Chantal Dessy

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.

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.

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.

Endothelial beta3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation beta-blocker nebivolol.

Background—The therapeutic effects of nonspecific β-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective β1-adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial β3-adrenoreceptors in human coronary microarteries that mediate endothelium- and NO-dependent relaxation and hypothesized that nebivolol activates these β3-adrenoreceptors. Methods and Results—Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 &mgr;mol/L, −86±6% of prostaglandin F2α contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the β1-2-blocker nadolol, and prevented by the β1-2-3-blocker bupranolol (P<0.05; n=3 to 8). Importantly, nebivolol failed to relax microarteries from β3-adrenoreceptor–deficient mice. Nebivolol (10 &mgr;mol/L) also relaxed human coronary microvessels (−71±5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to β1-2-blockade (all P<0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not β3-adrenoreceptor– or endothelial NOS–deficient mice. In cultured endothelial cells, 10 &mgr;mol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine495, and fura-2 calcium fluorescence increased by 91.8±23.7%; this effect was unaffected by β1-2-blockade but abrogated by β1-2-3-blockade (all P<0.05). Conclusions—Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial β3-adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.

Modulation of the Endothelial Nitric-oxide Synthase-Caveolin Interaction in Cardiac Myocytes IMPLICATIONS FOR THE AUTONOMIC REGULATION OF HEART RATE

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr−) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr− eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.

Role of Caveolar Compartmentation in Endothelium-Derived Hyperpolarizing Factor–Mediated Relaxation Ca2+ Signals and Gap Junction Function Are Regulated by Caveolin in Endothelial Cells

Background— In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)–mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Methods and Results— Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in nitric oxide– and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Conclusions— We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Caveolin-1 Expression Is Critical for Vascular Endothelial Growth Factor–Induced Ischemic Hindlimb Collateralization and Nitric Oxide–Mediated Angiogenesis

Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav−/−) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav−/− mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor l-NAME administered to operated Cav+/+ mice. We also isolated endothelial cells (ECs) from Cav−/− aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav+/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav−/− ECs. Interestingly, caveolin transfection in Cav−/− ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology.

Rosuvastatin Decreases Caveolin-1 and Improves Nitric Oxide–Dependent Heart Rate and Blood Pressure Variability in Apolipoprotein E−/− Mice In Vivo

Background—Decreased heart rate variability (HRV) and increased blood pressure variability (BPV), determined in part by nitric oxide (NO)–dependent endothelial dysfunction, are correlated with adverse prognosis in cardiovascular diseases. We examined potential alterations in BPV and HRV in genetically dyslipidemic, apolipoprotein (apo) E−/−, and control mice and the effect of chronic statin treatment on these parameters in relation to their NO synthase (NOS)–modifying properties. Methods and Results—BP and HR were recorded in unrestrained, nonanesthetized mice with implanted telemetry devices with or without rosuvastatin. Cardiac and aortic expression of endothelial NOS and caveolin-1 were measured by immunoblotting. Both systolic BP and HR were elevated in apoE−/− mice, with abolition of their circadian cycles. Spectral analysis showed an increase in their systolic BPV in the very-low-frequency (+17%) band and a decrease in HRV in the high-frequency (−57%) band, reflecting neurohumoral and autonomic dysfunction. Decreased sensitivity to acute injection of atropine or an NOS inhibitor indicated basal alterations in both parasympathetic and NOS regulatory systems in apoE−/− mice. Aortic caveolin-1 protein, an inhibitor of endothelial NOS, was also increased in these mice by 2.0-fold and correlated positively with systolic BPV in the very-low-frequency band. Rosuvastatin treatment corrected the hemodynamic and caveolin-1 expression changes despite persisting elevated plasma cholesterol levels. Conclusions—Rosuvastatin decreases caveolin-1 expression and promotes NOS function in apoE−/−, dyslipidemic mice in vivo, with concurrent improvements in BPV and HRV. This highlights the beneficial effects of rosuvastatin on cardiovascular function beyond those attributed to lipid lowering.

Preconditioning of the Tumor Vasculature and Tumor Cells by Intermittent Hypoxia: Implications for Anticancer Therapies

Hypoxia is a common feature in tumors associated with an increased resistance of tumor cells to therapies. In addition to O(2) diffusion-limited hypoxia, another form of tumor hypoxia characterized by fluctuating changes in pO(2) within the disorganized tumor vascular network is described. Here, we postulated that this form of intermittent hypoxia promotes endothelial cell survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We found that endothelial cell exposure to cycles of hypoxia reoxygenation not only rendered them resistant to proapoptotic stresses, including serum deprivation and radiotherapy, but also increased their capacity to migrate and organize in tubes. By contrast, prolonged hypoxia failed to exert protective effects and even seemed deleterious when combined with radiotherapy. The use of hypoxia-inducible factor-1alpha (HIF-1alpha)-targeting small interfering RNA led us to document that the accumulation of HIF-1alpha during intermittent hypoxia accounted for the higher resistance of endothelial cells. We also used an in vivo approach to enforce intermittent hypoxia in tumor-bearing mice and found that it was associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (versus normoxia or prolonged hypoxia). Radioresistance was further ascertained by an increased rate of tumor regrowth in irradiated mice preexposed to intermittent hypoxia and confirmed in vitro using distinctly radiosensitive tumor cell lines. In conclusion, we have documented that intermittent hypoxia may condition endothelial cells and tumor cells in such a way that they are more resistant to apoptosis and more prone to participate in tumor progression. Our observations also underscore the potential of drugs targeting HIF-1alpha to resensitize the tumor vasculature to anticancer treatments.