RESUMO
Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscular and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. We showed here an increase in oxidative stress levels in the hearts of mice carrying LMNA mutation, associated with a decrease of the key cellular antioxidant glutathione (GHS). Oral administration of N-acetyl cysteine, a GHS precursor, led to a marked improvement of GHS content, a decrease in oxidative stress markers including protein carbonyls and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our novel results provide therapeutic insights into LMNA cardiomyopathy.
Assuntos
Acetilcisteína/administração & dosagem , Cardiomiopatia Dilatada/genética , Insuficiência Cardíaca/genética , Lamina Tipo A/genética , Acetilcisteína/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Glutationa/metabolismo , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Humanos , Camundongos , Mutação , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
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.
Assuntos
Apoptose , Fígado Gorduroso/etiologia , Células de Kupffer/fisiologia , Fígado/citologia , Adulto , Animais , Arginase/metabolismo , Biomarcadores/metabolismo , Dieta Hiperlipídica , Ativação Enzimática , Etanol , Feminino , Humanos , Interleucina-10/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Obesidade Mórbida/metabolismo , Obesidade Mórbida/patologia , Comunicação Parácrina , Resveratrol , EstilbenosRESUMO
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.
Assuntos
Células de Kupffer/metabolismo , Hepatopatias Alcoólicas/metabolismo , Regeneração Hepática/fisiologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Etanol/farmacologia , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Células de Kupffer/citologia , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Valores de Referência , Estatísticas não ParamétricasRESUMO
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.
Assuntos
Cardiomiopatias/metabolismo , Distrofina/metabolismo , Glutationa/metabolismo , Miocárdio/metabolismo , Adulto , Análise de Variância , Animais , Cardiomiopatias/complicações , Cardiomiopatias/fisiopatologia , Distrofina/genética , Ecocardiografia , Coração/fisiopatologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
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.
Assuntos
Cardiomiopatias/etiologia , Fibroblastos/citologia , Traumatismo por Reperfusão Miocárdica/complicações , Miocárdio/patologia , Miócitos Cardíacos/citologia , Receptor CB2 de Canabinoide/fisiologia , Animais , Sobrevivência Celular , Peróxido de Hidrogênio , Camundongos , Camundongos Knockout , Substâncias Protetoras , Receptor CB2 de Canabinoide/deficiência , Disfunção Ventricular Esquerda/etiologiaRESUMO
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.
Assuntos
Acetilcisteína/farmacologia , Cálcio/metabolismo , Glutationa/análogos & derivados , Miócitos Cardíacos/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Glutationa/metabolismo , Glutationa/farmacologia , Masculino , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
BACKGROUND: Studies in isolated cardiomyocytes showed that replenishment in cellular glutathione, achieved with the glutathione precursor N-acetylcysteine (NAC), abrogated deleterious effects of tumor necrosis factor-alpha (TNF-alpha). METHODS AND RESULTS: We examined the ability of NAC to limit the progression of cardiac injury in the rat model of hypertension, induced by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) (50 mg/kg per day SC) and high-salt diet (HS) (8% NaCl). Four-week HS/L-NAME administration induced hypertension (193+/-8 versus 122+/-4 mm Hg for low-salt diet [LS] group) and left ventricular (LV) dysfunction, revealed by echocardiography and characterized by decreased LV shortening fraction (38+/-2% versus 49+/-4% for LS group; P<0.05) and decreased LV posterior wall thickening (49+/-3% versus 70+/-4% for LS group; P<0.05). LV dysfunction worsened further after 6-week HS/L-NAME administration. Importantly, increase in serum TNF-alpha level was strongly correlated with shortening fraction decrease and cardiac glutathione depletion. NAC (75 mg/d) was given as a therapeutic treatment in a subgroup of HS/L-NAME animals during weeks 5 and 6 of HS/L-NAME administration. NAC treatment, which replenished cardiac glutathione, had no effect on hypertension but reduced LV remodeling and dysfunction, normalized serum TNF-alpha level, and limited activation of matrix metalloproteinases -2 and -9 and collagen deposition in LV tissues. CONCLUSIONS: These findings suggest that glutathione status determines the adverse effects of TNF-alpha in cardiac failure and that TNF-alpha antagonism may be achieved by glutathione supplementation.
Assuntos
Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Glutationa/fisiologia , Hipertensão/tratamento farmacológico , Fator de Necrose Tumoral alfa/análise , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Colágeno/análise , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Glutationa/deficiência , Ventrículos do Coração/química , Hipertensão/sangue , Hipertensão/induzido quimicamente , Hipertensão/complicações , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Contração Miocárdica , Miocárdio/metabolismo , NG-Nitroarginina Metil Éster/toxicidade , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Cloreto de Sódio na Dieta/toxicidade , Esfingomielina Fosfodiesterase/metabolismo , Ultrassonografia , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/etiologiaRESUMO
OBJECTIVES: Severe myocyte alterations, characterized by enlarged myocytes and myolysis, is observed in fibrillating and dilated atria and contributes to atrial fibrillation. The aim of this study was to determine the nature of this cellular remodeling process and factors involved in its regulation. METHODS: In vivo, contractile proteins were studied in 24 human right atrial specimens by means of immunohistochemical techniques. In an attempt to reproduce in vitro the myocyte remodeling and to study its regulation, human atrial myocytes were cultured (n=27) and analyzed immunocytochemically; intracellular Ca(2+) transients (Ca(i)-tr) in response to electrical stimulation were monitored using Fura-2/AM. RESULTS: In diseased specimens, sarcomeres, seen at the periphery of myolytic myocytes, stained positively with antibodies against sarcomeric proteins of the Z-band (alpha-actinin and titin epitope T12) but not with antibodies against titin epitope T11 (I-band) or desmin (intermediate filament). beta-myosin heavy chain (MHC) and smooth muscle alpha-actin, two proteins of the fetal program, were re-expressed. In culture, diseased myocytes also showed myolysis and glycogen accumulation; their sarcomeres stained positively with anti-alpha-actinin, anti-T12, anti-beta-MHC and anti-smooth muscle alpha-actin but not with anti-titin T11 or anti-desmin antibodies. At confluence, myocytes regained a normal sarcomeric apparatus and were excitable, as shown by electrical Ca(i)-tr triggering. This redifferentiation process was inhibited by fibroblast proliferation. CONCLUSION: In diseased atria, myolytic myocytes are in a dedifferentiated state resembling that of immature muscle cells. In vitro, fibroblast proliferation prevents the reversibility of this cellular alteration.
Assuntos
Apêndice Atrial/patologia , Fibrilação Atrial/patologia , Actinina/análise , Actinas/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apêndice Atrial/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Divisão Celular , Células Cultivadas , Criança , Pré-Escolar , Conectina , Eletrofisiologia , Feminino , Fibroblastos/patologia , Fura-2 , Átrios do Coração , Humanos , Imuno-Histoquímica/métodos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas Musculares/análise , Proteínas Quinases/análise , Sarcômeros/metabolismo , Miosinas Ventriculares/análiseRESUMO
Dominant inherited Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy type 1B are due to mutations in the LMNA gene encoding lamin A/C and present similar life-threatening cardiac disease, the early diagnosis of which lacks reliable biomarkers. Glutathione depletion characterizes subjects with cardiac diseases of non-genetic aetiology. We examined blood glutathione in 22 LMNA-mutated subjects without altered left ventricular ejection fraction (LVEF>40%) measured by conventional echocardiography. Left and right ventricular (LV/RV) contractility was evaluated using echocardiography implemented with tissue-Doppler echography. Blood glutathione was positively correlated with LV and RV contractility (p<0.05), and was decreased by 23% in subjects with reduced LV/RV contractility compared to subjects with normal contractility. ROC analysis showed that blood glutathione reliably detected reduced LV/RV contractility (AUC-95% CI: 0.90 [0.76-1.04]; p=0.01). Blood glutathione decrease may allow the detection of reduced contractility in muscular dystrophic LMNA-mutated patients with still preserved LVEF.
Assuntos
Glutationa/sangue , Cardiopatias/sangue , Cardiopatias/genética , Lamina Tipo A/genética , Mutação de Sentido Incorreto/genética , Adolescente , Adulto , Estudos de Coortes , Estudos Cross-Over , Ecocardiografia , Ensaio de Imunoadsorção Enzimática/métodos , Saúde da Família , Feminino , Cardiopatias/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Estudos Retrospectivos , Estatísticas não Paramétricas , Volume Sistólico/fisiologia , Adulto JovemRESUMO
BACKGROUND: Recently, concerns have been raised about a possible lack of sensitivity of biomarkers to detect left ventricular (LV) dysfunction in patients with myopathies. We examined the ability of the N-terminal brain natriuretic peptide (NT-proBNP) to detect LV or right ventricular (RV) dysfunction in patients with lamin A/C (LMNA) gene mutations. METHODS: We prospectively measured plasma NT-proBNP in consecutive patients with documented LMNA mutations and age-sex matched controls. All patients underwent standard echocardiography implemented by pulsed tissue-Doppler echocardiography (TDE). RESULTS: Twenty-three patients were included (10 males, mean age 39.2 ± 18.9 years);10 had previous atrial arrhythmias, 8 had been implanted with cardioverter defibrillator for primary prevention of sudden death, 5 patients were of NYHA class II and 18 of NHYA class I. Sinus rhythm was recorded in all. NT-proBNP was increased in LMNA patients versus controls (123 ± 229 versus 26 ± 78 pg/ml, p=0.0004); 7 patients had depressed LV and/or RV contractility. Patients with reduced LV or RV contractility had increased mean NT-proBNP (341 ± 1032 pg/ml versus 80 ± 79 pg/ml in patients with normal myocardial contractility, p=0.004). Receiver-operating-characteristics analysis shows that NT-proBNP reliably detected depressed contractility (area under the curve 0.889 [0.697-1.000]). Sensitivity and specificity were 88% and 83% respectively, applying manufacturer's recommended cut-off concentration of 125 pg/ml. CONCLUSION: NT-proBNP reliably detected the presence of reduced LV/RV contractility in LMNA patients.
Assuntos
DNA/genética , Lamina Tipo A/genética , Mutação , Contração Miocárdica/genética , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Disfunção Ventricular Esquerda/genética , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Criança , Ecocardiografia Doppler em Cores , Ensaio de Imunoadsorção Enzimática , Feminino , Predisposição Genética para Doença , Humanos , Lamina Tipo A/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Esfingomielina Fosfodiesterase/fisiologia , Ceramidas/fisiologia , Doença da Artéria Coronariana/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Doenças Cardiovasculares/diagnóstico , Glutationa/deficiência , Átrios do Coração/química , Cardiopatias Congênitas/diagnóstico , Índice de Gravidade de Doença , Adulto , Idoso , Doenças Cardiovasculares/cirurgia , Estudos de Casos e Controles , Feminino , Glutationa/análise , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/química , Disfunção Ventricular EsquerdaRESUMO
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.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acetilcisteína/farmacologia , Animais , Anticorpos/farmacologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doença Crônica , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Miócitos Cardíacos/patologia , Fosfolipases A2 Citosólicas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Acetilcisteína/uso terapêutico , Cardiotônicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Ecocardiografia Doppler , Glutationa/deficiência , Glutationa/metabolismo , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Citosol/enzimologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfolipases A/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Caveolina 1 , Caveolina 3 , Caveolinas/metabolismo , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Immunoblotting , Imuno-Histoquímica , Indóis/farmacologia , Isoquinolinas/farmacologia , Lisofosfolipase/metabolismo , Microscopia Confocal , NG-Nitroarginina Metil Éster/farmacologia , Toxina Pertussis/farmacologia , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosforilação , Ratos , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Serina/química , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
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.
Assuntos
Ácido Araquidônico/fisiologia , Sinalização do Cálcio/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Amidoidrolases/antagonistas & inibidores , Animais , Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Separação Celular , Ceramidases , Inibidores de Ciclo-Oxigenase/farmacologia , Citosol/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Endocanabinoides , Ativação Enzimática/fisiologia , Etanolaminas/farmacologia , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Ácidos Oleicos , Fosfolipases A/metabolismo , Fosfolipases A2 , Ratos , Ratos WistarRESUMO
We have recently established that enhancement of intracellular calcium cycling and contraction in response to beta2-adrenergic receptor (beta2-AR) stimulation exclusively relies on the activation of the cytosolic phospholipase A2 (cPLA2) and arachidonic acid production, via a pertussis toxin-sensitive G protein (possibly Gi), in embryonic chick cardiomyocytes. We aimed to investigate the relevance of the beta2-AR/Gi/cPLA2 pathway in the human myocardium. In left ventricular biopsies obtained from explanted hearts, beta2-AR stimulation exerted either an inhibition of cPLA2 that was insensitive to pertussis toxin (PTX) treatment, or an activation of cPLA2, sensitive to PTX treatment. In right atrial appendages from patients who were undergoing open heart surgery, we demonstrated that beta2-AR-induced activation of cPLA2 was favored in situations of altered beta1-AR and/or beta2-AR/adenylyl cyclase (AC) stimulations. Alterations were characterized by an increase in EC50value of norepinephrine and a decrease in the maximal AC activation in response to zinterol, respectively. Quantitative reverse transcription-polymerase chain reaction analyses highlighted a positive correlation between the expression of AC5 and AC6 mRNAs in human cardiac atria, which suggested that functional alterations in AC responses were unlikely to be related to changes in the AC5/AC6 mRNA ratio. In addition, the shift from the cyclic AMP to the arachidonic acid pathway was not supported at the transcriptional level by opposite regulation of AC and cPLA2mRNAs expression. This study gives the first evidence of the recruitment of cPLA2by beta2-ARs in the human heart and suggests that the Gi/cPLA2pathway could substitute for a deficient Gs/AC pathway in mediating beta2-AR responses.