ± 526 vs. 6,374 ± 315 mmHg/s). Forskolin (25 μg·kg−1·min−1for 5 min) increased LVEF more ( P < 0.05) in AC6 TG mice (14.8 ± 1.0%) than in WT mice (7.7 ± 1.0%). Also, isoproterenol (0.04 μg·kg−1·min−1for 5 min) increased LVEF more ( P < 0.05) in AC6 TG mice (18.0 ± 1.2%) than in WT mice (11.6 ± 2.1%). Pressure overload, induced by 4 wk of transverse aortic constriction (TAC), increased the LV weight-to-body weight ratio and myocyte cross-sectional area similarly in both groups, but reduced LVEF more in AC6 TG mice (22%) compared with WT mice (9%), despite the higher starting level of LVEF in AC6 TG mice. LV systolic wall stress increased more in AC6 TG mice than in WT mice, which could be responsible for the reduced LVEF in AC6 TG mice with chronic pressure overload. In addition, LV dP/d t was no longer elevated in AC6 TG mice after TAC compared with WT mice. LV end-diastolic diameter was also greater ( P < 0.05) in AC6 TG mice (3.8 ± 0.07 mm) than in WT mice (3.6 ± 0.05 mm) after TAC. Thus, in contrast to other interventions previously reported to be salutary with cardiac AC6 overpression, the response to chronic pressure overload was not; actually, AC6 TG mice fared worse than WT mice. The mechanism may be due to the increased LV systolic wall stress in AC6 TG mice with chronic pressure overload.

By mimicking sympathetic stimulation in vivo, we previously reported that mice globally lacking serotonin 5-HT 2B receptors did not develop isoproterenol-induced left ventricular hypertrophy. However, the exact cardiac cell type(s) expressing 5-HT 2B receptors (cardiomyocytes versus noncardiomyocytes) involved in pathological heart hypertrophy was never addressed in vivo. We report here that mice expressing the 5-HT 2B receptor solely in cardiomyocytes, like global 5-HT 2B receptor–null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in cytokine plasma-levels. These data reveal a key role of noncardiomyocytes in isoproterenol-induced hypertrophy in vivo. Interestingly, we show that primary cultures of angiotensinogen null adult cardiac fibroblasts are releasing cytokines on stimulation with either angiotensin II or serotonin, but not in response to isoproterenol stimulation, demonstrating a critical role of angiotensinogen in adrenergic-dependent cytokine production. We then show a functional interdependence between AT 1 Rs and 5-HT 2B receptors in fibroblasts by revealing a transinhibition mechanism that may involve heterodimeric receptor complexes. Both serotonin- and angiotensin II–dependent cytokine production occur via a Src/heparin-binding epidermal growth factor–dependent transactivation of epidermal growth factor receptors in cardiac fibroblasts, supporting a common signaling pathway. Finally, we demonstrate that 5-HT 2B receptors are overexpressed in hearts from patients with congestive heart failure, this overexpression being positively correlated with cytokine and norepinephrine plasma levels. Collectively, these results reveal for the first time that interactions between AT 1 and 5-HT 2B receptors coexpressed by noncardiomyocytes are limiting key events in adrenergic agonist-induced, angiotensin-dependent cardiac hypertrophy. Accordingly, antagonists of 5-HT 2B receptors might represent novel therapeutics for sympathetic overstimulation-dependent heart failure.

We established previously that 5-HT 2B receptors are involved in cardiac hypertrophy through the regulation of hypertrophic cytokines in cardiac fibroblasts. Moreover, the generation of reactive oxygen species and tumor necrosis factor-α through the activation of reduced nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase has been implicated in cardiac hypertrophy. In this study, we investigated whether 5-HT 2B receptors could be involved in the development of cardiac hypertrophy associated with superoxide anion production. Therefore, we measured the effects of serotonergic 5-HT 2B receptor blockade on left-ventricular superoxide anion generation in 2 established pharmacological models of cardiac hypertrophy, ie, angiotensin II and isoproterenol infusions in mice. Angiotensin II infusion for 14 days increased superoxide anion concentration (+32%), NAD(P)H oxidase maximal activity (+84%), and p47 phox NAD(P)H oxidase subunit expression in the left ventricle together with hypertension (+37 mm Hg) and cardiac hypertrophy (+17% for heart weight:body weight). The 5-HT 2B receptor blockade by a selective antagonist (SB215505) prevented the increase in cardiac superoxide generation and hypertrophy. Similarly, infusion for 5 days of isoproterenol increased left-ventricular NAD(P)H oxidase activity (+48%) and cardiac hypertrophy (+31%) that were prevented by the 5-HT 2B receptor blockade. Finally, in the primary culture of left-ventricular cardiac fibroblasts, angiotensin II and isoproterenol stimulated NAD(P)H oxidase activity. This activation was prevented by SB215505. These findings suggest that the 5-HT 2B receptor may represent a new target to reduce cardiac hypertrophy and oxidative stress. Its blockade affects both angiotensin II and β-adrenergic trophic responses without significant hemodynamic alteration.

Background— The serotonergic 5-HT 2B receptor regulates cardiomyocyte development and growth. A putative contribution of this receptor to fibroblast-dependent cardiac function has not been identified. Methods and Results— By mimicking sympathetic stimulation with chronic isoproterenol perfusion in vivo, we found that mice developed a cardiac hypertrophy, which was prevented by exposure to the 5-HT 2B receptor antagonists SB206553 or SB215505 or in 5-HT 2B receptor–knockout mice. The isoproterenol-induced hypertrophy was associated with an increase in the plasma levels of interleukin-1β and tumor necrosis factor-α but not interleukin-6. In contrast, the plasma isoproterenol-induced cytokine increase was not observed in either 5-HT 2B receptor–mutant or wild-type mice perfused with isoproterenol+SB206553. We demonstrated that stimulation of wild-type cardiac fibroblasts by isoproterenol markedly increased the production of the interleukin-6, interleukin-1β, and tumor necrosis factor-α cytokines. Strikingly, we found that this isoproterenol-induced cytokine production was abolished by SB206553 or in 5-HT 2B receptor–knockout fibroblasts. Serotonin also stimulated production of the 3 cytokines in wild-type fibroblasts, which was effectively reduced in 5-HT 2B receptor–knockout fibroblasts. Conclusions— Our results demonstrate for the first time that 5-HT 2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1β, and tumor necrosis factor-α cytokine production by cardiac fibroblasts.

The aim of the present study was to study the influence of fatty acids on the adrenergic control of lipolysis both in vitro and in vivo. Human subcutaneous adipose tissue explants were cultured for 48 h in the presence of 100 microM bromopalmitate (BrPal), and lipolysis was measured in isolated adipocytes. In control conditions, beta-AR-dependent activation of lipolysis by epinephrine was almost undetectable, and could be fully restored by pharmacological blockade of alpha2-AR-dependent antilipolysis. After BrPal treatment, epinephrine became fully lipolytic and was no longer influenced by alpha2-AR-blockade. Radioligand binding analysis revealed that BrPal treatment led to a significant reduction in the coupling of alpha2-AR to G proteins. In parallel, a chronic and significant increase in plasma fatty acids resulting from a 4-day high-fat diet (HFD) was accompanied by an impairment of the amplifying effect of the alpha2-AR antagonist phentolamine on exercise-induced lipolysis (measured in the subcutaneous adipose tissue with the use of a microdialysis probe) normally observed after a low-fat diet. In conclusion, in vitro and in vivo studies showed that fatty acids impair alpha2-AR-dependent antilipolysis.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. The activity of HSL is thought to be primarily regulated by reversible phosphorylation. However, the regulation of HSL activity by pretranslational mechanisms has been poorly studied. The present studies were undertaken to explore the relationship between the levels of HSL protein and mRNA expressions and the lipolytic capacity. The study was performed in human abdominal subcutaneous adipocytes with identical sizes but having either a high (HL) or low (LL) lipolytic capacity (n = 16). Basal and maximal lipolysis induced by catecholamines, an adenylyl cyclase activator forskolin, and a cyclic AMP analogue dibutyryl cAMP were 50% lower in LL- in comparison with HL-fat cells (P < 0.05 or better). No differences in drug sensitivity were found. HSL activity and quantity were about 50% lower in LL- compared with HL-fat cells (P < 0.05). Moreover, the mRNA ratio between HSL and γ-actin was 35% lower in LL-compared with HL-fat cells (P < 0.05). There was a strong linear correlation between the protein and enzymatic HSL measurements (r2 = 0.91). In addition, the maximum lipolytic capacity was significantly correlated with HSL activity (r 2 = 0.75) and HSL protein amount (r 2 = 0.64). It is concluded that hormone-sensitive lipase (HSL) expression, measured either as total HSL protein by Western blot analysis or as total amount of activatable HSL enzyme, is a major determinant of the maximum lipolytic capacity of human fat cells. In addition, HSL protein expression is at least, in part, determined by HSL mRNA expression.—Large, V., P. Arner, S. Reynisdottir, J. Grober, V. Van Harmelen, C. Holm, and D. Langin. Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells.

International audience; The selectivity of rat auditory cortex neurons for pure tone frequency was studied during and after ionophoretic application (5-40 nA) of noradrenaline in urethane-anaesthetized rats. The dominant effect induced by noradrenaline was a significant decrease in spontaneous (93/268 cells) and evoked activity (133/268 cells) which outlasted the application. In the whole population of cells (n = 268) the signal-to-noise ratio, computed using as the signal either the mean evoked response or the response at the best frequency, was unchanged during noradrenaline application. It was significantly increased only for cells showing significantly decreased spontaneous activity, and was significantly decreased for cells showing increased spontaneous activity. Frequency selectivity was significantly increased for the whole population during and after noradrenaline application. It was also significantly increased for cells showing significantly decreased evoked activity, and was significantly decreased for cells showing increased evoked activity. The noradrenaline-induced inhibition was not blocked by propranolol (beta antagonist); it was blocked by prazosin (alpha1 antagonist) and partly mimicked by phenylephrine (alpha1 agonist). GABA, which also inhibited spontaneous and evoked activity, slightly increased the signal-to-noise ratio and significant increased frequency selectivity. However, when noradrenaline was ejected in the presence of bicuculline at doses that were able to block GABAergic inhibition, the inhibitory effects of noradrenaline on spontaneous and evoked activity were still observed. The possible function of noradrenaline-induced inhibitions in sensory cortices is briefly discussed.

International audience; 1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half-maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)-free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 microM) to the remote part had no effect on ICa, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation.

International audience; Acute exposure of isolated adipocytes to isoproterenol induces the desensitization of lipolytic responses to norepinephrine and selective beta 1-, beta 2- and beta 3-adrenoceptor agonists, as well as the adrenocorticotropic hormone 1-24 fragment (ACTH). Forskolin and 8-bromo-cAMP responses are also desensitized. When lipolysis was measured in the presence of OPC 3911 [N-cyclohexyl-N-2-hydroxyethyl-4(6-(1,2-dihydro-2- oxoquinolyloxy))butyramide], a specific inhibitor of the cAMP phosphodiesterase of adipocytes, the desensitization of all lipolytic agents--except the beta 2-adrenoceptor agonist--was abolished. Isoproterenol induced a similar loss (35%) of both membrane beta 1- and beta 2-adrenoceptors and an uncoupling of beta 1-adrenoceptors, but did not modify the weak coupling of control beta 2-adrenoceptors. These data suggest that isoproterenol induced (i) an activation of the cAMP phosphodiesterase, which is solely responsible for the desensitization of norepinephrine response as well as beta 1- and beta 3-adrenoceptor mediated responses and (ii) an additional desensitization of the sole beta 2-adrenergic signaling system which suggests a subtype-selective pattern of regulating processes.

International audience; The effects of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the L-type Ca2+ current (ICa) were examined in frog ventricular myocytes under basal and phosphorylated conditions. SIN-1 was found to exert insignificant effects on basal ICa but to induce a biphasic action on stimulated ICa. Indeed, in the nanomolar range of concentrations (0.1-10 nM), SIN-1 induced a pronounced (approximately 40%) stimulation of ICa elevated by a non-maximal concentration of forskolin (0.3 microM). However, the stimulatory effects of SIN-1 on ICa were not additive with those of maximal concentrations (10 microM) of forskolin or intracellular cAMP. In contrast, at higher concentrations (100 nM to 1 mM), SIN-1 strongly reduced ICa (by up to 85%) which had been previously stimulated by cAMP, forskolin, or isoprenaline. All the effects of SIN-1 appeared to be mediated by the liberation of NO since they were suppressed by methylene blue and LY83583 and were not mimicked by SIN-1C, a metabolite of SIN-1. The stimulatory or inhibitory effects of SIN-1 were absent, respectively, in the presence of milrinone (10 microM) or when the hydrolysis-resistant cAMP analog 8-bromo-cAMP was used instead of cAMP to stimulate ICa. In addition to its effects on ICa, SIN-1 induced a dose-dependent stimulation of guanylyl cyclase activity in the cytosolic and membrane fractions of frog ventricle. The membrane form of guanylyl cyclase displayed a higher sensitivity to SIN-1 than the cytosolic form, which correlated with SIN-1 sensitivity of ICa. Our data suggest that the activatory and inhibitory effects of NO donors on ICa result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively, both linked to the activation of guanylyl cyclase, possibly a membrane form of the enzyme.

A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (ICa) to rapid exposures to beta-adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of approximately 5 cm/s. Movement from one capillary to the adjacent one occurred in20 ms and complete exchange of extracellular solution was achieved in50 ms as demonstrated by the block of ICa by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of ICa at [Iso]1 nM. The response of ICa to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically approximately 3 s at 10 microM Iso. The rising phase of ICa increase was monophasic and independent of [Iso]100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of ICa occurred approximately 20 s and approximately 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), IBa never increased during that pulse. The kinetics of the ICa response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (-)Bay K 8644 on ICa was approximately 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (-)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of beta-adrenergic agonists.

International audience; In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.