The Platelet-Derived Growth Factor Pathway in Pulmonary Arterial Hypertension: Still an Interesting Target?

The lack of curative options for pulmonary arterial hypertension drives important research to understand the mechanisms underlying this devastating disease. Among the main identified pathways, the platelet-derived growth factor (PDGF) pathway was established to control vascular remodeling and anti-PDGF receptor (PDGFR) drugs were shown to reverse the disease in experimental models. Four different isoforms of PDGF are produced by various cell types in the lung. PDGFs control vascular cells migration, proliferation and survival through binding to their receptors PDGFRα and ß. They elicit multiple intracellular signaling pathways which have been particularly studied in pulmonary smooth muscle cells. Activation of the PDGF pathway has been demonstrated both in patients and in pulmonary hypertension (PH) experimental models. Tyrosine kinase inhibitors (TKI) are numerous but without real specificity and Imatinib, one of the most specific, resulted in beneficial effects. However, adverse events and treatment discontinuation discouraged to pursue this therapy. Novel therapeutic strategies are currently under experimental evaluation. For TKI, they include intratracheal drug administration, low dosage or nanoparticles delivery. Specific anti-PDGF and anti-PDGFR molecules can also be designed such as new TKI, soluble receptors, aptamers or oligonucleotides.

Early Protective Role of Inflammation in Cardiac Remodeling and Heart Failure: Focus on TNFα and Resident Macrophages.

Cardiac hypertrophy, initiated by a variety of physiological or pathological stimuli (hemodynamic or hormonal stimulation or infarction), is a critical early adaptive compensatory response of the heart. The structural basis of the progression from compensated hypertrophy to pathological hypertrophy and heart failure is still largely unknown. In most cases, early activation of an inflammatory program reflects a reparative or protective response to other primary injurious processes. Later on, regardless of the underlying etiology, heart failure is always associated with both local and systemic activation of inflammatory signaling cascades. Cardiac macrophages are nodal regulators of inflammation. Resident macrophages mostly attenuate cardiac injury by secreting cytoprotective factors (cytokines, chemokines, and growth factors), scavenging damaged cells or mitochondrial debris, and regulating cardiac conduction, angiogenesis, lymphangiogenesis, and fibrosis. In contrast, excessive recruitment of monocyte-derived inflammatory macrophages largely contributes to the transition to heart failure. The current review examines the ambivalent role of inflammation (mainly TNFα-related) and cardiac macrophages (Mφ) in pathophysiologies from non-infarction origin, focusing on the protective signaling processes. Our objective is to illustrate how harnessing this knowledge could pave the way for innovative therapeutics in patients with heart failure.

Platelet-Derived Growth Factor Receptor Type α Activation Drives Pulmonary Vascular Remodeling Via Progenitor Cell Proliferation and Induces Pulmonary Hypertension.

Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1+) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1+ cells expressing PDGFRα. PW1nLacZ reporter mice were used to follow the fate of pulmonary PW1+ progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1+ progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1+ progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1+ progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.

Transcriptomic and Lipidomic Mapping of Macrophages in the Hub of Chronic Beta-Adrenergic-Stimulation Unravels Hypertrophy-, Proliferation-, and Lipid Metabolism-Related Genes as Novel Potential Markers of Early Hypertrophy or Heart Failure.

Sympathetic nervous system overdrive with chronic release of catecholamines is the most important neurohormonal mechanism activated to maintain cardiac output in response to heart stress. Beta-adrenergic signaling behaves first as a compensatory pathway improving cardiac contractility and maladaptive remodeling but becomes dysfunctional leading to pathological hypertrophy and heart failure (HF). Cardiac remodeling is a complex inflammatory syndrome where macrophages play a determinant role. This study aimed at characterizing the temporal transcriptomic evolution of cardiac macrophages in mice subjected to beta-adrenergic-stimulation using RNA sequencing. Owing to a comprehensive bibliographic analysis and complementary lipidomic experiments, this study deciphers typical gene profiles in early compensated hypertrophy (ECH) versus late dilated remodeling related to HF. We uncover cardiac hypertrophy- and proliferation-related transcription programs typical of ECH or HF macrophages and identify lipid metabolism-associated and Na+ or K+ channel-related genes as markers of ECH and HF macrophages, respectively. In addition, our results substantiate the key time-dependent role of inflammatory, metabolic, and functional gene regulation in macrophages during beta-adrenergic dependent remodeling. This study provides important and novel knowledge to better understand the prevalent key role of resident macrophages in response to chronically activated beta-adrenergic signaling, an effective diagnostic and therapeutic target in failing hearts.

Cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR2- and Orai3- dependent signaling.

Early adaptive cardiac hypertrophy (EACH) is initially a compensatory process to optimize pump function. We reported the emergence of Orai3 activity during EACH. This study aimed to characterize how inflammation regulates store-independent activation of Orai3-calcium influx and to evaluate the functional role of this influx. Isoproterenol infusion or abdominal aortic banding triggered EACH. TNFα or conditioned medium from cardiac CD11b/c cells activated either in vivo [isolated from rats displaying EACH], or in vitro [isolated from normal rats and activated with lipopolysaccharide], were added to adult cardiomyocytes before measuring calcium entry, cell hypertrophy and cell injury. Using intramyocardial injection of siRNA, Orai3 was in vivo knockdown during EACH to evaluate its protective activity in heart failure. Inflammatory CD11b/c cells trigger a store-independent calcium influx in hypertrophied cardiomyocytes, that is mimicked by TNFα. Pharmacological or molecular (siRNA) approaches demonstrate that this calcium influx, depends on TNFR2, is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3ß phosphorylation, impairs EACH and accelerates heart failure. Orai3 exerts a pathophysiological protective impact in EACH promoting hypertrophy and resistance to oxidative stress. We highlight inflammation arising from CD11b/c cells as a potential trigger of TNFR2- and Orai3-dependent signaling pathways.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling.

BACKGROUND: Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. METHODS AND RESULTS: To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload-induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3ß molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of Akt(S473), a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. CONCLUSIONS: These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3ß activity.

Emergence of Orai3 activity during cardiac hypertrophy.

AIMS: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.

M2 kupffer cells promote hepatocyte senescence: an IL-6-dependent protective mechanism against alcoholic liver disease.

Alcoholic liver disease is a predominant cause of liver-related mortality in Western countries. The early steps of alcohol-induced steatosis and liver injury involve several mechanisms, including inflammation and oxidative stress. The inflammatory process is initiated by polarization of Kupffer cells toward a proinflammatory M1 phenotype, and we recently found that promoting anti-inflammatory M2 Kupffer cell polarization protects against alcohol-induced hepatocyte steatosis and apoptosis. Alcohol-induced oxidative stress is a potential trigger of senescence, and senescent cells exhibit characteristic functional resistance to apoptosis. We sought to evaluate induction of hepatocyte senescence as an early protective mechanism against alcoholic liver disease. Combining in vivo and in vitro studies, we show that M2 macrophages trigger hepatocyte senescence and enhance alcohol-induced hepatocyte senescence, as indicated by increased ß-galactosidase activity, elevated CDKN1A mRNA expression, and induction of nuclear p21. We identify IL-6 as the mediator of M2-induced hepatocyte senescence. Senescent hepatocytes display characteristic resistance to apoptosis but also to steatosis, thus arguing for an early protective effect against alcoholic liver disease. These findings further suggest that pharmacologic interventions targeting M2 polarization during the early stages of alcoholic liver disease may represent an attractive strategy for the limitation of inflammation, hepatocyte apoptosis, and steatosis.

M2 Kupffer cells promote M1 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease.

UNLABELLED: Alcoholic and nonalcoholic fatty liver disease (ALD and NAFLD) are the predominant causes of liver-related mortality in Western countries. We have shown that limiting classical (M1) Kupffer cell (KC) polarization reduces alcohol-induced liver injury. Herein, we investigated whether favoring alternatively activated M2 KCs may protect against ALD and NAFLD. Ongoing alcohol drinkers and morbidly obese patients, with minimal hepatic injury and steatosis, displayed higher hepatic expression of M2 genes, as compared to patients with more severe liver lesions; individuals with limited liver lesions showed negligible hepatocyte apoptosis but significant macrophage apoptosis. Experiments in mouse models of ALD or NAFLD further showed that BALB/c or resveratrol-treated mice fed alcohol or a high-fat diet displayed preponderant M2 KC polarization, M1 KC apoptosis, and resistance to hepatocyte steatosis and apoptosis, as compared to control C57BL6/J mice. In vitro experiments in isolated KC, peritoneal, and Raw264.7 macrophages demonstrated that M1 macrophage apoptosis was promoted by conditioned medium from macrophages polarized into an M2 phenotype by either interleukin (IL)4, resveratrol, or adiponectin. Mechanistically, IL10 released from M2 cells promoted M1 death, and anti-IL10 antibodies blunted the proapoptic effects of M2-conditioned media. IL10 secreted by M2 KCs promoted selective M1 death by a mechanism involving activation of arginase in high inducible nitric oxide synthase-expressing M1 KCs. In alcohol-exposed mice, neutralization of IL10 impaired M1 apoptosis. CONCLUSION: These data uncover a novel mechanism regulating the M1/M2 balance that relies on apoptotic effects of M2 KCs towards their M1 counterparts. They suggest that promoting M2-induced M1 KC apoptosis might prove a relevant strategy to limit alcohol- and high fat-induced inflammation and hepatocyte injury.

Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice.

Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti-inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis. Wild-type (WT) mice fed an alcohol diet showed an induction of hepatic classical (M1) and alternative (M2) markers. Cotreatment of alcohol-fed mice with the CB2 agonist, JWH-133, decreased hepatic M1 gene expression without affecting the M2 profile. In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol-induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH-133-treated mice and its enhancement in CB2-/- animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned-medium experiments showed that preventing M1 polarization in CB2-activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase-1 (HO-1) mediated the anti-inflammatory effects of CB2 receptors. Indeed, alcohol-fed mice treated with JWH-133 showed increased hepatic expression of macrophage HO-1, as compared to vehicle-treated counterparts. In keeping with this, JWH-133 induced HO-1 expression in cultured macrophages, and the HO-1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH-133 on lipopolysaccharide-induced nuclear factor-kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol-induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2 agonists as potential therapeutic agents for the management of alcoholic liver disease.

Delayed cardiomyopathy in dystrophin deficient mdx mice relies on intrinsic glutathione resource.

Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in beta-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer.

Glutathione deficiency in cardiac patients is related to the functional status and structural cardiac abnormalities.

BACKGROUND: The tripeptide glutathione (L-gamma-glutamyl-cysteinyl-glycine) is essential to cell survival, and deficiency in cardiac and systemic glutathione relates to heart failure progression and cardiac remodelling in animal models. Accordingly, we investigated cardiac and blood glutathione levels in patients of different functional classes and with different structural heart diseases. METHODS: Glutathione was measured using standard enzymatic recycling method in venous blood samples obtained from 91 individuals, including 15 healthy volunteers and 76 patients of New York Heart Association (NYHA) functional class I to IV, undergoing cardiac surgery for coronary artery disease, aortic stenosis or terminal cardiomyopathy. Glutathione was also quantified in right atrial appendages obtained at the time of surgery. RESULTS: In atrial tissue, glutathione was severely depleted (-58%) in NYHA class IV patients compared to NYHA class I patients (P = 0.002). In patients with coronary artery disease, this depletion was related to the severity of left ventricular dysfunction (P = 0.006). Compared to healthy controls, blood glutathione was decreased by 21% in NYHA class I patients with structural cardiac disease (P<0.01), and by 40% in symptomatic patients of NYHA class II to IV (P<0.0001). According to the functional NYHA class, significant depletion in blood glutathione occurred before detectable elevation in blood sTNFR1, a marker of symptomatic heart failure severity, as shown by the exponential relationship between these two parameters in the whole cohort of patients (r = 0.88). CONCLUSIONS: This study provides evidence that cardiac and systemic glutathione deficiency is related to the functional status and structural cardiac abnormalities of patients with cardiac diseases. These data also suggest that blood glutathione test may be an interesting new biomarker to detect asymptomatic patients with structural cardiac abnormalities.

The cannabinoid receptor type 2 promotes cardiac myocyte and fibroblast survival and protects against ischemia/reperfusion-induced cardiomyopathy.

Post-myocardial infarction (MI) heart failure is a major public health problem in Western countries and results from ischemia/reperfusion (IR)-induced cell death, remodeling, and contractile dysfunction. Ex vivo studies have demonstrated the cardioprotective anti-inflammatory effect of the cannabinoid type 2 (CB2) receptor agonists within hours after IR. Herein, we evaluated the in vivo effect of CB2 receptors on IR-induced cell death, fibrosis, and cardiac dysfunction and investigated the target role of cardiac myocytes and fibroblasts. The infarct size was increased 24 h after IR in CB2(-/-) vs. wild-type (WT) hearts and decreased when WT hearts were injected with the CB2 agonist JWH133 (3 mg/kg) at reperfusion. Compared with WT hearts, CB2(-/-) hearts showed widespread injury 3 d after IR, with enhanced apoptosis and remodeling affecting the remote myocardium. Finally, CB2(-/-) hearts exhibited exacerbated fibrosis, associated with left ventricular dysfunction 4 wk after IR, whereas their WT counterparts recovered normal function. Cardiac myocytes and fibroblasts isolated from CB2(-/-) hearts displayed a higher H(2)O(2)-induced death than WT cells, whereas 1 microM JWH133 triggered survival effects. Furthermore, H(2)O(2)-induced myofibroblast activation was increased in CB2(-/-) fibroblasts but decreased in 1 microM JWH133-treated WT fibroblasts, compared with that in WT cells. Therefore, CB2 receptor activation may protect against post-IR heart failure through direct inhibition of cardiac myocyte and fibroblast death and prevention of myofibroblast activation.

Sphingomyelinases: their regulation and roles in cardiovascular pathophysiology.

Sphingomyelinases (SMases) hydrolyse sphingomyelin, releasing ceramide and creating a cascade of bioactive lipids. These lipids include sphingosine and sphingosine-1-phosphate, all of which have a specific signalling capacity. Sphingomyelinase activation occurs in different cardiovascular system cell types, namely cardiac myocytes, endothelial and vascular smooth muscle cells, mediating cell proliferation, cell death, and contraction of cardiac and vascular myocytes. Three main types of SMases contribute to cardiovascular physiology: the lysosomal and secreted acidic SMases (L- and S-ASMases, respectively) and the membrane neutral SMase (NSMase). These three enzymes have common activators, including ischaemia/reperfusion stress and proinflammatory cytokines, but they differ in their enzymatic properties and subcellular locations that determine the final effect of enzyme activation. This review focuses on the recent advances in the understanding of ASMase and NSMase pathways and their specific contribution to cardiovascular pathophysiology. Current knowledge indicates that the inhibitors of the different SMase types are potential tools for the treatment of cardiovascular diseases. Acid SMase inhibitors could be tools against post-ischaemia reperfusion injury and in the treatment of atherosclerosis. Neutral SMase inhibitors could be tools for the treatment of atherosclerosis, heart failure, and age-related decline in vasomotion. However, the design of bioavailable and more specific SMase-type inhibitors remains a challenge.

TNFR1 and TNFR2 signaling interplay in cardiac myocytes.

Tumor necrosis factor alpha (TNFalpha) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFalpha induced ROS production, exerted a dual positive and negative action on [Ca(2+)] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFalpha responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFalpha on [Ca(2+)] transient and cell fractional shortening. The positive effect of TNFalpha relied on TNFR2-dependent activation of the cPLA(2) activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA(2) redistribution and AA release, TNFalpha induced a time-dependent phosphorylation of ERK, MSK1, PKCzeta, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFalpha while releasing TNFR2 pathways.

Neutral sphingomyelinase inhibition participates to the benefits of N-acetylcysteine treatment in post-myocardial infarction failing heart rats.

Deficiency in cellular thiol tripeptide glutathione (L-gamma glutamyl-cysteinyl-glycine) determines the severity of several chronic and inflammatory human diseases that may be relieved by oral treatment with the glutathione precursor N-acetylcysteine (NAC). Here, we showed that the left ventricle (LV) of human failing heart was depleted in total glutathione by 54%. Similarly, 2-month post-myocardial infarction (MI) rats, with established chronic heart failure (CHF), displayed deficiency in LV glutathione. One-month oral NAC treatment normalized LV glutathione, improved LV contractile function and lessened adverse LV remodelling in 3-month post-MI rats. Biochemical studies at two time-points of NAC treatment, 3 days and 1 month, showed that inhibition of the neutral sphingomyelinase (N-SMase), Bcl-2 depletion and caspase-3 activation, were key, early and lasting events associated with glutathione repletion. Attenuation of oxidative stress, downregulation of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and its TNF-R1 receptor were significant after 1-month NAC treatment. These data indicate that, besides glutathione deficiency, N-SMase activation is associated with post-MI CHF progression, and that blockade of N-SMase activation participates to post-infarction failing heart recovery achieved by NAC treatment. NAC treatment in post-MI rats is a way to disrupt the vicious sTNF-alpha/TNF-R1/N-SMase cycle.

Structural localization and expression of CXCL12 and CXCR4 in rat heart and isolated cardiac myocytes.

CXCL12 (SDF-1), which binds CXCR4, is involved in several physiological and pathophysiological processes. In heart, this axis seems to play a key role in cardiogenesis and is involved in the neovascularization of ischemic tissues. Rats have three known CXCL12 mRNA isoforms, of which only alpha and gamma are present in the normal heart. However, little is known about CXCL12 protein expression and localization. We investigated the pattern of protein expression and the localization of both CXCR4 and CXCL12 in the heart, using isolated cardiomyocytes and a rat myocardial infarction model. Western blots showed that cardiomyocytes contained a specific 67-kDa CXCR4 isoform and a 12-kDa CXCL12 isoform. Confocal and electron microscopy clearly showed that CXCR4 was present at the plasmalemma and CXCL12 in continuity of the Z-line, in the proximal part of T-tubules. In conclusion, we provide the first description of the expression and fine localization of CXCR4 and CXCL12 proteins in normal rat heart and cardiomyocytes. These results suggest that the CXCL12/CXCR4 axis may be involved in cardiomyocyte calcium homeostasis regulation. Our work and the well-known chemoattraction properties of the CXCL12/CXCR4 axis highlight the importance of deciphering the function of this axis in both normal and pathological hearts.

Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells.

Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1-3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.

The cytosolic phospholipase A2 pathway, a safeguard of beta2-adrenergic cardiac effects in rat.

We have recently demonstrated that in human heart, beta2-adrenergic receptors (beta2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The beta2-AR/cPLA2 inhibitory pathway involved G(i) and MSK1. Potentiation of beta2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the beta2-AR/cPLA2 pathway on the beta2-AR/AC/PKA-induced Ca2+ responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta2-AR signaling pathway that limits beta2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.

The cardiac beta2-adrenergic signalling a new role for the cPLA2.

The cardiac actions of catecholamines have long been attributed to the predominant beta(1)-AR subtype that couples to the classical Gs/AC/cAMP pathway. Recent research clearly indicates that cardiac beta(2)-ARs play a functional role in healthy heart and assume increasing importance in failing and aged heart. beta(2)-ARs are primarily coupled to an atypical compartmentalized cAMP pathway, regulated by phosphorylation and/or oligomerization of beta(2)-ARs, and under the control of additional beta(2)-AR/Gi-coupled lipidic pathways, the impact of which seems to vary depending on the animal species, the developmental and pathophysiological state. This review focuses, more especially, on one of the last identified beta(2)-AR/Gi pathway, namely the cPLA(2).