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1.
Artigo em Inglês | MEDLINE | ID: mdl-32940892

RESUMO

PURPOSE: Metformin is the first-line antidiabetic drug and shown to reduce cardiovascular risk independent from its glucose lowering action. Particularly in poorly controlled diabetes, tissue factor (TF) is expressed in the vasculature and accounts for thromboembolic complications. Here, we aimed to assess the effect of metformin on TF activity and markers of vascular inflammation in poorly controlled type 2 diabetes. METHODS: In a cohort of patients with uncontrolled type 2 diabetes (glycosylated hemoglobin 8.39 ± 0.24%, 68.1 ± 2.6 mmol/mol, n = 46) of whom half of the individuals were treated with metformin and the other half did not receive metformin as part of an anti-diabetic combination therapy, we assessed TF activity and markers of vascular inflammation. In vitro, human monocytic cells (THP-1) were exposed to metformin and TF expression measured in the presence and absence of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the AMPK inhibitor compound C. RESULTS: In the patients, metformin treatment was associated with lower levels of TF protein (241.5 ± 19 vs. 315.4 ± 25 pg/mL, p = 0.03) and reduced TF activity (408.9 ± 49 vs. 643.8 ± 47 U/mL, p = 0.001) compared with controls. Moreover, the patients on metformin showed lower levels of vascular cell adhesion molecule (VCAM)1 (26.6 ± 1.4 vs. 35.03 ± 3.1 ng/mL, p = 0.014) and higher expression of miR-126-3p/U6sno (11.39 ± 2.8 vs. 4.26 ± 0.9, p = 0.006), a known post-transcriptional down regulator of TF and VCAM1. In vitro, metformin dose-dependently reduced lipopolysaccharide (LPS)-induced TF expression in THP-1 cells. The AMPK activator AICAR alone lowered TF expression in THP-1, while the AMPK inhibitor compound C abrogated the metformin-dependent reduction in TF expression. CONCLUSIONS: Our data are the first to report that metformin is associated with reduced plasma TF procoagulant activity possibly explaining-at least in part-the vasculoprotective properties of metformin.

2.
Cardiovasc Diabetol ; 19(1): 20, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066445

RESUMO

BACKGROUND: Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. METHODS: In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. RESULTS: In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS. CONCLUSIONS: miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.


Assuntos
Coagulação Sanguínea , Diabetes Mellitus Tipo 2/complicações , Células Endoteliais/metabolismo , Inflamação/etiologia , MicroRNAs/metabolismo , Tromboplastina/metabolismo , Trombose/etiologia , Idoso , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , NF-kappa B/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Células THP-1 , Tromboplastina/genética , Trombose/genética , Trombose/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , alfa Carioferinas/metabolismo
3.
Cells ; 8(12)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817787

RESUMO

The cardiac-specific overexpression of the adenine nucleotide translocase 1 (ANT1) has cardioprotective effects in various experimental heart disease models. Here, we analyzed the link between ANT1 expression and heat shock protein 27 (HSP27)-mediated toll-like receptor 4 (TLR4) signaling, which represents a novel communication pathway between mitochondria and the extracellular environment. The interaction between ANT1 and HSP27 was identified by co-immunoprecipitation from neonatal rat cardiomyocytes. ANT1 transgenic (ANT1-TG) cardiomyocytes demonstrated elevated HSP27 expression levels. Increased levels of HSP27 were released from the ANT1-TG cardiomyocytes under both normoxic and hypoxic conditions. Extracellular HSP27 stimulated TLR4 signaling via protein kinase B (AKT). The HSP27-mediated activation of the TLR4 pathway was more pronounced in ANT1-TG cardiomyocytes than in wild-type (WT) cardiomyocytes. HSP27-specific antibodies inhibited TLR4 activation and the expression of HSP27. Inhibition of the HSP27-mediated TLR4 signaling pathway with the TLR4 inhibitor oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) reduced the mitochondrial membrane potential (∆ψm) and increased caspase 3/7 activity, which are both markers for cell stress. Conversely, treating cardiomyocytes with recombinant HSP27 protein stimulated TLR4 signaling, induced HSP27 and ANT1 expression, and stabilized the mitochondrial membrane potential. The activation of HSP27 signaling was verified in ischemic ANT1-TG heart tissue, where it correlated with ANT1 expression and the tightness of the inner mitochondrial membrane. Our study shows a new mechanism by which ANT1 is part of the cardioprotective HSP27-mediated TLR4 signaling.

4.
Eur Heart J ; 40(40): 3318-3332, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31004144

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). METHODS AND RESULTS: Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = -0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent α-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-ß)-dependent signalling. This enhanced TGF-ß/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). CONCLUSIONS: Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-ß signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-ß-axis might be a promising therapeutic approach to reduce HFpEF.

5.
Cardiovasc Diabetol ; 17(1): 34, 2018 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-29477147

RESUMO

BACKGROUND: Diabetes mellitus is characterized by chronic vascular disorder and presents a main risk factor for cardiovascular mortality. In particular, hyperglycaemia and inflammatory cytokines induce vascular circulating tissue factor (TF) that promotes pro-thrombotic conditions in diabetes. It has recently become evident that alterations of the post-transcriptional regulation of TF via specific microRNA(miR)s, such as miR-126, contribute to the pathogenesis of diabetes and its complications. The endothelial miR-19a is involved in vascular homeostasis and atheroprotection. However, its role in diabetes-related thrombogenicity is unknown. Understanding miR-networks regulating procoagulability in diabetes may help to develop new treatment options preventing vascular complications. METHODS AND RESULTS: Plasma of 44 patients with known diabetes was assessed for the expression of miR-19a, TF protein, TF activity, and markers for vascular inflammation. High miR-19a expression was associated with reduced TF protein, TF-mediated procoagulability, and vascular inflammation based on expression of vascular adhesion molecule-1 and leukocyte count. We found plasma expression of miR-19a to strongly correlate with miR-126. miR-19a reduced the TF expression on mRNA and protein level in human microvascular endothelial cells (HMEC) as well as TF activity in human monocytes (THP-1), while anti-miR-19a increased the TF expression. Interestingly, miR-19a induced VCAM expression in HMEC. However, miR-19a and miR-126 co-transfection reduced total endothelial VCAM expression and exhibited additive inhibition of a luciferase reporter construct containing the F3 3'UTR. CONCLUSIONS: While both miRs have differential functions on endothelial VCAM expression, miR-19a and miR-126 cooperate to exhibit anti-thrombotic properties via regulating vascular TF expression. Modulating the post-transcriptional control of TF in diabetes may provide a future anti-thrombotic and anti-inflammatory therapy.


Assuntos
Coagulação Sanguínea/genética , Diabetes Mellitus/genética , Epigênese Genética , MicroRNAs/genética , Tromboplastina/genética , Trombose/genética , Regiões 3' não Traduzidas , Idoso , Sítios de Ligação , Diabetes Mellitus/sangue , Diabetes Mellitus/diagnóstico , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Células THP-1 , Tromboplastina/metabolismo , Trombose/sangue , Trombose/diagnóstico , Trombose/prevenção & controle , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
6.
J Mol Med (Berl) ; 94(6): 645-53, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27080394

RESUMO

UNLABELLED: Ischemia impairs the adenine nucleotide translocase (ANT), which transports ADP and ATP across the inner mitochondrial membrane. We investigated whether ANT1 overexpression has protective effects on ischemic hearts. Myocardial infarction was induced in wild-type (WT) and heart-specific ANT1-transgenic (ANT1-TG) rats, and hypoxia was set in isolated cardiomyocytes. ANT1 overexpression reduced the myocardial infarct area and increased the survival rate of infarcted rats. Reduced ANT1 expression and increased 4-hydroxynonenal modification of ANT paralleled to impaired ANT function in infarcted WT hearts. ANT1 overexpression improved ANT expression and function. This was accompanied by reduced mitochondrial cytochrome C release and caspase-3 activation. ANT1-TG hearts suffered less from oxidative stress, as shown by lower protein carbonylation and 4-hydroxynonenal modification of ANT. ANT1 overexpression also increased cell survival of hypoxic cardiomyocytes and attenuated reactive oxygen species (ROS) production. This was linked to higher stability of mitochondrial membrane potential and lower activity of ROS detoxifying catalase. ANT1-TG cardiomyocytes also showed higher resistance against H2O2 treatment, which was independent of catalase activity. In conclusion, ANT1 overexpression compensates impaired ANT activity under oxygen-restricted conditions. It reduces ROS production and oxidative stress, stabilizes mitochondrial integrity, and increases survival, making ANT1 a component in ROS management and heart protection during ischemia. KEY MESSAGES: ANT1 overexpression reduces infarct size and increases survival after infarction. ANT1 overexpression compensates restricted ANT expression and function in infarcted hearts. Increased ANT1 expression enhances mitochondrial integrity. ANT1-overexpressing hearts reduce oxidative stress by decreasing ROS generation. ANT1 is a component in ROS management and heart protection.


Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Mitocôndrias Cardíacas/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Translocador 1 do Nucleotídeo Adenina/metabolismo , Aldeídos/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Catalase/genética , Catalase/metabolismo , Hipóxia Celular , Sobrevivência Celular , Citocromos c/metabolismo , Regulação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias Cardíacas/efeitos dos fármacos , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Estresse Oxidativo , Cultura Primária de Células , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Transgênicos , Transdução de Sinais , Análise de Sobrevida
7.
Cell Signal ; 28(1): 152-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26548633

RESUMO

The influence of mitochondrial function on intracellular signalling is currently under intense investigation. In this regard, we analysed the effect of adenine nucleotide translocase 1 (ANT1), which facilitates the exchange of ADP and ATP across the mitochondrial membrane, on cell-protective survival signalling under hypoxia. ANT1 overexpression enhanced the survival rate in hypoxic cardiomyocytes. The effect was related to stabilization of the mitochondrial membrane potential, suppression of caspase 3 activity, and a reduction in DNA fragmentation. Activation of the cell-protective signalling proteins extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (AKT) was substantially higher in hypoxic ANT1-transgenic (ANT1-TG) cardiomyocytes than in wild-type cardiomyocytes. Kinase activation was associated with significantly higher expression of hypoxia-inducible factor 1α, which induces glycolytic pathway to stabilize ATP production. Accordingly, ANT1-TG cardiomyocytes exhibited earlier and stronger activation of lactate dehydrogenase and a higher ATP content. Treatment with PD980559 and triciribine, inhibitors of ERK1/2 and AKT activation, respectively, abolished cell protection in hypoxic ANT1-TG cardiomyocytes. Inhibition of ANT by carboxyatractyloside prevented the increase in ERK1/2 and AKT phosphorylation and eliminated the cell protective program in hypoxic ANT1-TG cardiomyocytes. In conclusion, the cytoprotective effect observed in hypoxic ANT1-overexpressing cardiomyocytes involves an interdependence between ANT1, activation of ERK1/ERK2 and AKT, and induction of the survival processes regulated by these kinases.


Assuntos
Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley
9.
J Neurosci ; 32(11): 3917-30, 2012 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-22423112

RESUMO

We have identified the adenine nucleotide translocator (ANT) isoforms ANT1 and ANT2 that are present in the plasma membrane of mouse cerebellar neurons as novel binding partners of the cell adhesion molecule L1. The direct interaction between ANT and L1 is mediated by sites within the fibronectin type III domains of L1 and the first and third extracellular loops of the ANT proteins. We also show that L1 interacts with the ANT binding partner matrix metalloprotease 14 (MMP14) and that the ANT proteins bind directly to the L1 interaction partner glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, we provide evidence that the functional interplay between L1, ANT proteins, MMP14, and GAPDH at the plasma membrane mediates L1-induced neurite outgrowth of cerebellar neurons. Disruption of this interplay by ANT inhibitors, ANT-derived synthetic peptides, and/or function-blocking MMP14 and ANT antibodies leads to alterations in L1-dependent neurite outgrowth. Stimulation of L1-mediated signaling in cerebellar neurons triggers transient ATP secretion via ANT proteins and leads to transient src family-dependent tyrosine phosphorylation of L1, ANT1, ANT2, and MMP14. Thus, our results indicate that plasma membrane-localized ANT1 and ANT2 regulate L1-mediated neurite outgrowth in conjunction with MMP14.


Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/metabolismo , Cerebelo/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Neuritos/fisiologia , Animais , Células Cultivadas , Cerebelo/citologia , Feminino , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Ligação Proteica/fisiologia
10.
Lab Invest ; 92(1): 125-34, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21968812

RESUMO

Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5 ± 1.4 × 10(4) plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4 ± 0.8 × 10(7) p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.


Assuntos
Infecções por Coxsackievirus/imunologia , Transporte de Elétrons/fisiologia , Enterovirus Humano B , Mitocôndrias Cardíacas/fisiologia , Miocardite/imunologia , Animais , Apoptose , Resistência à Doença , Complexo I de Transporte de Elétrons/fisiologia , Coração/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Carga Viral
11.
J Virol ; 85(24): 13409-19, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21976640

RESUMO

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.


Assuntos
Evolução Biológica , Enterovirus Humano B/crescimento & desenvolvimento , Enterovirus Humano B/genética , Miócitos Cardíacos/virologia , Animais , Sobrevivência Celular , Células Cultivadas , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/patogenicidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Virulência
12.
Cell Physiol Biochem ; 27(2): 121-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21325829

RESUMO

BACKGROUND/AIMS: The adenine nucleotide translocase (ANT) exchanges ATP and ADP over the inner mitochondrial membrane, supplying the cells with energy. Interestingly, myocardial ANT1 overexpression preserves cardiac structure and function under pathophysiological conditions. To ascertain whether the contractile system is directly affected by increased ANT1 expression, we analyzed cell morphology, contraction and relaxation parameters of ANT1 transgenic (ANT1-TG) cardiomyocytes, myofibrillar protein expression, and Ca(2+) handling in ANT1-TG rat hearts. RESULTS: ANT1-TG cardiomyoycytes displayed an elevation in cell volume (52.6 ± 12.0%; p<0.0001) in comparison to wildtype (WT) cells. Concurrently, contractile function in ANT1-TG cells was significantly increased, measured by a decline in time to peak contraction (TTP) and RT50, the time from peak contraction to 50% relaxation, during stimulation with 0.5, 1, and 2 Hz. Quantification of myofibrillar proteins exhibited a marked increase in total cardiac myosin heavy chain (51.8 ± 12.8%) (p<0.03), beta myosin heavy chain (22.9 ± 5.0%; p<0.03), actin (23.8 ± 8.8%; p<0.05), and troponin I (51.5 ± 13.7%; p<0.01). Regarding intracellular Ca(2+) handling, ANT1-TGs revealed a significant elevation in sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) protein level (22.2 ± 4.7%; p<0.01) associated with increased Ca(2+) uptake into the SR (34%; p<0.01). Moreover, the plasmalemmal Ca(2+) ATPase (PMCA) indicated advanced protein expression (23.8 ± 4.8%; p<0.01), whereas the protein amount of the Na(+)/Ca(2+) exchanger was not altered in ANT1 overexpressing hearts. CONCLUSION: These data reveal a close association of elevated mitochondrial ATP/ADP transportation via ANT1 with increased contractile function. Furthermore, the ANT1-TGs exhibit an elevation in SR Ca(2+) transport that contributes to increased cardiac work, which may protect the heart under pathophysiological conditions.


Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Miócitos Cardíacos/fisiologia , Actinas/metabolismo , Translocador 1 do Nucleotídeo Adenina/genética , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Tamanho Celular , Células Cultivadas , Masculino , Mitocôndrias/metabolismo , Contração Muscular/fisiologia , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Troponina I/metabolismo
13.
J Immunol ; 186(4): 2561-70, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21239721

RESUMO

TRIF is a member of the innate immune system known to be involved in viral recognition and type I IFN activation. Because IFNs are thought to play an important role in viral myocarditis, we investigated the role of TRIF in induced myocarditis in mice. Whereas C57BL/6 (wild-type) mice showed only mild myocarditis, including normal survival postinfection with coxsackievirus group B serotype 3 (CVB3), infection of TRIF(-/-) mice led to the induction of cardiac remodeling, severe heart failure, and 100% mortality (p < 0.0001). These mice showed markedly reduced virus control in cardiac tissues and cardiomyocytes. This was accompained with dynamic cardiac cytokine activation in the heart, including a suppression of the antiviral cytokine IFN-ß in the early viremic phase. TRIF(-/-) myocytes displayed a TLR4-dependent suppression of IFN-ß, and pharmacological treatment of CVB3-infected TRIF(-/-) mice with murine IFN-ß led to improved virus control and reduced cardiac inflammation. Additionally, this treatment within the viremic phase of myocarditis showed a significant long-term outcome indexed by reduced mortality (20 versus 100%; p < 0.001). TRIF is essential toward a cardioprotection against CVB3 infection.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Cardiomiopatia Dilatada/imunologia , Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/imunologia , Miocardite/imunologia , Disfunção Ventricular Esquerda/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Cardiomiopatia Dilatada/mortalidade , Cardiomiopatia Dilatada/terapia , Células Cultivadas , Infecções por Coxsackievirus/mortalidade , Infecções por Coxsackievirus/terapia , Enterovirus Humano B/patogenicidade , Células HeLa , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/terapia , Humanos , Interferon beta/biossíntese , Interferon beta/fisiologia , Interferon beta/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/mortalidade , Miocardite/terapia , Sorotipagem , Disfunção Ventricular Esquerda/mortalidade , Disfunção Ventricular Esquerda/terapia , Replicação Viral/imunologia
14.
Circulation ; 120(23): 2358-66, 2009 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-19933937

RESUMO

BACKGROUND: Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach. METHODS AND RESULTS: We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59+/-3.8 versus 45.4+/-2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dt(max) 3645.1+/-443.6 versus 2057.9+/-490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dt(min) -2125.5+/-330.5 versus -1310.2+/-330.3 mm Hg/s, median -2083.7 versus -1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4+/-19.2 versus 56.8+/-10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dt(max) 5214.2+/-1786.2 versus 3011.6+/-918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation. CONCLUSIONS: AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.


Assuntos
Infecções por Coxsackievirus/prevenção & controle , Regulação Viral da Expressão Gênica , Miocardite/prevenção & controle , Receptores Virais/biossíntese , Receptores Virais/genética , Doença Aguda , Animais , Cardiomiopatias/genética , Cardiomiopatias/prevenção & controle , Cardiomiopatias/virologia , Infecções por Coxsackievirus/genética , Terapia Genética/métodos , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/genética , Miocardite/virologia , Receptores Virais/administração & dosagem
15.
Basic Res Cardiol ; 104(3): 247-57, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19190956

RESUMO

The disturbance of myocardial energy metabolism has been discussed as contributing to the progression of heart failure. Little however is known about the cardiac mitochondrial/cytosolic energy transfer in murine and human inflammatory heart disease. We examined the myocardial creatine kinase (CK) system, which connects mitochondrial ATP-producing and cytosolic ATP-consuming processes and is thus of central importance to the cellular energy homeostasis. The time course of expression and enzymatic activity of mitochondrial (mtCK) and cytosolic CK (cytCK) was investigated in Coxsackievirus B3 (CVB3)-infected SWR mice, which are susceptible to the development of chronic myocarditis. In addition, cytCK activity and isoform expression were analyzed in biopsies from patients with chronic inflammatory heart disease (n = 22). Cardiac CVB3 titer in CVB3-infected mice reached its maximum at 4 days post-infection (pi) and became undetectable at 28 days pi; cardiac inflammation cumulated 14 days pi but persisted through the 28-day survey. MtCK enzymatic activity was reduced by 40% without a concurrent decrease in mtCK protein during early and acute MC. Impaired mtCK activity was correlated with virus replication and increased level of interleukine 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and elevated catalase expression, a marker for intracellular oxidative stress. A reduction in cytCK activity of 48% was observed at day 14 pi and persisted to day 28 pi. This restriction was caused by a decrease in cytCK subunit expression but also by direct inhibition of specific cytCK activity. CytCK activity and expression were also reduced in myocardial biopsies from enterovirus genome-negative patients with inflammatory heart disease. The decrease in cytCK activity correlated with the number of infiltrating macrophages. Thus, viral infection and myocardial inflammation significantly influence the myocardial CK system via restriction of specific CK activity and down-regulation of cytCK protein. These changes may contribute to the progression of chronic inflammatory heart disease and malfunction of the heart.


Assuntos
Creatina Quinase Forma MB/metabolismo , Creatina Quinase Mitocondrial/metabolismo , Citoplasma/enzimologia , Miocardite/enzimologia , Adulto , Animais , Western Blotting , Infecções por Coxsackievirus/enzimologia , Enterovirus Humano B , Feminino , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Miocardite/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Exp Physiol ; 94(2): 220-7, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18945756

RESUMO

Mitochondrial dysfunction is implicated in the pathogenesis of diabetic cardiomyopathy, a common complication of diabetes. Adenosine nucleotide translocase (ANT) translocates ADP/ATP across the inner mitochondrial membrane. Our study aimed to test the hypothesis that overexpression of ANT1 in cardiomyocytes has cardioprotective effects in diabetic cardiomyopathy induced by streptozotocin (STZ). Mice specifically overexpressing murine ANT1 in the heart were generated using alpha-myosin heavy chain promoter. Expression of ANT1 mRNA and protein in hearts was characterized by real-time polymerase chain reaction and Western blot analysis. Five- to 6-month-old male transgenic mice and their age-matched wild-type littermates were subjected to type 1 diabetes induced by STZ. Six weeks later, haemodynamic measurement was performed to assess cardiac function. Ventricular mRNA expression of atrial natriuretic peptide, a molecular marker of heart failure, was characterized by RNase-protection assay. Both ANT1 mRNA and ANT1 protein were specifically overexpressed in the heart of transgenic mice. Heart weight was decreased and cardiac function was dramatically impaired in wild-type mice 6 weeks after induction of diabetes, but ANT1 overexpression prevented these significant changes. The mRNA expression level of atrial natriuretic peptide confirmed the haemodynamic findings, being upregulated in wild-type mice receiving STZ, but showing no statistical differences in ANT1 transgenic mice. Cardiomyocyte-restricted overexpression of ANT1 prevents the development of diabetic cardiomyopathy; therefore, accelerated ADP/ATP exchange could be a new promising target to treat diabetic cardiomyopathy.


Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Cardiomiopatias/prevenção & controle , Complicações do Diabetes/prevenção & controle , Miocárdio/metabolismo , Translocador 1 do Nucleotídeo Adenina/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cardiomiopatias/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Modelos Animais de Doenças , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Estreptozocina
17.
J Clin Invest ; 118(8): 2758-70, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18636119

RESUMO

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in beta-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.


Assuntos
Nó Atrioventricular/metabolismo , Conexinas/metabolismo , Coração , Miocárdio/metabolismo , Receptores Virais/metabolismo , Animais , Conexinas/ultraestrutura , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Eletrocardiografia , Embrião de Mamíferos , Técnica Direta de Fluorescência para Anticorpo , Células HeLa , Ventrículos do Coração/ultraestrutura , Humanos , Camundongos , Camundongos Knockout , Miocárdio/ultraestrutura , Receptores Virais/ultraestrutura , Telemetria
18.
J Mol Cell Cardiol ; 45(1): 118-26, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18495150

RESUMO

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.


Assuntos
Infecções por Coxsackievirus/enzimologia , Enterovirus , Miocardite/enzimologia , Trombofilia/enzimologia , Tromboplastina/biossíntese , Animais , Antígenos de Diferenciação/metabolismo , Coagulação Sanguínea , Complexo CD3/metabolismo , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/fisiopatologia , Fibrina/metabolismo , Hemodinâmica , Humanos , Camundongos , Miocardite/patologia , Miocardite/fisiopatologia , Miocardite/virologia , Trombofilia/patologia , Trombofilia/fisiopatologia , Trombofilia/virologia , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/metabolismo
19.
Trends Cardiovasc Med ; 17(8): 284-90, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18021939

RESUMO

Adenine nucleotide translocase (ANT) facilitates the exchange of extramitochondrial adenosine diphosphate and intramitochondrial adenosine triphosphate across the inner mitochondrial membrane and appears to be a member of the mitochondrial permeability transition pore whose opening induces apoptosis. Genetically or physiologically restricted ANT function associated with insufficient energy supply and induced apoptosis leads to severe cardiac disturbance. In contrast, to counter myocardial stress, heart tissue developed cell protecting gene programs including ANT1 up-regulation to stabilize energy supply and concurrently suppress apoptotic processes. This review describes characteristics of ANT function and expression in cardiovascular diseases and ANT's role in cardioprotection.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Difosfato de Adenosina , Trifosfato de Adenosina , Apoptose , Expressão Gênica , Humanos , Isquemia Miocárdica/fisiopatologia , Miocárdio , Fosforilação Oxidativa
20.
Clin Res Cardiol ; 96(7): 469-80, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17503113

RESUMO

During recent years, increasing evidence has been obtained that cellular as well as humoral autoimmunity is involved in the pathogenesis of dilated cardiomyopathy (DCM). The immune system is generally activated by viral infections with the objective of virus elimination from the myocardium. However, a relevant number of patients demonstrate viral persistence and/or chronic inflammation in the myocardium. This chronic myocardial inflammation, defined by chronic inflammation, is termed "inflammatory cardiomyopathy" according to the WHO classification of cardiomyopathies. Chronic inflammation is frequently followed by the development of autoimmunity. A breakdown in the control mechanisms protecting against autoimmune reactions by both presentation of normally not accessible self-antigens and bystander- activation, induced by the pathogen, leads to the formation of autoreactive antibodies and T cells. The auto-reactive antibodies interact directly with heart tissue resulting in altered signal transduction or complement activation, whereas the T cell-mediated mechanisms include direct attack by cytotoxic T cells or indirect effects of cytotoxic cytokines released by stimulated T cells or macrophages.


Assuntos
Autoanticorpos/imunologia , Autoimunidade/imunologia , Miocardite/imunologia , Citocinas/metabolismo , Humanos , Imunidade Celular/imunologia , Miocardite/metabolismo
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