Jean-Luc Balligand

Nitric Oxide and Cardiac Function

Nitric oxide (NO) is produced from virtually all cell types composing the myocardium and regulates cardiac function through both vascular-dependent and -independent effects. The former include regulation of coronary vessel tone, thrombogenicity, and proliferative and inflammatory properties as well as cellular cross-talk supporting angiogenesis. The latter comprise the direct effects of NO on several aspects of cardiomyocyte contractility, from the fine regulation of excitation-contraction coupling to modulation of (presynaptic and postsynaptic) autonomic signaling and mitochondrial respiration. This multifaceted involvement of NO in cardiac physiology is supported by a tight molecular regulation of the three NO synthases, from cellular spatial confinement to posttranslational allosteric modulation by specific interacting proteins, acting in concert to restrict the influence of NO to a particular intracellular target in a stimulus-specific manner. Loss of this specificity, such as produced on excessive NO delivery from inflammatory cells (or cytokine-stimulated cardiomyocytes themselves), may result in profound cellular disturbances leading to heart failure. Future therapeutic manipulations of cardiac NO synthesis will necessarily draw on additional characterization of the cellular and molecular determinants for the net effect of this versatile radical on the cardiomyocyte biology.

A Cathepsin D-Cleaved 16 kDa Form of Prolactin Mediates Postpartum Cardiomyopathy

Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.

Nitric oxide and cardiac function: ten years after, and continuing.

Abstract— Nitric oxide (NO) is produced from virtually all cell types composing the myocardium and regulates cardiac function through both vascular-dependent and -independent effects. The former include regulation of coronary vessel tone, thrombogenicity, and proliferative and inflammatory properties as well as cellular cross-talk supporting angiogenesis. The latter comprise the direct effects of NO on several aspects of cardiomyocyte contractility, from the fine regulation of excitation-contraction coupling to modulation of (presynaptic and postsynaptic) autonomic signaling and mitochondrial respiration. This multifaceted involvement of NO in cardiac physiology is supported by a tight molecular regulation of the three NO synthases, from cellular spatial confinement to posttranslational allosteric modulation by specific interacting proteins, acting in concert to restrict the influence of NO to a particular intracellular target in a stimulus-specific manner. Loss of this specificity, such as produced on excessive NO delivery from inflammatory cells (or cytokine-stimulated cardiomyocytes themselves), may result in profound cellular disturbances leading to heart failure. Future therapeutic manipulations of cardiac NO synthesis will necessarily draw on additional characterization of the cellular and molecular determinants for the net effect of this versatile radical on the cardiomyocyte biology.

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium.

The mechanism by which soluble mediators of immune cell origin depress myocardial contractility, either globally as in systemic sepsis, or regionally in areas of inflammatory myocardial infiltrates, remains unclear. When freshly isolated ventricular myocytes from adult rat hearts were preincubated for at least 24 h in medium conditioned by endotoxin (LPS)-activated rat alveolar macrophages, their subsequent inotropic response to the beta-adrenergic agonist isoproterenol was reduced from 225 +/- 19% to 155 +/- 10% of the baseline amplitude of shortening (mean +/- SEM, P < 0.05). Neither baseline contractile function nor the contractile response to high extracellular calcium were affected. To determine whether an endogenous nitric-oxide (NO)-signaling pathway within ventricular myocytes was responsible for their decreased responsiveness to isoproterenol, the L-arginine analogue L-NMMA was added to the preincubation medium. While L-NMMA did not affect baseline contractile function or the response of control myocytes to isoproterenol, it completely restored the positive inotropic response to isoproterenol in myocytes preincubated in LPS-activated macrophage medium. Release of NO by ventricular myocytes following exposure to activated macrophage medium was detected as an increase in cGMP content in a reporter-cell (RFL-6) bioassay and also as increased nitrite content in myocyte-conditioned medium. Thus, the depressed contractile response of adult rat ventricular myocytes to beta-adrenergic agonists by a 24-h exposure to soluble inflammatory mediators is mediated at least in party by induction of an autocrine NO signaling pathway.

Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance.

Background—Hypercholesterolemia is causally associated with defects of endothelial nitric oxide (NO)–dependent vasodilation. Increased uptake of cholesterol by endothelial cells (ECs) upregulates the abundance of the structural protein caveolin-1 and impairs NO release through the stabilization of the inhibitory heterocomplex between caveolin-1 and endothelial NO synthase (eNOS). Therefore, we examined whether the hydroxy-methylglutaryl–coenzyme A reductase inhibitor atorvastatin modulates caveolin abundance, eNOS activity, and NO release through a reduction in endogenous cholesterol levels. Methods and Results—ECs were incubated with increasing doses of atorvastatin in the absence or in the presence of human LDL cholesterol (LDL-Chol) fractions in the presence of antioxidants. Our results show that atorvastatin (10 nmol/L to 1 &mgr;mol/L) reduced caveolin-1 abundance in the absence (−75%) and in the presence (−20% to 70%) of LDL-Chol. This was paralleled by a decreased inhibitory interaction between caveolin-1 and eNOS and a restoration and/or potentiation of the basal (+45%) and agonist-stimulated (+107%) eNOS activity. These effects were observed in the absence of changes in eNOS abundance and were reversed with mevalonate. In the presence of LDL-Chol, atorvastatin also promoted the agonist-induced association of eNOS and the chaperone Hsp90, resulting in the potentiation of eNOS activation. Conclusions—We provide biochemical and functional evidence that atorvastatin promotes NO production by decreasing caveolin-1 expression in ECs, regardless of the level of extracellular LDL-Chol. These findings highlight the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.

Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle.

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.

Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes.

Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K–Akt–endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.

Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology

The reductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti‐cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under‐represent older patients and those with significant co‐morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre‐clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross‐disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.

Upregulation of β3-Adrenoceptors and Altered Contractile Response to Inotropic Amines in Human Failing Myocardium

BackgroundContrary to &bgr;1- and &bgr;2-adrenoceptors, &bgr;3-adrenoceptors mediate a negative inotropic effect in human ventricular muscle. To assess their functional role in heart failure, our purpose was to compare the expression and contractile effect of &bgr;3-adrenoceptors in nonfailing and failing human hearts. Methods and ResultsWe analyzed left ventricular samples from 29 failing (16 ischemic and 13 dilated cardiomyopathic) hearts (ejection fraction 18.6±2%) and 25 nonfailing (including 12 innervated) explanted hearts (ejection fraction 64.2±3%). &bgr;3-Adrenoceptor proteins were identified by immunohistochemistry in ventricular cardiomyocytes from nonfailing and failing hearts. Contrary to &bgr;1-adrenoceptor mRNA, Western blot analysis of &bgr;3-adrenoceptor proteins showed a 2- to 3-fold increase in failing compared with nonfailing hearts. A similar increase was observed for G&agr;i-2 proteins that couple &bgr;3-adrenoceptors to their negative inotropic effect. Contractile tension was measured in electrically stimulated myocardial samples ex vivo. In failing hearts, the positive inotropic effect of the nonspecific amine isoprenaline was reduced by 75% compared with that observed in nonfailing hearts. By contrast, the negative inotropic effect of &bgr;3-preferential agonists was only mildly reduced. ConclusionsOpposite changes occur in &bgr;1- and &bgr;3-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.