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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 β. 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.

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.

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. PW1 nLacZ 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.

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.

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 TNFR 2 , is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3β 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 TNFR 2 - and Orai3-dependent signaling pathways.

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-3β 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-3β activity., (© 2016 American Heart Association, Inc.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

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 β-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., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

Background: The negative effect of tumor necrosis factor-alpha (TNF-alpha) on heart contraction, which is mediated by sphingosine, is a major component in heart failure. Because the cellular level of glutathione may limit sphingosine production via the inhibition of the Mg-dependent neutral sphingomyelinase (N-SMase), we hypothesized that cardiac glutathione status might determine the negative contractile response to TNF-alpha., Methods and Results: We examined the effects of TNF-alpha in isolated cardiomyocytes obtained from control rats or rats that were given the glutathione precursor N-acetylcysteine (NAC, 100 mg IP per animal). In cardiomyocytes obtained from control rats, 25 ng/mL TNF-alpha increased reactive oxygen species generation and N-SMase activity (500% and 34% over basal, respectively) and decreased the amplitude of [Ca(2+)](i) in response to electrical stimulation (22% below basal). NAC treatment increased cardiac glutathione content by 42%. In cardiomyocytes obtained from NAC-treated rats, 25 ng/mL TNF-alpha had no effect on reactive oxygen species production or N-SMase activity but increased the amplitude of [Ca(2+)](i) transients and contraction in response to electrical stimulation by 40% to 50% over basal after 20 minutes. This was associated with a hastened relaxation (20% reduction in t(1/2) compared with basal) and an increased phosphorylation of both Ser(16)- and Thr(17)-phospholamban residues (260% and 115% of maximal isoproterenol effect, respectively)., Conclusions: It is concluded that cardiac glutathione status, by controlling N-SMase activation, determines the severity of the adverse effects of TNF-alpha on heart contraction. Glutathione supplementation may therefore provide therapeutic benefits for vulnerable hearts.

Tumor necrosis factor (TNF)-alpha has a biphasic effect on heart contractility and stimulates phospholipase A2 (PLA2) in cardiomyocytes. Because arachidonic acid (AA) exerts a dual effect on intracellular Ca2+ concentration ([Ca2+]i) transients, we investigated the possible role of AA as a mediator of TNF-alpha on [Ca2+]i transients and contraction with electrically stimulated adult rat cardiac myocytes. At a low concentration (10 ng/ml) TNF-alpha produced a 40% increase in the amplitude of both [Ca2+]i transients and contraction within 40 min. At a high concentration (50 ng/ml) TNF-alpha evoked a biphasic effect comprising an initial positive effect peaking at 5 min, followed by a sustained negative effect leading to 50-40% decreases in [Ca2+]i transients and contraction after 30 min. Both the positive and negative effects of TNF-alpha were reproduced by AA and blocked by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2. Lipoxygenase and cyclooxygenase inhibitors reproduced the high-dose effects of TNF-alpha and AA. The negative effects of TNF-alpha and AA were also reproduced by sphingosine and were abrogated by the ceramidase inhibitor n-oleoylethanolamine. These results point out the key role of the cytosolic PLA2/AA pathway in mediating the contractile effects of TNF-alpha.

We have recently reported that arachidonic acid mediates beta(2)-adrenergic receptor (AR) stimulation of [Ca(2+)](i) cycling and cell contraction in embryonic chick ventricular cardiomyocytes (Pavoine, C., Magne, S., Sauvadet, A., and Pecker, F. (1999) J. Biol. Chem. 274, 628-637). In the present work, we demonstrate that beta(2)-AR agonists trigger arachidonic acid release via translocation and activation of cytosolic phospholipase A(2) (cPLA(2)) and increase caffeine-releasable Ca(2+) pools from Fura-2-loaded cells. We also show that beta(2)-AR agonists trigger a rapid and dose-dependent phosphorylation of both p38 and p42/44 MAPKs. Translocation and activation of cPLA(2), as well as Ca(2+) accumulation in sarcoplasmic reticulum stores sensitive to caffeine and amplification of [Ca(2+)](i) cycling in response to beta(2)-AR agonists, were blocked by inhibitors of the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively), suggesting a role of both MAPK subtypes in beta(2)-AR stimulation. In contrast, beta(1)-AR stimulation of [Ca(2+)](i) cycling was rather limited by the MAPKs, clearly proving the divergence between beta(2)-AR and beta(1)-AR signaling systems. This study presents the first evidence for the coupling of beta(2)-AR to cardiac cPLA(2) and points out the key role of the MAPK pathway in the intracellular signaling elicited by positive inotropic beta(2)-AR agonists in heart.

Objective: Cardiomyocytes can be transplanted successfully into skeletal and cardiac muscle. Our goal was to determine the feasibility of grafting cardiomyocytes onto various synthetic supports to create an excitable and viable tissue for implantation., Methods: Adult rat cardiomyocytes were cultured over an 8-week period onto different substitutes, including human glutaraldehyde-treated pericardium (n = 3), equine glutaraldehyde-treated pericardium (n = 3), polytetrafluoroethylene (n = 8), Dacron polyester (n = 16), and Vicryl polyglactin (n = 8)., Results: Only the cells seeded on the Dacron survived, with the synthetic fibers colonized at 8 weeks. On the other supports, the number of myocytes progressively decreased from the first week, with their density (number of cells per square millimeter) being, after 20 days, 17 +/- 2 on the polytetrafluoroethylene and 5 +/- 1 on the human or equine pericardium compared with 45 +/- 3 on the Dacron. After 8 weeks of culture on Dacron, the sarcomeric protein (sarcomeric alpha-actinin) was detected in all cells. In addition, the staining was regularly arranged and well aligned in a striated pattern. Spontaneous beating activity was obtained. Moreover, electrical stimulation of the cell preparation resulted in the generation of calcium transients, the frequency of which followed the frequency of the electrical stimulation., Conclusions: These results suggest that adult cardiac myocytes remain viable and excitable during long-term culture on a 3-dimensional Dacron support, which might constitute a new synthetic cardiac tissue.

It has been recently shown that the physiological processing of glucagon into its C-terminal (19-29) fragment, miniglucagon, by cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlying the effects of miniglucagon remained undetermined. In the present study, we assessed the effects of miniglucagon on Ca2+ homeostasis in embryonic chick ventricular myocytes. In quiescent cells, short-term applications of 0.1 nmol/L miniglucagon markedly increased the accumulation of 45Ca into intracellular compartments resistant to digitonin lysis and sensitive to caffeine. Ca2+ accumulation into the sarcoplasmic reticular (SR) store was further attested by fura 2 imaging studies on quiescent or prestimulated cells: miniglucagon potentiated Ca2+ release from the SR compartment triggered by caffeine and evoked a rise in cytosolic Ca2+ when applied on cells pretreated with 1 mumol/L thapsigargin, a specific inhibitor of the SR Ca2+ pump. Glucagon alone produced a small cytosolic Ca2+ signal that was considerably amplified by miniglucagon. The action of glucagon was mimicked by 8-bromo-cAMP and was blocked by isradipine, suggesting that it relied on the activation of L-type Ca2+ channels, via phosphorylation. We conclude that the combined actions of miniglucagon and glucagon on Ca2+ accumulation into SR stores and Ca2+ release from the same stores are likely to support the positive inotropic effect elicited in vivo by glucagon on heart contraction.

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.

The effect of human parathyroid hormone-(1-34) (hPTH) and human calcitonin (hCT) on the activity of the Ca2(+)-extrusion pump in liver plasma membranes was studied. Both hormones were found to be potent inhibitors of Ca2+ transport and the related high-affinity (Ca2(+)-Mg2+)-ATPase activity, causing maximal inhibition of 25-30% at concentrations of 100 nM. Half-maximal inhibition was observed with 20 nM-hPTH and with 0.5 nM-hCT. By comparison, salmon calcitonin and intact bovine parathyroid hormone-(1-84) were inhibitory only at 10 microM. The effects of hCT and hPTH on the Ca2+ pump activity were not mimicked by cyclic AMP. Also, 10 microM of either hPTH-(1-34) or hCT did not alter the 45Ca2+ influx rate into isolated hepatocytes. We conclude that inhibition of Ca2+ efflux, rather than the stimulation of Ca2+ influx, may play a functional role in the control of hepatic calcium homeostasis by hPTH-(1-34) and hCT.

Glucagon specifically inhibits the Ca2+ pump in liver plasma membranes independently of adenylate cyclase activation. However, this inhibition is only observed at high concentrations of glucagon (Ki = 0.7 microM). Moreover, in the presence of bacitracin, an inhibitor of glucagon degradation, the Ca2+ pump is no longer sensitive to glucagon. These findings suggest that a fragment of glucagon might be the true effector of the liver Ca2+ pump. Pairs of basic amino acids are recognized as potential cleavage sites in post-translational processing of peptide hormones. The glucagon molecule includes a dibasic doublet (Arg 17-Arg 18). Therefore, we have examined the action of glucagon(19-29) on the liver Ca2+ pump. This peptide was obtained from glucagon by tryptic cleavage and separated by reverse-phase high-performance liquid chromatography. We found that glucagon(19-29), which is totally ineffective in activating adenylate cyclase, inhibited both the Ca2+-activated and Mg2+-dependent ATPase activity [Ca2+-Mg2+) ATPase) and Ca2+ transport in liver plasma membranes with an efficiency 1,000-fold higher than that of glucagon. Glucagon(1-21) was completely inactive; glucagon(18-29) and glucagon(22-29) acted only as partial agonists of glucagon(19-29). These results indicate that glucagon(19-29), obtained by proteolytic cleavage of glucagon, is likely to be the active peptide involved in the inhibition of the liver Ca2+ pump. We suggest that glucagon may be a precursor of at least one biologically active peptide.