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1.
Sci Transl Med ; 11(493)2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118294

RESUMO

Atrial fibrillation (AF), the most common sustained heart rhythm disorder worldwide, is linked to dysfunction of the intrinsic cardiac autonomic nervous system (ICNS). The role of ICNS damage occurring during catheter-based treatment of AF, which is the therapy of choice for many patients, remains controversial. We show here that the neuronal injury marker S100B is expressed in cardiac glia throughout the ICNS and is released specifically upon catheter ablation of AF. Patients with higher S100B release were more likely to be AF free during follow-up. Subsequent in vitro studies revealed that murine intracardiac neurons react to S100B with diminished action potential firing and increased neurite growth. This suggests that release of S100B from cardiac glia upon catheter-based treatment of AF is a hallmark of acute neural damage that contributes to nerve sprouting and can be used to assess ICNS damage.

2.
J Mol Cell Cardiol ; 131: 53-65, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31005484

RESUMO

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

3.
Basic Res Cardiol ; 114(3): 19, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30887214

RESUMO

Heart failure is a consequence of various cardiovascular diseases and associated with poor prognosis. Despite progress in the treatment of heart failure in the past decades, prevalence and hospitalisation rates are still increasing. Heart failure is typically associated with cardiac remodelling. Here, inflammation and fibrosis are thought to play crucial roles. During cardiac inflammation, immune cells invade the cardiac tissue and modulate tissue-damaging responses. Cardiac fibrosis, however, is characterised by an increased amount and a disrupted composition of extracellular matrix proteins. As evidence exists that cardiac inflammation and fibrosis are potentially reversible in experimental and clinical set ups, they are interesting targets for innovative heart failure treatments. In this context, animal models are important as they mimic clinical conditions of heart failure patients. The advantages of mice in this respect are short generation times and genetic modifications. As numerous murine models of heart failure exist, the selection of a proper disease model for a distinct research question is demanding. To facilitate this selection, this review aims to provide an overview about the current understanding of the pathogenesis of cardiac inflammation and fibrosis in six frequently used murine models of heart failure. Hence, it compares the models of myocardial infarction with or without reperfusion, transverse aortic constriction, chronic subjection to angiotensin II or deoxycorticosterone acetate, and coxsackievirus B3-induced viral myocarditis in this context. It furthermore provides information about the clinical relevance and the limitations of each model, and, if applicable, about the recent advancements in their methodological proceedings.


Assuntos
Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Animais , Fibrose , Insuficiência Cardíaca/patologia , Camundongos , Miocardite/complicações , Miocárdio/patologia
4.
Biomolecules ; 9(2)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30678084

RESUMO

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to play a major role in inflammatory diseases such as myocardial infarction (MI), where its expression increases. Cardio protective functions of MIF during ischemia have been reported. Recently, the structurally related MIF-2 was identified and similar effects are assumed. We wanted to further investigate the role of MIF and MIF-2 on inflammatory processes during MI. Therefore, we subjected mice to experimentally induced MI by coronary occlusion for one and five days. During the acute phase of MI, the gene expression of Mif was upregulated in the infarct zone, whereas Mif-2 was downregulated, suggesting a minor role of MIF-2. Simulating ischemic conditions or mechanical stress in vitro, we demonstrated that Mif expression was induced in resident cardiac cells. To investigate possible auto /paracrine effects, cardiomyocytes and cardiac fibroblasts were individually treated with recombinant murine MIF, which in turn induced Mif expression and the expression of pro-inflammatory genes in cardiac fibroblasts. Cardiomyocytes did not respond to recombinant MIF with pro-inflammatory gene expression. While MIF stimulation alone did not change the expression of pro-fibrotic genes in cardiac fibroblasts, ischemia reduced their expression. Mimicking the increased MIF levels during MI, we exposed cardiac fibroblasts to simulated ischemia in the presence of MIF, which led to further reduced expression of pro-fibrotic genes. The presented data show that MIF was expressed by resident cardiac cells during MI. In vitro, Mif expression was induced by different external stimuli in cardiomyocytes and cardiac fibroblasts. Addition of recombinant MIF protein increased the expression of pro-inflammatory genes in cardiac fibroblasts including Mif expression itself. Thereby, cardiac fibroblasts may amplify Mif expression during ischemia promoting cardiomyocyte survival.


Assuntos
Fibroblastos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Fibroblastos/patologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais/genética
6.
Proc Natl Acad Sci U S A ; 115(37): E8727-E8736, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30166452

RESUMO

Increased adrenomedullin (ADM) levels are associated with various cardiac diseases such as myocardial infarction (MI). ADM is cleaved off from the full-length precursor protein proadrenomedullin (ProADM) during its posttranslational processing. To date, no biological effect of ProADM is reported, while ADM infusion leads to antiapoptotic effects and improved cardiac function. Using an MI mouse model, we found an induction of ProADM gene as well as protein expression during the early phase of MI. This was accompanied by apoptosis and increasing inflammation, which substantially influence the post-MI remodeling processes. Simulating ischemia in vitro, we demonstrate that ProADM expression was increased in cardiomyocytes and cardiac fibroblasts. Subsequently, we treated ischemic cardiomyocytes with either ProADM or ADM and found that both proteins increased survival. This effect was diminishable by blocking the ADM1 receptor. To investigate whether ProADM and ADM play a role in the regulation of cardiac inflammation, we analyzed chemokine expression after treatment of cells with both proteins. While ProADM induced an expression of proinflammatory cytokines, thus promoting inflammation, ADM reduced chemokine expression. On leukocytes, both proteins repressed chemokine expression, revealing antiinflammatory effects. However, ProADM but not ADM dampened concurrent activation of leukocytes. Our data show that the full-length precursor ProADM is biologically active by reducing apoptosis to a similar extent as ADM. We further assume that ProADM induces local inflammation in affected cardiac tissue but attenuates exaggerated inflammation, whereas ADM has low impact. Our data suggest that both proteins are beneficial during MI by influencing apoptosis and inflammation.


Assuntos
Adrenomedulina/genética , Inflamação/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Precursores de Proteínas/genética , Adrenomedulina/metabolismo , Adrenomedulina/farmacologia , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células Cultivadas , Citocinas/metabolismo , Feminino , Expressão Gênica/genética , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacologia
7.
Int J Cardiol ; 270: 278-286, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30082120

RESUMO

BACKGROUND: Myeloid differentiation factor-2 (MD-2) has been shown to be an important modulator of the innate immune system, but its role in cardiac diseases is unknown. We investigated whether MD-2 plays a role as risk predictor and contributor in dilated cardiomyopathy (DCM). METHODS AND RESULTS: We included 174 patients with reduced left ventricular (LV) ejection fraction (LVEF <45%) due to DCM. Coronary artery disease and severe valvular diseases were excluded in all patients by angiography or echocardiography. Cardiac inflammation, viral infection and MD-2 expression were analyzed from right ventricular endomyocardial biopsies. MD-2 was quantified by ELISA in serum upon first hospital admission. Myocyte contractility and inflammatory response after stimulation with recombinant MD-2 protein were analyzed in isolated rat cardiomyocytes. Median follow-up of the patients was 3.51 years (2.73; 4.48) with 34 deaths. Absolute mortality risk increases in patients displaying a MD-2 serum concentration greater than the median (302 ng/ml) was 23% (P < 0.0001). Age- and sex-adjusted Cox regression analyses demonstrated that mortality risk was highly related to MD-2 concentrations (P < 0.001), but not to age or sex. An increase of 100 ng/ml in the MD-2 level was associated with an absolute mortality risk increase of 50.4%. Receiver operating characteristic (ROC) analyses showed no difference between MD-2 and nterminal-pro brain natriuretic peptide (NT-pro-BNP), while the combination of both MD-2 and NT-pro-BNP resulted in a significantly increased capability of risk prediction when compared to NT-pro-BNP alone (P = 0.014). In-vitro, recombinant MD-2 decreases cell shortening and modulates cytokine activation in isolated cardiomyocytes. CONCLUSION: MD-2 predicts long-term outcome in DCM patients and improves mortality risk prediction capability compared to NT-pro-BNP alone. In addition, MD-2 exerts direct negative inotropic effects on isolated cardiomyocytes in-vitro. Further randomized trials should confirm MD-2 as a diagnostic and therapeutic target.

8.
PLoS One ; 13(3): e0193844, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29538462

RESUMO

Heart failure (HF) is a leading cause of morbidity and mortality in the western world. Although optimal medical care and treatment is widely available, the prognosis of patients with HF is still poor. Toll-like receptors (TLRs) are important compartments of the innate immunity. Current studies have identified TLRs as critical mediators in cardiovascular diseases. In the present study, we investigated the involvement of TLRs and interferon (IFN) regulatory factors (IRFs) in different experimental HF models including viral myocarditis, myocardial ischemia, diabetes mellitus, and cardiac hypertrophy. In addition, we investigated for the first time comprehensive TLR and IRF gene and protein expression under basal conditions in murine and human cardiac tissue. We found that Tlr4, Tlr9 and Irf7 displayed highest gene expression under basal conditions, indicating their significant role in first-line defense in the murine and human heart. Moreover, induction of TLRs and IRFs clearly differs between the various experimental HF models of diverse etiology and the concomitant inflammatory status. In the HF model of acute viral-induced myocarditis, TLR and IRF activation displayed the uppermost gene expression in comparison to the remaining experimental HF models, indicating the highest amount of myocardial inflammation in myocarditis. In detail, Irf7 displayed by far the highest gene expression during acute viral infection. Interestingly, post myocardial infarction TLR and IRF gene expression was almost exclusively increased in the infarct zone after myocardial ischemia (Tlr2, Tlr3, Tlr6, Tlr7, Tlr9, Irf3, Irf7). With one exception, Irf3 showed a decreased gene expression in the remote zone post infarction. Finally, we identified Irf7 as novel cardiovascular stress-inducible factor in the pathologically stressed heart. These findings on TLR and IRF function in the inflamed heart highlight the complexity of inflammatory immune response and raise more interesting questions for future investigation.


Assuntos
Insuficiência Cardíaca/metabolismo , Fatores Reguladores de Interferon/metabolismo , Miocárdio/metabolismo , Receptores Toll-Like/metabolismo , Animais , Cardiomegalia/metabolismo , Membrana Celular/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Insuficiência Cardíaca/etiologia , Humanos , Inflamação/metabolismo , Espaço Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/metabolismo , Miocardite/metabolismo , Proteoma , Distribuição Aleatória
9.
J Immunol Res ; 2017: 6590609, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28352641

RESUMO

Background. Infection with Coxsackievirus B3 induces myocarditis. We aimed to compare the acute and chronic phases of viral myocarditis to identify the immediate effects of cardiac inflammation as well as the long-term effects after resolved inflammation on cardiac fibrosis and consequently on cardiac function. Material and Methods. We infected C57BL/6J mice with Coxsackievirus B3 and determined the hemodynamic function 7 as well as 28 days after infection. Subsequently, we analyzed viral burden and viral replication in the cardiac tissue as well as the expression of cytokines and matrix proteins. Furthermore, cardiac fibroblasts were infected with virus to investigate if viral infection alone induces profibrotic signaling. Results. Severe cardiac inflammation was determined and cardiac fibrosis was consistently colocalized with inflammation during the acute phase of myocarditis. Declined cardiac inflammation but no significantly improved hemodynamic function was observed 28 days after infection. Interestingly, cardiac fibrosis declined to basal levels as well. Both cardiac inflammation and fibrosis were reversible, whereas the hemodynamic function remains impaired after healed viral myocarditis in C57BL/6J mice.


Assuntos
Miocardite/patologia , Miocardite/fisiopatologia , Miocardite/virologia , Disfunção Ventricular , Remodelação Ventricular , Doença Aguda , Animais , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Enterovirus Humano B , Fibroblastos/metabolismo , Fibrose , Hemodinâmica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Fatores de Tempo , Replicação Viral
10.
Physiol Rep ; 4(2)2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811054

RESUMO

The contribution of human atrial fibroblasts to cardiac physiology and pathophysiology is poorly understood. Fibroblasts may contribute to arrhythmogenesis through fibrosis, or by directly altering electrical activity in cardiomyocytes. The objective of our study was to uncover phenotypic differences between cells from patients in sinus rhythm (SR) and chronic atrial fibrillation (AF), with special emphasis on electrophysiological properties. We isolated fibroblasts from human right atrial tissue for patch-clamp experiments, proliferation, migration, and differentiation assays, and gene expression profiling. In culture, proliferation and migration of AF fibroblasts were strongly impaired but differentiation into myofibroblasts was increased. This was associated with a higher number of AF fibroblasts expressing functional Nav1.5 channels. Strikingly Na(+) currents were considerably larger in AF cells. Blocking Na(+) channels in culture with tetrodotoxin did not affect proliferation, migration, or differentiation in neither SR nor AF cells. While freshly isolated fibroblasts showed mostly weak rectifier currents, fibroblasts in culture developed outward rectifier K(+) currents of similar amplitude between the SR and AF groups. Adding the K(+) channel blockers tetraethylammonium and 4-aminopyridin in culture reduced current amplitude and inhibited proliferation in the SR group only. Analysis of gene expression revealed significant differences between SR and AF in genes encoding for ion channels, collagen, growth factors, connexins, and cadherins. In conclusion, this study shows that under AF conditions atrial fibroblasts undergo phenotypic changes that are revealed in culture. Future experiments should be performed in situ to understand the nature of those changes and whether they affect cardiac electrical activity.


Assuntos
Fibrilação Atrial/fisiopatologia , Fibroblastos/metabolismo , Átrios do Coração/metabolismo , Canais Iônicos/metabolismo , Idoso , Fibrilação Atrial/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Doença Crônica , Feminino , Fibroblastos/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Patch-Clamp , Transcriptoma
12.
Mediators Inflamm ; 2014: 519528, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25374444

RESUMO

Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3) and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-ß was significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase) compared to cardiomyocytes (14-fold increase) between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.


Assuntos
Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/fisiologia , Enterovirus Humano B/patogenicidade , Miocardite/patologia , Miocardite/virologia , Miocárdio/patologia , Animais , Células Cultivadas , Infecções por Coxsackievirus/fisiopatologia , Citocinas/genética , Enterovirus Humano B/genética , Fibroblastos/imunologia , Fibroblastos/patologia , Fibroblastos/virologia , Expressão Gênica , Genoma Viral , Coração/virologia , Macrófagos/imunologia , Macrófagos/patologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/fisiopatologia , Miocárdio/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Função Ventricular Esquerda , Carga Viral , Replicação Viral
13.
Basic Res Cardiol ; 109(5): 428, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25086637

RESUMO

Cardiac remodeling and inflammation are hallmarks of cardiac failure and correlate with outcome in patients. However, the basis for the development of both remains unclear. We have previously reported that cardiac inflammation triggers transdifferentiation of fibroblasts to myofibroblasts and therefore increase accumulation of cardiac collagen, one key pathology in cardiac remodeling. Hence, identifying key pathways for inflammation would be beneficial for patients suffering from heart failure also. Besides their well-characterized function in matrix regulation, we here investigate the role of fibroblasts in the inflammatory process. We address for the first time the role of fibroblasts as inflammatory supporter cells in heart failure. Using endomyocardial biopsies from patients with heart failure and dilated cardiomyopathy, we created a primary human cardiac fibroblast cell culture system. We found that mechanical stretch mimicking cardiac dilation in heart failure induces activation of fibroblasts and not only stimulates production of extracellular matrix but more interestingly up-regulates chemokine production and triggers typical inflammatory pathways in vitro. Moreover, the cell culture supernatant of stretched fibroblasts activates inflammatory cells and induces further recruitment of monocytes by allowing transendothelial migration into the cardiac tissue. Our findings reveal that cardiac fibroblasts provide pro-inflammatory mediators and may act as sentinel cells activated by mechanical stress. Those cells are able to recruit inflammatory cells into the cardiac tissue, a process known to aggravate outcome of patients. This might be important in different forms of heart failure and therefore may be one general mechanism specific for fibroblasts.


Assuntos
Fibroblastos/imunologia , Insuficiência Cardíaca/patologia , Inflamação/patologia , Miocárdio/imunologia , Miocárdio/patologia , Animais , Células Cultivadas , Insuficiência Cardíaca/imunologia , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Estresse Mecânico
14.
J Biol Chem ; 289(9): 5846-59, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24375409

RESUMO

Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr(2.64) interacts with Tyr(27) of hPP through hydrophobic contacts. Asn(7.32) is affected by modifications on position Arg(33) of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe(7.35) is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6-TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.


Assuntos
Polipeptídeo Pancreático/química , Receptores de Neuropeptídeo Y/química , Animais , Sítios de Ligação , Células COS , Cercopithecus aethiops , Cristalografia por Raios X , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Pancreático/genética , Polipeptídeo Pancreático/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo
15.
Circ Heart Fail ; 7(1): 161-71, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24300243

RESUMO

BACKGROUND: CD4+ cells are implicated in the healing process after myocardial infarction (MI). We sought to investigate the role of interleukin-23 (IL-23) deficiency, a cytokine important in differentiation of CD4+ cells, in scar formation of the ischemic heart. METHODS AND RESULTS: MI was performed in wild-type and IL23p19-/- mice. Thirty-day mortality, hemodynamic function 4 days after MI and myocardial inflammation, and remodeling 4 and 30 days after MI were examined. Differentiation of fibroblasts from infarcted and noninfarcted hearts into myofibroblasts was examined under basal conditions and after stimulation with interferon-γ, IL-17α and IL-23. Interleukin-23p19-/- mice showed higher expression of proinflammatory cytokines and immune cell infiltration in the scar early after MI compared with wild-type mice. A stronger interferon-γ/Th1 reaction seemed to be responsible for the increased inflammation under IL-23 deficiency. Expression of α-smooth muscle actin (α-SMA), collagen I and III was significantly higher in the heart tissue and isolated cardiac fibroblasts 4 days after MI in the wild-type mice. Interleukin-23p19-/- mice showed impaired healing compared with wild-type mice, as seen by significantly higher mortality because of ventricular rupture (40% higher after 30 days) and stronger left ventricular dilation early after MI. Stimulation of cardiac fibroblasts with interferon-γ, the main Th1 cytokine, but not with IL-23 or IL-17α, led to a significant downregulation of α-smooth muscle actin, collagen I and III and decreased migration and differentiation to myofibroblasts. CONCLUSIONS: IL-23 deficiency leads to increased myocardial inflammation and decreased cardiac fibroblast activation, associated with impaired scar formation and adverse remodeling after MI.


Assuntos
Interleucina-23/deficiência , Interleucina-23/fisiologia , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/fisiopatologia , Cicatrização/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cicatriz/patologia , Cicatriz/fisiopatologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Interferon gama/farmacologia , Interleucina-17/farmacologia , Interleucina-23/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/mortalidade , Prognóstico
16.
J Mol Cell Cardiol ; 66: 141-56, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24239602

RESUMO

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fibroblastos/metabolismo , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Adenoviridae/genética , Animais , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/patologia , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Inativação Gênica , Vetores Genéticos , Humanos , Inflamação/prevenção & controle , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Terapia de Alvo Molecular , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo
17.
Expert Opin Med Diagn ; 7(5): 463-74, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23930995

RESUMO

INTRODUCTION: Heart failure with preserved ejection fraction (HFPEF) is a common syndrome, accounting for about 50% of all patients with heart failure (HF). Morbidity and mortality are similar to patients with HF with reduced ejection fraction (HFREF), yet no effective treatment has been identified in randomized clinical trials. AREAS COVERED: This article provides an overview of the available literature regarding diagnosing established HFPEF and potential new therapeutic targets for the early diagnosis of HFPEF. Vascular dysfunction, ventricular-arterial coupling, oxidative stress, extracellular matrix regulation, chronotropic incompetence, pulmonary hypertension, exercise testing and biomarkers were taken into consideration next to conventional measurements of diastolic dysfunction. EXPERT OPINION: Measuring diastolic dysfunction in HFPEF is considered important in many patients. Nevertheless, today we know that other causes besides diastolic dysfunction are also involved in the pathophysiology of many HFPEF patients and need to be investigated in order to make a correct diagnosis. Therefore, further research is required to allow better and more specific diagnostic and treatment options to reduce the morbidity and mortality for this ever-expanding HF population.


Assuntos
Insuficiência Cardíaca/diagnóstico , Volume Sistólico/fisiologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Testes de Função Cardíaca , Humanos
18.
Cardiovasc Res ; 99(1): 111-20, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23619422

RESUMO

AIMS: We investigated whether the pro-fibrotic matricellular protein osteopontin (OPN) is associated with the enzymes involved in the extracellular synthesis of fibril-forming collagen type I (i.e. procollagen C-proteinase, PCP) and its cross-linking to form insoluble fibrils (i.e. lysyl oxidase, LOX) in heart failure (HF) of hypertensive origin. METHODS AND RESULTS: OPN, PCP, and LOX were assessed by histochemical and molecular methods in the myocardium of 21 patients with hypertensive heart disease (HHD) and HF. Whereas the myocardial expression of OPN was very scarce in control hearts (n = 10), it was highly expressed in HF patients (P < 0.0001). OPN was directly correlated with LOX (r = 0.460, P = 0.041), insoluble collagen (r = 0.534, P = 0.015), pulmonary capillary wedge pressure (r = 0.558; P = 0.009), and left-ventricular (LV) chamber stiffness (r = 0.458, P = 0.037), and inversely correlated with LV ejection fraction (r = -0.513, P = 0.017) in all patients. OPN did not correlate with PCP and other parameters assessing collagen synthesis by fibroblasts or degradation by matrix metalloproteinases. In vitro studies showed that OPN significantly (P < 0.05) increases the expression and activity of LOX in human cardiac and dermal fibroblasts. CONCLUSION: An excess of OPN is associated with increased LOX and insoluble collagen, as well as with LV stiffness and systolic dysfunction in patients with HHD and HF. In addition, OPN up-regulates LOX in human fibroblasts. It is suggested that the OPN-LOX axis might facilitate the formation of insoluble collagen (i.e. stiff and resistant to degradation) and the subsequent alteration in LV mechanical properties and function in patients with HHD and HF.


Assuntos
Insuficiência Cardíaca/enzimologia , Miocárdio/enzimologia , Osteopontina/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Idoso , Proteína Morfogenética Óssea 1/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Colágeno/metabolismo , Elasticidade , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão/complicações , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Pressão Propulsora Pulmonar , Volume Sistólico , Função Ventricular Esquerda
19.
Basic Res Cardiol ; 107(6): 308, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23117837

RESUMO

Sildenafil inhibits cyclic GMP-specific phosphodiesterase type-5A (PDE5A) and can prevent cardiac hypertrophy and left ventricular (LV) dysfunction in mice subjected to severe pressure-overload. The pathophysiological role of sildenafil in adverse remodeling in the hypertensive heart after chronic renin-angiotensin aldosterone system stimulation is unknown. Therefore, we studied the efficacy of the PDE5A inhibitor sildenafil for treating advanced cardiac hypertrophy and LV remodeling due to angiotensin (Ang)II-induced heart failure (HF) in vivo. C57BL6/J mice were subjected to AngII-induced cardiac hypertrophy for 3 weeks and cardiac dysfunction, cardiac inflammatory stress response, adverse remodeling as well as apoptosis were documented. Mice were subsequently treated with sildenafil (100 mg/kg/day) or placebo with delay of 5 days for treating AngII infusion-induced adverse events. Compared to controls, AngII infusion resulted in impaired systolic (dP/dt (max) -46 %, SV -16 %, SW -43 %, E (a) +51 %, EF -37 %, CO -36 %; p < 0.05) and diastolic (dP/dt (min) -36 %, LV end diastolic pressure +73 %, Tau +21 %, stiffness constant ß +74 %; p < 0.05) LV function. This was associated with a significant increase in cardiac hypertrophy and fibrosis. Increased inflammatory response was also indicated by an increase in immune cell infiltration and apoptosis. Treatment with sildenafil led to a significant improvement in systolic and diastolic LV performance. This effect was associated with less LV hypertrophy, remodeling, cardiac inflammation and apoptosis. PDE5A inhibition with sildenafil may provide a new treatment strategy for cardiac hypertrophy and adverse remodeling in the hypertensive heart.


Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Inibidores da Fosfodiesterase 5/uso terapêutico , Piperazinas/uso terapêutico , Sulfonas/uso terapêutico , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II , Animais , Anti-Hipertensivos , Apoptose/efeitos dos fármacos , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Citocinas/metabolismo , Matriz Extracelular/efeitos dos fármacos , Insuficiência Cardíaca/induzido quimicamente , Testes de Função Cardíaca , Hidralazina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/tratamento farmacológico , Miocárdio/metabolismo , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Purinas/uso terapêutico , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Citrato de Sildenafila , Sulfonas/farmacologia , Vasoconstritores , Disfunção Ventricular Esquerda/tratamento farmacológico
20.
J Biol Chem ; 287(38): 32181-94, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22778259

RESUMO

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.


Assuntos
Mutação , Hormônio Liberador de Prolactina/química , Prolactina/química , Receptores Acoplados a Proteínas-G/química , Sequência de Aminoácidos , Animais , Células COS , Cercopithecus aethiops , Clonagem Molecular , Desenho de Drogas , Vetores Genéticos , Células HEK293 , Humanos , Concentração Inibidora 50 , Ligantes , Dados de Sequência Molecular , Mutagênese , Peptídeos/química , Ligação Proteica , Homologia de Sequência de Aminoácidos , Transdução de Sinais
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