RESUMO
Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca²âº release as well as prolonged duration of relengthening and intracellular Ca²âº decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Dieta Hiperlipídica , Deleção de Genes , Miocárdio/patologia , Obesidade/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Western Blotting , Cardiomegalia/complicações , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Comportamento Alimentar , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Obesidade/complicações , Obesidade/patologia , Obesidade/fisiopatologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Serina-Treonina Quinases TOR/metabolismoRESUMO
Phosphatase and tensin homolog (PTEN) deleted from chromosome 10 has been implicated in the maintenance of cardiac homeostasis although the underlying mechanism(s) remains elusive. We generated a murine model of cardiomyocyte-specific knockout of PTEN to evaluate cardiac geometry and contractile function, as well as the effect of metformin on PTEN deficiency-induced cardiac anomalies, if any. Cardiac histology, autophagy and related signaling molecules were evaluated. Cardiomyocyte-specific PTEN deletion elicited cardiac hypertrophy and contractile anomalies (echocardiographic and cardiomyocyte contractile dysfunction) associated with compromised intracellular Ca(2+) handling. PTEN deletion-induced cardiac hypertrophy and contractile anomalies were associated with dampened phosphorylation of PTEN-inducible kinase 1 (Pink1) and AMPK. Interestingly, administration of AMPK activator metformin (200mg/kg/d, in drinking H2O for 4weeks) rescued against PTEN deletion-induced geometric and functional defects as well as interrupted autophagy and autophagic flux in the heart. Moreover, metformin administration partially although significantly attenuated PTEN deletion-induced accumulation of superoxide. RNA interference against Pink1 in H9C2 myoblasts overtly increased intracellular ATP levels and suppressed AMPK phosphorylation, confirming the role of AMPK as a downstream target for PTEN-Pink1. Further scrutiny revealed that activation of AMPK and autophagy using metformin and rapamycin, respectively, rescued against PTEN deletion-induced mechanical anomalies with little additive effect. These data demonstrated that cardiomyocyte-specific deletion of PTEN leads to the loss of Pink1-AMPK signaling, development of cardiac hypertrophy and contractile defect. Activation of AMPK rescued against PTEN deletion-induced cardiac anomalies associated with restoration of autophagy and autophagic flux. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Deleção de Genes , Contração Miocárdica , Miócitos Cardíacos/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Autofagia/efeitos dos fármacos , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Técnicas de Inativação de Genes , Espaço Intracelular/metabolismo , Metformina/farmacologia , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , PTEN Fosfo-Hidrolase/deficiência , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr(3+) to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.
Assuntos
Cromo/farmacologia , Oligopeptídeos/farmacologia , Animais , Células Cultivadas , Cromo/administração & dosagem , Cromo/química , Glucose/administração & dosagem , Glucose/metabolismo , Teste de Tolerância a Glucose , Injeções Intravenosas , Resistência à Insulina , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Espectrometria de Fluorescência , Espectrofotometria InfravermelhoRESUMO
Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca²âº properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1ß, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca²âº release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca²âº properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3ß. In vitro study using the GSK3ß inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca²âº anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3ß-dependent mechanism.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Lipopolissacarídeos/toxicidade , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Cálcio/metabolismo , Caspase 3/biossíntese , Caspase 3/genética , Caspase 9/biossíntese , Caspase 9/genética , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fator de Iniciação 2 em Eucariotos/biossíntese , Fator de Iniciação 2 em Eucariotos/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Contração Miocárdica/genética , Miocárdio/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genética , Fator de Transcrição TFIIH , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genética , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
Apelin has been recognized as an adipokine that plays an important role in regulating energy metabolism and is credited with antiobesity and antidiabetic properties. This study was designed to examine the effect of exogenous apelin on obesity-associated cardiac dysfunction. Oral glucose tolerance test, echocardiography, cardiomyocyte contractile and intracellular Ca(2+) properties were assessed in adult C57BL/6J mice fed - low or a - high-fat diet for 24weeks followed by apelin treatment (100nmol/kg, i.p. for 2weeks). High-fat diet resulted in increased left ventricular diastolic and systolic diameters, and wall thickness, compromised fractional shortening, impaired cardiomyocyte mechanics (peak-shortening, maximal velocity of shortening/relengthening, and duration of shortening and relengthening) and compromised intracellular Ca(2+) handling, all of which were reconciled by apelin. Apelin treatment also reversed high fat diet-induced changes in intracellular Ca(2+) regulatory proteins, ER stress, and autophagy. In addition, microRNAs (miR) -133a, miR-208 and miR-1 which were elevated following high-fat feeding were attenuated by apelin treatment. In cultured cardiomyocytes apelin reconciled palmitic acid-induced cardiomyocyte contractile anomalies. Collectively, these data depict a pivotal role of apelin in obesity-associated cardiac contractile dysfunction, suggesting a therapeutic potential of apelin in the management of cardiac dysfunction associated with obesity.
Assuntos
Cardiomegalia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Contração Miocárdica/efeitos dos fármacos , Adipocinas , Animais , Apelina , Autofagia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Cardiomegalia/etiologia , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Leptina/sangue , Camundongos , MicroRNAs/genética , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ácido Palmítico/farmacologia , Triglicerídeos/sangueRESUMO
[This corrects the article DOI: 10.1016/j.apsb.2021.07.006.].
RESUMO
BACKGROUND: Lethal and edema toxins secreted by Bacillus anthracis during anthrax infection were found to incite serious cardiovascular complications. However, the underlying mechanisms in anthrax lethal toxin-induced cardiac anomalies remain unknown. This study was designed to evaluate the impact of antioxidant enzyme catalase in anthrax lethal toxin-induced cardiomyocyte contractile dysfunction. METHODS: Wild type (WT) and cardiac-specific catalase overexpression mice were challenged with lethal toxin (2 µg/g, intraperotineally (i.p.)). Cardiomyocyte contractile and intracellular Ca(2+) properties were assessed 18 h later using an IonOptix edge-detection system. Proteasome function was assessed using chymotrypsin-like and caspase-like activities. GFP-LC3 puncta and Western blot analysis were used to evaluate autophagy and protein ubiquitination. RESULTS: Lethal toxin exposure suppressed cardiomyocyte contractile function (suppressed peak shortening, maximal velocity of shortening/re-lengthening, prolonged duration of shortening/re-lengthening, and impaired intracellular Ca(2+) handling), the effects of which were alleviated by catalase. In addition, lethal toxin triggered autophagy, mitochondrial and ubiquitin-proteasome defects, the effects of which were mitigated by catalase. Pretreatment of cardiomyocytes from catalase mice with the autophagy inducer rapamycin significantly attenuated or ablated catalase-offered protection against lethal toxin-induced cardiomyocyte dysfunction. On the other hand, the autophagy inhibitor 3-MA ablated or significantly attenuated lethal toxin-induced cardiomyocyte contractile anomalies. CONCLUSIONS: Our results suggest that catalase is protective against anthrax lethal toxin-induced cardiomyocyte contractile and intracellular Ca(2+) anomalies, possibly through regulation of autophagy and mitochondrial function.
Assuntos
Antígenos de Bactérias/toxicidade , Autofagia , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/toxicidade , Catalase/metabolismo , Contração Miocárdica/efeitos dos fármacos , Estresse Oxidativo , Animais , Cálcio/análise , Citoplasma/química , Masculino , Camundongos , Camundongos Transgênicos , Peptídeo Hidrolases/análise , UbiquitinaçãoRESUMO
AMPK, a metabolic sensor, protects against ischemic injury and cardiac hypertrophy although its role in obesity is unclear. This study was designed to examine the impact of AMPK deficiency on cardiac dysfunction following high fat feeding. Adult WT and transgenic mice overexpressing a kinase dead (KD) α2 isoform (K45R mutation) of AMPK were fed a low or high fat diet for 20 weeks. DEXA was used to confirm adiposity. Wheat germ agglutinin immunostaining was used to evaluate myocardial histology. Myocardial function was evaluated using echocardiography and edge-detection. AMPK activity was analyzed using fluorescence polarization assays. [1-(14)C] oleate was used to determine fatty acid oxidation. Expression of AMPK, α1, α2, ACC, Akt, the Glut-4 translocation mediator Akt substrate of 160KD (AS160), mTOR, total and membrane Glut-4 was evaluated using Western blot. AMPK activity was decreased in KD mice regardless of diet regimen. High fat diet led to obesity, glucose intolerance and cardiac hypertrophy with accentuated glucose intolerance, dampened fatty acid oxidation and cardiac hypertrophy in KD mice. High fat feeding triggered lower fractional shortening, increased LV mass, left ventricular end diastolic/systolic diameter, decreased PS, ± dL/dt, prolonged TR(90) and intracellular Ca(2+) mishandling with a more pronounced effect in KD mice. High fat diet and AMPK KD lessened AMPKα2 isoform activity and ACC phosphorylation. AMPK deficiency unveiled or accentuated high fat diet-induced decrease in phosphorylation of Akt and AS160, membrane fraction of Glut-4 and mTOR expression (a greater mTOR phosphorylation). Taken together, these data suggest that AMPK deficiency exacerbates obesity-induced cardiac hypertrophy and contractile dysfunction, possibly associated with AS160 and mTOR signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/metabolismo , Gorduras na Dieta/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Ecocardiografia , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2-Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2 -/- and Ampkα2 -/- mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2 -/- and Ampkα2 -/- mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2 -/- and Ampkα2 -/- mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser485), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser485), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.
RESUMO
BACKGROUND/AIM: Recent evidence suggests that endoplasmic reticulum (ER) stress provoked under diabetic conditions augments the expression of scavenger receptors on macrophages, promoting the uptake of oxidized low-density lipoprotein uptake and atherogenesis. The aim of the present study was to test the hypothesis that the chemical chaperone tauroursodeoxycholic acid (TUDCA) attenuates lipid accumulation in macrophages subjected to ER stress. METHODS: Cultured human macrophages were subjected to ER stress by treating them with tunicamycin. Lipid uptake by macrophages subjected to ER stress in the presence or absence of TUDCA was assessed by oil red O staining and by assessing the cellular uptake of Dil-oxidized low-density lipoprotein by fluorescence measurement. Protein levels and phosphorylation status of ER stress markers, insulin-signaling molecules, and scavenger receptor were assessed by Western blotting. RESULTS: Treatment of cultured human macrophages with the ER stressor tunicamycin caused an increase in the protein levels of cluster of differentiation 36 (CD-36) and augmentation of lipid uptake both of which were inhibited by TUDCA. TUDCA treatment inhibited tunicamycin-induced ER stress as evidenced by the attenuation of phosphorylation of eukaryotic translation initiation factor-2a and glucose reactive protein-78. In addition, TUDCA improved insulin signaling in macrophages by augmenting Akt phosphorylation and blunting c-Jun N-terminal kinase activity. CONCLUSIONS: Inhibition of macrophage ER stress may represent a potential strategy in preventing atherogenesis under diabetic conditions.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Western Blotting , Antígenos CD36/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Insulina/metabolismo , Lipoproteínas LDL/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tunicamicina/farmacologiaRESUMO
Vascular smooth muscle cells (VSMCs) perform essential smooth muscle contractile and synthetic functions including migration, differentiation and proliferation under physiological and pathological conditions. In response to pathological stimuli, VMSCs undergo phenotypic change resulting in abnormal migration and proliferation, which may contribute to a "pathogenesis-like" atherosclerosis. Intracellular signaling mechanisms governing this phenotypic switch are of great significance not only for better understanding of atherosclerotic plaque formation but also for strategy for pertinent therapeutic remedies. Src Homology 2 Containing Protein Tyrosine Phosphatase 2 (SHP2) is a ubiquitous tyrosine phosphatase containing Src Homology 2 domains which plays major biological functions in response to various growth factors, hormones or cytokines. In particular, SHP2 is implicated in cell signaling pathways controlling cell cycle progression, growth and migration. In this review we will mainly discuss the recent literature demonstrating the role of SHP2 in VSMC migration and proliferation.
Assuntos
Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Movimento Celular , Proliferação de Células , Humanos , Transdução de Sinais , Domínios de Homologia de srcRESUMO
High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca2+ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca2+ handling), ultrastructural disarray, apoptosis, O2-, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser757 and Ser777), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hipertrofia Ventricular Esquerda/imunologia , Miocárdio/patologia , Obesidade/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Autofagia/genética , Autofagia/imunologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Mitofagia/genética , Mitofagia/imunologia , Miocárdio/citologia , Miocárdio/imunologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Nucleotidiltransferases/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Remodelação Ventricular/genéticaRESUMO
Increased age often leads to a gradual deterioration in cardiac geometry and contractile function although the precise mechanism remains elusive. Both Akt and AMPK play an essential role in the maintenance of cardiac homeostasis. This study examined the impact of ablation of Akt2 (the main cardiac isoform of Akt) and AMPKα2 on development of cardiac aging and the potential mechanisms involved with a focus on autophagy. Cardiac geometry, contractile, and intracellular Ca2+ properties were evaluated in young (4-month-old) and old (12-month-old) wild-type (WT) and Akt2-AMPK double knockout mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy were evaluated using western blot. Our results revealed that increased age (12â¯months) did not elicit any notable effects on cardiac geometry, contractile function, morphology, ultrastructure, autophagy and mitophagy, although Akt2-AMPK double knockout predisposed aging-related unfavorable changes in geometry (heart weight, LVESD, LVEDD, cross-sectional area and interstitial fibrosis), TEM ultrastructure, and function (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, intracellular Ca2+ release and clearance rate). Double knockout of Akt2 and AMPK unmasked age-induced cardiac autophagy loss including decreased Atg5, Atg7, Beclin1, LC3BII-to-LC3BI ratio and increased p62. Double knockout of Akt2 and AMPK also unmasked age-related loss in mitophagy markers PTEN-induced putative kinase 1 (Pink1), Parkin, Bnip3, and FundC1, the mitochondrial biogenesis cofactor PGC-1α, and lysosomal biogenesis factor TFEB. In conclusion, our data indicate that Akt2-AMPK double ablation predisposes cardiac aging possibly related to compromised autophagy and mitophagy. This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Envelhecimento , Coração/fisiopatologia , Contração Miocárdica , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Autofagia , Deleção de Genes , Coração/fisiologia , Cardiopatias/genética , Cardiopatias/fisiopatologia , Longevidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , MitofagiaRESUMO
Endothelin (ET)-1 is implicated in the pathophysiology of cardiovascular diseases although its role in obesity anomalies has not been fully elucidated. This study was designed to examine the impact of ET-1 receptor A (ETA) ablation on obesity-induced changes in cardiac geometry and contractile function, as well as the mechanisms involved with a focus on autophagy. Cardiomyocyte-specific ETA receptor knockout (ETAKO) and WT mice were fed either low-fat (10% calorie from fat) or high-fat (45% calorie from fat) diet for 24â¯weeks. Glucose tolerance test was examined to confirm insulin resistance. High-fat diet intake compromised myocardial geometry (enlarged left ventricular diameters in systole and diastole), morphology (cardiac hypertrophy, increased wall thickness and interstitial fibrosis), contractile function (reduced fractional shortening, ejection fraction and cardiomyocyte shortening) and intracellular Ca2+ handling, the effect of which was significantly attenuated by ETAKO. TUNEL staining revealed overt apoptosis in high-fat-fed group, the effect was reverted by ETAKO. Western blot analysis noted that high-fat intake downregulated leptin receptor and PPARγ, insulin signaling (elevated basal/dampened insulin-stimulated phosphorylation of Akt and IRS1), phosphorylation of AMPK, ACC, upregulated GATA-4, ANP, NFATc3, PPARα, m-TOR/p70s6k signaling, which were attenuated by ETAKO with the exception of AMPK/ACC. Furthermore, high-fat intake suppressed cardiac autophagy, which was abrogated by ETAKO. In cultured murine cardiomyocytes, palmitic acid challenged mimicked high-fat diet-induced hypertrophic and autophagic responses, the effect of which were abolished by the ETA receptor antagonist BQ123 or mTOR inhibitor rapamycin. These results suggest that inhibition of ETA rescues high-fat intake-induced cardiac anomalies possibly through autophagy regulation.
Assuntos
Cardiomiopatias/genética , Miócitos Cardíacos/citologia , Obesidade/complicações , Receptor de Endotelina A/genética , Animais , Autofagia , Cardiomiopatias/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Resistência à Insulina , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Obesidade/genética , Obesidade/metabolismo , PPAR gama/metabolismo , Ácido Palmítico/farmacologia , Receptor de Endotelina A/metabolismo , Receptores para Leptina/metabolismo , Transdução de SinaisRESUMO
Leptin hormone plays a vital role in the pathophysiological changes in heart geometry and function. Nonetheless, the precise mechanism(s) triggering leptin-induced cardiomyocyte contractile dysfunction is not well understood. The present study was designed to examine if autophagy plays a role in leptin-induced cardiac contractile anomalies. Cardiomyocyte contractile function was evaluated using an IonOptix edge detection system in cardiomyocytes following treatment with leptin in the presence or absence of the autophagy inhibiting chemical 3-methyladenine (3-MA). Immunoblotting was employed to evaluate expression of AMPK, Beclin1, Atg 5, p62 and LC3-II. GFP-LC3 puncta was used to assess autophagosome formation. Leptin suppressed cardiac contractile function as evidenced by decreased peak shortening, maximal velocity of shortening and relengthening, increased time-to-90% relengthening, all the observed effects were reduced or obliterated by autophagy inhibition. Leptin promoted superoxide generation, AMPK activation and overt autophagy induction. Leptin promoted autophagy as evidenced by enhanced LC3-II, Beclin, Atg 5 and decreased p62 levels. Pharmacological inhibition of reactive oxygen species (ROS) using tempol significantly attenuated leptin-induced autophagosome formation and cardiac contractile anomalies. In addition, genetic deletion of AMPKα2 or pharmacological inhibition of AMPK using compound C abrogated leptin or superoxide induced cardiac contractile dysfunction and autophagosome formation. In summary, our data revealed that leptin impairs cardiac contractile function through a superoxide generation-AMPK activation-and autophagy dependent mechanism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Cardiotônicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Leptina/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Leptina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fagossomos/enzimologia , Fagossomos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
This study was designed to evaluate the role of ULK1 in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge. Wild-type and AMPK knockout mice were challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial function was evaluated using echocardiography and edge-detection. Western blot analysis was employed to evaluate the levels of AMPK, Raptor, mTOR, the AMPK downstream signal ULK1 and autophagy markers Beclin-1 and LC3-II. siRNA was used to knockdown ULK1 in H9C2 myoblasts. GFP-LC3 puncta was used to evaluate autophagosome formation. Alcohol challenge compromised cardiac function as evidenced by decreased fractional shortening, peak shortening and intracellular Ca²âº rise, prolonged relengthening and intracellular Ca²âº decay in WT mice, the effects of which were mitigated by AMPK knockout. Ethanol exposure facilitated myocardial autophagy as evidenced by enhanced LC3-II level, as well as phosphorylation of AMPK, Raptor, and dephosphorylation of mTOR and ULKI in WT hearts, which were alleviated by AMPK knockout. Pharmacological inhibition of AMPK using compound C attenuated ethanol-induced autophagosome formation, AMPK phosphorylation, ULK1 dephosphorylation and apoptosis. Ethanol exposure-induced cardiomyocyte contractile defects and autophagosome accumulation were reversed by the autophagy inhibitor 3-MA. Similarly, knockdown of ULK1 using siRNA in H9C2 cells ablated ethanol-induced autophagosome accumulation, LC3-II expression and cell death. Lysosomal inhibition using bafilomycin, E64-D and pepstatin A potentiated ethanol-induced increase in autophagosome formation. Taken together, our results suggest that ULK1 may play a critical role in AMPK-mediated myocardial autophagy, apoptosis and contractile dysfunction following acute alcohol challenge.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Cardiomiopatia Alcoólica/metabolismo , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miocárdio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Sinalização do Cálcio , Cardiomiopatia Alcoólica/patologia , Cardiomiopatia Alcoólica/fisiopatologia , Linhagem Celular , Inativação Gênica , Coração/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Mioblastos Cardíacos/enzimologia , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/patologia , Contração Miocárdica , Miocárdio/patologia , Fagossomos/metabolismo , Fagossomos/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno , RatosRESUMO
Maternal overnutrition is associated with predisposition of offspring to cardiovascular disease in later life. Since maternal overnutrition may promote fetal and placental inflammatory responses, we hypothesized that maternal overnutrition/obesity increases expression of fetal cardiac proinflammatory mediators and alter cardiac morphometry. Multiparous ewes were fed either 150% of National Research Council (NRC) nutrient recommendations (overfed) or 100% of NRC requirement (control) from 60 days prior to mating to gestation Day 75 (D75), when ewes were euthanized. An additional cohort of overfed and control ewes were necropsied on D135. Cardiac morphometry, histology, mRNA and protein expression of toll-like receptor 4, iNOS, IL-1a, IL-1b, IL-6, IL-18, CD-14, CD-68, M-CSF and protein levels of phosphorylated I-κB and nuclear factor κB (NF-κB) were examined. Immunohistochemistry was performed to assess neutrophil and monocyte infiltration. Crown rump length, left and right ventricular free wall weights as well as left and right ventricular wall thickness were significantly increased in D75 fetuses of overfed mothers. Hematoxylin and eosin staining revealed irregular myofiber orientation and increased interstitial space in fetal ventricular tissues born to overfed mothers. Oil red O staining exhibited marked lipid droplet accumulation in the overfed fetuses. Overfeeding significantly enhanced TLR4, IL-1a, IL-1b IL-6 expression, promoted phosphorylation of IκB, decreased cytoplasmic NF-κB levels and increased neutrophil and monocyte infiltration. Collectively, these data suggest that maternal overfeeding prior to and throughout gestation leads to inflammation in the fetal heart and alters fetal cardiac morphometry.
Assuntos
Coração Fetal/fisiopatologia , Mediadores da Inflamação/metabolismo , Hipernutrição/fisiopatologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Feminino , Ventrículos do Coração/metabolismo , Miocardite/embriologia , Gravidez , OvinosRESUMO
Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.
Assuntos
Autofagia , Etanol/metabolismo , Coração/fisiopatologia , Contração Miocárdica , Miócitos Cardíacos/patologia , Acetaldeído/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Álcool Desidrogenase/metabolismo , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Etanol/administração & dosagem , Imunofluorescência , Coração/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Proteína bcl-X/metabolismoRESUMO
BACKGROUND AND PURPOSE: Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction. EXPERIMENTAL APPROACH: Wild-type (WT) and TLR4 knockout (TLRâ»/â») mice were challenged with lethal toxin (2 µg·g⻹, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP-LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2α and calreticulin). KEY RESULTS: In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca²âº derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells. CONCLUSION AND IMPLICATIONS: Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.