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1.
PLoS Pathog ; 15(4): e1007674, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30958867

RESUMO

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNß pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that-in contrast to cardiomyocytes-other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection.


Assuntos
Proteínas de Transporte/fisiologia , Infecções por Coxsackievirus/prevenção & controle , Enterovirus Humano B/patogenicidade , Miocardite/prevenção & controle , Miócitos Cardíacos/enzimologia , Animais , Células Cultivadas , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/metabolismo , Miocardite/virologia , Proteínas de Ligação a RNA , Replicação Viral
2.
Nature ; 568(7752): 351-356, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971818

RESUMO

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.


Assuntos
Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Estresse Nitrosativo , Volume Sistólico , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Endorribonucleases/metabolismo , Insuficiência Cardíaca/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
3.
Toxicol Lett ; 307: 11-16, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817976

RESUMO

Aldo-Keto Reductase Family 7 Member A2 (AKR7A2) is the most abundant anthracycline metabolizing enzyme in human myocardium. Myocardial AKR7A2 contributes to the synthesis of cardiotoxic C-13 anthracycline alcohol metabolites (e.g., doxorubicinol). The factors that govern the transcription of human AKR7A2 in cardiomyocytes remain largely unexplored. In this study, we performed the functional characterization of the AKR7A2 gene promoter in human AC16 cardiomyocytes. Experiments with gene reporter constructs and chromatin immunoprecipitation assays suggest that NF-κB binds to specific regions in the AKR7A2 promoter. Doxorubicin treatment modified the cellular levels of NF-κB and the expression of AKR7A2. Moreover, doxorubicin treatment led to changes in the pattern of AKR7A2 phosphorylation status. Our results suggest that AKR7A2 expression in human cardiomyocytes is mediated by NF-κB through conserved response elements in the proximal gene promoter region. This study provides the first insights into the functional characteristics of the human AKR7A2 gene promoter.


Assuntos
Aldeído Redutase/metabolismo , Miócitos Cardíacos/metabolismo , Imunoprecipitação da Cromatina , Doxorrubicina/farmacologia , Regulação da Expressão Gênica , Humanos , Miócitos Cardíacos/enzimologia , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas
4.
Nat Commun ; 10(1): 1307, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898999

RESUMO

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF.


Assuntos
Fibrilação Atrial/metabolismo , Fibrilação Atrial/prevenção & controle , Modelos Cardiovasculares , Miócitos Cardíacos/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Benzimidazóis/farmacologia , Células Cultivadas , Dano ao DNA , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Ativação Enzimática/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Humanos , Larva/efeitos dos fármacos , Larva/metabolismo , Camundongos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/patologia , Niacinamida/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Marca-Passo Artificial/efeitos adversos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Pupa/efeitos dos fármacos , Pupa/metabolismo , Ratos , Ratos Wistar
5.
Circ Arrhythm Electrophysiol ; 12(3): e007071, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30879335

RESUMO

BACKGROUND: A structural, electrical and metabolic atrial remodeling is central in the development of atrial fibrillation (AF) contributing to its initiation and perpetuation. In the heart, HDACs (histone deacetylases) control remodeling associated processes like hypertrophy, fibrosis, and energy metabolism. Here, we analyzed, whether the HDAC class I/IIa inhibitor valproic acid (VPA) is able to attenuate atrial remodeling in CREM-IbΔC-X (cAMP responsive element modulator isoform IbΔC-X) transgenic mice, a mouse model of extensive atrial remodeling with age-dependent progression from spontaneous atrial ectopy to paroxysmal and finally long-lasting AF. METHODS: VPA was administered for 7 or 25 weeks to transgenic and control mice. Atria were analyzed macroscopically and using widefield and electron microscopy. Action potentials were recorded from atrial cardiomyocytes using patch-clamp technique. ECG recordings documented the onset of AF. A proteome analysis with consecutive pathway mapping identified VPA-mediated proteomic changes and related pathways. RESULTS: VPA attenuated many components of atrial remodeling that are present in transgenic mice, animal AF models, and human AF. VPA significantly ( P<0.05) reduced atrial dilatation, cardiomyocyte enlargement, atrial fibrosis, and the disorganization of myocyte's ultrastructure. It significantly reduced the occurrence of atrial thrombi, reversed action potential alterations, and finally delayed the onset of AF by 4 to 8 weeks. Increased histone H4-acetylation in atria from VPA-treated transgenic mice verified effective in vivo HDAC inhibition. Cardiomyocyte-specific genetic inactivation of HDAC2 in transgenic mice attenuated the ultrastructural disorganization of myocytes comparable to VPA. Finally, VPA restrained dysregulation of proteins in transgenic mice that are involved in a multitude of AF relevant pathways like oxidative phosphorylation or RhoA (Ras homolog gene family, member A) signaling and disease functions like cardiac fibrosis and apoptosis of muscle cells. CONCLUSIONS: Our results suggest that VPA, clinically available, well-tolerated, and prescribed to many patients for years, has the therapeutic potential to delay the development of atrial remodeling and the onset of AF in patients at risk.


Assuntos
Antiarrítmicos/farmacologia , Fibrilação Atrial/prevenção & controle , Remodelamento Atrial/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Ácido Valproico/farmacologia , Potenciais de Ação , Animais , Fibrilação Atrial/enzimologia , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modelos Animais de Doenças , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Átrios do Coração/ultraestrutura , Frequência Cardíaca , Masculino , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/ultraestrutura , Fatores de Tempo
6.
Molecules ; 24(3)2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717322

RESUMO

Arsenic trioxide (ATO) has been verified as a breakthrough with respect to the management of acute promyelocytic leukemia (APL) in recent decades but associated with some serious adverse phenomena, particularly cardiac functional abnormalities. Salvianolic acid A (Sal A) is a major effective component in treating ATO-induced cardiotoxicity. Therefore, the objective of our study was to assess whether Sal A had protective effects by the regulation of calcium homeostasis and endoplasmic reticulum (ER) stress. For the in vivo study, BALB/c mice were treated with ATO and/or Sal A via daily tail vein injections for two weeks. For the in vitro study, we detected the effects of ATO and/or Sal A in real time using adult rat ventricular myocytes (ARVMs) and an IonOptix MyoCam system. Our results showed that Sal A pretreatment alleviated cardiac dysfunction and Ca2+ overload induced by ATO in vivo and vitro. Moreover, Sal A increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity and expression, alleviated [Ca2+]ER depletion, and decreased ER stress-related protein expression. Sal A protects the heart from ATO-induced injury and its administration correlates with the modulation of SERCA, the recovery of Ca2+ homeostasis, and the down-regulation of ER stress-mediated apoptosis.


Assuntos
Trióxido de Arsênio/efeitos adversos , Ácidos Cafeicos/administração & dosagem , Cardiotoxicidade/tratamento farmacológico , Lactatos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/administração & dosagem , Cálcio/metabolismo , Cardiotoxicidade/etiologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/tratamento farmacológico , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Ratos , Retículo Sarcoplasmático/efeitos dos fármacos
7.
Toxicol Appl Pharmacol ; 368: 26-36, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776389

RESUMO

Cardiac dysfunction is a vital complication during endotoxemia (ETM). Accumulating evidence suggests that enhanced glycolytic metabolism promotes inflammatory and myocardial diseases. In this study, we performed deep mRNA sequencing analysis on the hearts of control and lipopolysaccharide (LPS)-challenged mice (40 mg/kg, i.p.) and identified that the glycolytic enzyme, 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase 3 (PFKFB3) might play an indispensable role in ETM-induced cardiac damage. Quantitative real-time PCR validated the transcriptional upregulation of PFKFB3 in the myocardium of LPS-challenged mice and immunoblotting and immunostaining assays confirmed that LPS stimulation markedly increased the expression of PFKFB3 at the protein level both in vivo and in vitro. The potent antagonist 3-(3pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) was used to block PFKFB3 activity in vivo (50 mg/kg, i.p.) and in vitro (10 µM). Echocardiographic analysis and TUNEL staining showed that 3PO significantly alleviated LPS-induced cardiac dysfunction and apoptotic injury in vivo. 3PO also suppressed the LPS-induced secretion of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 and lactate in the serum, in addition to lactate in the myocardium. PFKFB3 inhibition also diminished the nuclear translocation and phosphorylation of transcription factor nuclear factor-κB (NF-κB) in both adult cardiomyocytes and HL-1 cells. Furthermore, immunoblotting analysis showed that 3PO inhibited LPS-induced apoptotic induction in cardiomyocytes. Taken together, these findings demonstrate that PFKFB3 participates in LPS-induced cardiac dysfunction via mediating inflammatory and apoptotic signaling pathway.


Assuntos
Apoptose , Endotoxemia/enzimologia , Cardiopatias/enzimologia , Mediadores da Inflamação/metabolismo , Miócitos Cardíacos/enzimologia , Fosfofrutoquinase-2/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/patologia , Endotoxemia/prevenção & controle , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Cardiopatias/prevenção & controle , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/genética , Piridinas/farmacologia , Transdução de Sinais
8.
Mol Cell Biochem ; 456(1-2): 85-93, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30712071

RESUMO

Chymases, a family of serine proteases with chymotryptic activity, play a significant role in cardiac angiotensin II (Ang II) formation from its substrate Ang-(1-12) in both human and rodent models. No studies, to date, have assessed the differences in enzymatic activity among these isoforms in Ang II formation, particularly in the cardiomyocyte (CM). Using PCR and DNA sequencing, we demonstrated that MCP-1, MCP-2, MCP-4, and MCP-5 mRNAs are expressed in the CM of both spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). While rMCP-1 and rMCP-5 gene transcripts were higher than that of other isoforms in both rat strains, WKY CM exhibits higher levels of rMCP-1 and rMCP-5 mRNAs compared to the SHR CM. Ovariectomy (OVX) increased the expression of rMCP-1 and rMCP-5 mRNAs in WKY. In SHR, OVX was associated with a blunted increase in rMCP-1 mRNA compared to OVX normotensive WKY. Chymase activity, measured as Ang II formation from Ang-(1-12), significantly correlated with rMCP-1 and rMCP-5 mRNA expression in both rat strains. Both rMCP-1 and rMCP-5 mRNA expressions were positively correlated with progressive diastolic dysfunction (increasing the ratio of early mitral inflow velocity-to-early mitral annular velocity, E/e') and expanding chamber dimensions or increasing left ventricular internal diameter end diastole. These data show rMCP-1 and rMCP-5 as the Ang II forming chymase isoforms participating in the loss of normal cardiac function due to OVX in rodents.


Assuntos
Quimases/biossíntese , Diástole/efeitos dos fármacos , Estrogênios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Musculares/biossíntese , Miócitos Cardíacos/enzimologia , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Feminino , Miócitos Cardíacos/citologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
9.
Acta Biochim Biophys Sin (Shanghai) ; 51(3): 323-330, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30721967

RESUMO

Protein phosphatase 1 isoforms α, ß, and γ (PP1α, PP1ß, and PP1γ) are highly homologous in the catalytic domains but have distinct subcellular localizations. In this study, we utilized both primary cell culture and knockout mice to investigate the isoform-specific roles of PP1s in the heart. In both neonatal and adult cardiac myocytes, PP1ß was mainly localized in the nucleus, compared to the predominant presence of PP1α and PP1γ in the cytoplasm. Adenovirus-mediated overexpression of PP1α led to decreased phosphorylation of phospholamban, which was not influenced by overexpression of either PP1ß or PP1γ. Interestingly, only cardiac-specific knockout of PP1ß resulted in increased HDAC7 phosphorylation, consistent with the predominant nuclear localization of PP1ß. Functionally, deletion of either PP1 isoform resulted in reduced fractional shortening in aging mice, however only PP1ß deletion resulted in interstitial fibrosis in mice as early as 3 weeks of age. Deletion of neither PP1 isoform had any effect on pathological cardiac hypertrophy induced by 2 weeks of pressure overload stimulation. Together, our data suggest that PP1 isoforms have differential localizations to regulate the phosphorylation of their specific substrates for the physiological function in the heart.


Assuntos
Miócitos Cardíacos/enzimologia , Proteína Fosfatase 1/fisiologia , Animais , Células Cultivadas , Feminino , Coração/fisiologia , Isoenzimas/fisiologia , Masculino , Camundongos , Fosforilação , Proteína Fosfatase 1/análise
10.
Clin Sci (Lond) ; 133(2): 225-238, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30610007

RESUMO

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.


Assuntos
Proteína ADAM17/genética , Cardiomegalia/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Células-Tronco Embrionárias Humanas/enzimologia , Mutação com Perda de Função , Mutação de Sentido Incorreto , Miócitos Cardíacos/enzimologia , Tetralogia de Fallot/genética , Proteína ADAM17/metabolismo , Animais , Células COS , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Estudos de Casos e Controles , Diferenciação Celular , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Células HEK293 , Células-Tronco Embrionárias Humanas/patologia , Humanos , Lactente , Masculino , Miócitos Cardíacos/patologia , Fenótipo , Transdução de Sinais , Tetralogia de Fallot/diagnóstico , Tetralogia de Fallot/enzimologia
11.
Cardiovasc Drugs Ther ; 33(1): 13-23, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30637549

RESUMO

PURPOSE: Necroptosis is an important form of cell death following myocardial ischemia/reperfusion (I/R) and phosphoglycerate mutase 5 (PGAM5) functions as the convergent point for multiple necrosis pathways. This study aims to investigate whether inhibition of PGAM5 could reduce I/R-induced myocardial necroptosis and the underlying mechanisms. METHODS: The SD rat hearts (or H9c2 cells) were subjected to 1-h ischemia (or 10-h hypoxia) plus 3-h reperfusion (or 4-h reoxygenation) to establish the I/R (or H/R) injury model. The myocardial injury was assessed by the methods of biochemistry, H&E (hematoxylin and eosin), and PI/DAPI (propidium iodide/4',6-diamidino-2-phenylindole) staining, respectively. Drug interventions or gene knockdown was used to verify the role of PGAM5 in I/R (or H/R)-induced myocardial necroptosis and possible mechanisms. RESULTS: The I/R-treated heart showed the injuries (increase in infarct size and creatine kinase release), upregulation of PGAM5, dynamin-related protein 1 (Drp1), p-Drp1-S616, and necroptosis-relevant proteins (RIPK1/RIPK3, receptor-interacting protein kinase 1/3; MLKL, mixed lineage kinase domain-like); these phenomena were attenuated by inhibition of PGAM5 or RIPK1. In H9c2 cells, H/R treatment elevated the levels of PGAM5, RIPK1, RIPK3, MLKL, Drp1, and p-Drp1-S616 and induced mitochondrial dysfunctions (elevation in mitochondrial membrane potential and ROS level) and cellular necrosis (increase in LDH release and the ratio of PI+/DAPI+ cells); these effects were blocked by inhibition or knockdown of PGAM5. CONCLUSIONS: Inhibition of PGAM5 can reduce necroptosis in I/R-treated rat hearts through suppression of Drp1; there is a positive feedback between RIPK1 and PGAM5, and PGAM5 might serve as a novel therapeutic target for prevention of myocardial I/R injury.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Inibidores Enzimáticos/farmacologia , Glicolatos/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fosfoglicerato Mutase/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
13.
Circulation ; 139(14): 1725-1740, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30636447

RESUMO

BACKGROUND: Hypertrophic response to pathological stimuli is a complex biological process that involves transcriptional and epigenetic regulation of the cardiac transcriptome. Although previous studies have implicated transcriptional factors and signaling molecules in pathological hypertrophy, the role of RNA-binding protein in this process has received little attention. METHODS: Here we used transverse aortic constriction and in vitro cardiac hypertrophy models to characterize the role of an evolutionary conserved RNA-binding protein Lin28a in pathological cardiac hypertrophy. Next-generation sequencing, RNA immunoprecipitation, and gene expression analyses were applied to identify the downstream targets of Lin28a. Epistatic analysis, metabolic assays, and flux analysis were further used to characterize the effects of Lin28a and its downstream mediator in cardiomyocyte hypertrophic growth and metabolic remodeling. RESULTS: Cardiac-specific deletion of Lin28a attenuated pressure overload-induced hypertrophic growth, cardiac dysfunction, and alterations in cardiac transcriptome. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 ( Pck2) mRNA and increased its transcript level. Increasing Pck2 was sufficient to promote hypertrophic growth similar to that caused by increasing Lin28a, whereas knocking down Pck2 attenuated norepinephrine-induced cardiac hypertrophy. Epistatic analysis demonstrated that Pck2 mediated, at least in part, the role of Lin28a in cardiac hypertrophic growth. Furthermore, metabolomic analyses highlighted the role for Lin28a and Pck2 in promoting cardiac biosynthesis required for cell growth. CONCLUSIONS: Our study demonstrates that Lin28a promotes pathological cardiac hypertrophy and glycolytic reprograming, at least in part, by binding to and stabilizing Pck2 mRNA.


Assuntos
Proliferação de Células , Metabolismo Energético , Hipertrofia Ventricular Esquerda/enzimologia , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Glicólise , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos Sprague-Dawley , Função Ventricular Esquerda , Remodelação Ventricular
14.
Circ Res ; 124(5): 696-711, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30620686

RESUMO

RATIONALE: Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease. OBJECTIVE: To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease. METHODS AND RESULTS: Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure. CONCLUSIONS: Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.


Assuntos
Estresse do Retículo Endoplasmático , Insuficiência Cardíaca/enzimologia , Miócitos Cardíacos/enzimologia , Quinases Ativadas por p21/metabolismo , Animais , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Células-Tronco Pluripotentes Induzidas/enzimologia , Macaca mulatta , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Miócitos Cardíacos/patologia , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Quinases Ativadas por p21/deficiência , Quinases Ativadas por p21/genética
15.
Circ Res ; 124(5): 737-746, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30602331

RESUMO

RATIONALE: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. OBJECTIVE: To define phosphatase pathways that regulate INa,L in vivo. METHODS AND RESULTS: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. CONCLUSIONS: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.


Assuntos
Arritmias Cardíacas/enzimologia , Frequência Cardíaca , Ativação do Canal Iônico , Miócitos Cardíacos/enzimologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína Fosfatase 2/metabolismo , Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Fosforilação , Proteína Fosfatase 2/deficiência , Proteína Fosfatase 2/genética , Fatores de Tempo
16.
Thorac Cardiovasc Surg ; 67(6): 494-501, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30452075

RESUMO

BACKGROUND: It has been demonstrated that remote ischemic preconditioning (RIPC) increases ribonuclease (RNase) levels and protects the heart by reducing extracellular ribonucleic acid (eRNA). As medication-induced preconditioning (MIPC) is also a powerful tool for cardioprotection, we examined the influence of both types of preconditioning on the eRNA/RNase system. METHODS: In 17 male rats, RIPC (3 × 5 minute hind-leg ischemia) or MIPC (isoflurane and buprenorphine anesthesia) was performed. Five rats served as control and did not undergo preconditioning (non-MIPC). After preconditioning, eRNA levels and RNase activity were determined in plasma, and the hearts were mounted on a blood-perfused Langendorff ischemia/reperfusion apparatus. Hemodynamic, metabolic, and electron microscopic parameters were determined. Furthermore, MIPC with one anesthetic drug only (isoflurane, buprenorphine, or etomidate) was induced in another five rats. After 30 minutes, eRNA levels and RNase activity were determined and compared with an RIPC group (n = 5). RESULTS: The plasma of RIPC-treated rats had higher RNase activity and lower eRNA levels than that of MIPC-treated rats. In addition, RIPC increased RNase activity more than MIPC with one drug alone. The RNase activity and eRNA levels in these MIPC groups differed considerably. Hemodynamic parameters of RIPC- and MIPC-treated hearts were better preserved after 90-minute ischemia than those of non-MIPC hearts. No obvious differences were noted between MIPC and RIPC regarding hemodynamics, metabolism, or structural parameters. CONCLUSIONS: Our results suggest that RIPC does not have any additional cardioprotective benefit in this experimental system. However, the influence of RIPC on the eRNA/RNase system was greater than that of MIPC.


Assuntos
Anestésicos/administração & dosagem , Buprenorfina/administração & dosagem , Ácidos Nucleicos Livres/sangue , Membro Posterior/irrigação sanguínea , Precondicionamento Isquêmico/métodos , Isoflurano/administração & dosagem , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Ribonucleases/sangue , Animais , Hemodinâmica/efeitos dos fármacos , Preparação de Coração Isolado , Masculino , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/ultraestrutura , Ratos Endogâmicos Lew , Oclusão Terapêutica , Fator de Necrose Tumoral alfa/sangue
17.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30514824

RESUMO

Heart failure (HF) is a serious disease with high mortality. The incidence of this disease has continued to increase over the past decade. All cardiovascular diseases causing dysfunction of various physiological processes can result in HF. AMP-activated protein kinase (AMPK), an energy sensor, has pleiotropic cardioprotective effects and plays a critical role in the progression of HF. In this review, we highlight that AMPK can not only improve the energy supply in the failing heart by promoting ATP production, but can also regulate several important physiological processes to restore heart function. In addition, we discuss some aspects of some potential clinical drugs which have effects on AMPK activation and may have value in treating HF. More studies, especially clinical trials, should be done to evaluate manipulation of AMPK activation as a potential means of treating HF.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia
18.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30237226

RESUMO

To evaluate the role of glycogen synthase kinase-3ß (GSK-3ß) in the apoptosis of cardiomyocytes in diabetic cardiomyopathy (DCM). Diabetes mellitus (DM) in rats was induced by intraperitoneal injection of 1% streptozotocin (STZ), and lithium chloride (LiCl) was used to decrease the expression of GSK-3ß. Hematoxylin/eosin (HE) staining and the terminal deoxyribonucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay was conducted to evaluate the pathological injury and apoptosis of cardiomyocytes respectively. Western blot was applied to detect the protein expressions of Cleaved-caspase 3, caspase 3, Bax and Bcl-2 in rat cardiomyocytes. Real-time polymerase chain reaction (RT-PCR) was applied to detect the gene expressions of phosphoinositide 3-kinases (PI3K), Akt, and GSK-3ß in rat cardiomyocytes. DM-induced cardiomyocyte injuries, which were presented as capillary basement membrane thickening, interstitial fibrosis, cardiomyocyte hypertrophy and necrosis in HE staining and increased apoptosis detected by TUNEL assay. When comparing with the control group, the mRNA expression of PI3K and Akt in DM group obviously decreased but the mRNA expression of GSK-3ß obviously elevated (P < 0.05). In addition, the ratio of Cleaved-caspase 3/caspase 3 and Bax/Bcl-2 were notably increased in DM group compared with control group (P < 0.05). LiCl, as an inhibitor of GSK-3 apparently reduced the expression of GSK-3ß mRNA (P < 0.05) but not the PI3K and Akt comparing with the DM group. LiCl also attenuated the myocardial injury and apoptosis induced by DM. The myocardial injury induced by DM is associated with the up-regulation of GSK-3ß. LiCl inhibited the expression of GSK-3ß and myocardial apoptosis in diabetic myocardium.


Assuntos
Apoptose/efeitos dos fármacos , Cardiotônicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/tratamento farmacológico , Cloreto de Lítio/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/genética , Glicemia/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/genética , Dieta Hiperlipídica/métodos , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Estreptozocina , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
19.
Cardiovasc Res ; 115(1): 20-30, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30321309

RESUMO

With an estimated 38 million current patients, heart failure (HF) is a leading cause of morbidity and mortality worldwide. Although the aetiology differs, HF is largely a disease of cardiomyocyte (CM) death or dysfunction. Due to the famously limited amount of regenerative capacity of the myocardium, the only viable option for advanced HF patients is cardiac transplantation; however, donor's hearts are in very short supply. Thus, novel regenerative strategies are urgently needed to reconstitute the injured hearts. Emerging data from our lab and others have elucidated that CM-specific deletion of glycogen synthase kinase (GSK)-3 family of kinases induces CM proliferation, and the degree of proliferation is amplified in the setting of cardiac stress. If this proliferation is sufficiently robust, one could induce meaningful regeneration without the need for delivering exogenous cells to the injured myocardium (i.e. cardiac regeneration in situ). Herein, we will discuss the emerging role of the GSK-3s in CM proliferation and differentiation, including their potential implications in cardiac regeneration. The underlying molecular interactions and cross-talk among signalling pathways will be discussed. We will also review the specificity and limitations of the available small molecule inhibitors targeting GSK-3 and their potential applications to stimulate the endogenous cardiac regenerative responses to repair the injured heart.


Assuntos
Proliferação de Células/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Insuficiência Cardíaca/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Regeneração/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Neuregulina-1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
20.
J Cardiovasc Pharmacol Ther ; 24(1): 78-89, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30033751

RESUMO

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na+, K+-ATPase inhibition mediates CS-induced increase in heart contractility. Recently, the involvement of intracellular signal transduction was implicated in this effect. The aim of the present study was to test the hypothesis that combined treatment with ouabain and Akt inhibitor (MK-2206) augments ouabain-induced inotropy in mammalian models. We demonstrate that the combined treatment led to an ouabain-induced increase in contractility at concentrations at which ouabain alone was ineffective. This was shown in 3 experimental systems: neonatal primary rat cardiomyocytes, a Langendorff preparation, and an in vivo myocardial infarction induced by left anterior descending coronary artery (LAD) ligation. Furthermore, cell viability experiments revealed that this treatment protected primary cardiomyocytes from MK-2206 toxicity and in vivo reduced the size of scar tissue 10 days post-LAD ligation. We propose that Akt activity imposes a constant inhibitory force on muscle contraction, which is attenuated by low concentrations of MK-2206, resulting in potentiation of the ouabain effect. This demonstration of the increase in the CS effect advocates the development of the combined treatment in CHF.


Assuntos
Cardiotônicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Preparação de Coração Isolado , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Transdução de Sinais
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