RESUMO
BACKGROUND: Granulation tissue cells at the subacute stage of myocardial infarction (MI) are eliminated by apoptosis to finally make a scar at the chronic stage. We hypothesized that postinfarct inhibition of apoptosis might preserve myofibroblasts and endothelial cells in granulation and modulate chronic left ventricular (LV) remodeling and heart failure. METHODS AND RESULTS: A pancaspase inhibitor, Boc-Asp-fmk (BAF, 10 micromol/kg per day), or vehicle (control) was given to rats with experimental large MI. The treatment was started on the third day after MI and continued until 4-week-old MI. Two weeks later, the apoptosis of granulation tissue cells was significantly reduced and conversely, the cell population was greater in BAF. Twelve weeks later, BAF showed significantly greater survival rates (84% versus 42%) with significantly smaller LV cavity, lower LV end-diastolic pressure and central venous pressure, and higher LV dP/dt, which indicated improvement of LV remodeling and dysfunction. A scar was established in old infarct of control subjects, but in BAF, the infarct wall was thicker because of greater old infarct area, which contained abundant myofibroblasts and vessels. Surprisingly, many of the alpha-smooth muscle actin-positive myofibroblast-like cells in BAF, making bundles and running parallel to the survived cardiomyocytes, were ultrastructurally mature smooth muscle cells with contractile phenotype. Cardiomyocyte apoptosis in the infarct area was equally rare in each group. CONCLUSIONS: The postinfarct treatment with BAF improved LV remodeling and dysfunction through inhibition of granulation tissue cell apoptosis. These findings imply a new therapeutic strategy against postinfarct heart failure.
Assuntos
Apoptose , Tecido de Granulação/patologia , Infarto do Miocárdio/patologia , Disfunção Ventricular/fisiopatologia , Remodelação Ventricular , Clorometilcetonas de Aminoácidos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Doença Crônica , Inibidores de Cisteína Proteinase/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Tecido de Granulação/efeitos dos fármacos , Coração/efeitos dos fármacos , Masculino , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Ratos , Ratos Wistar , Taxa de Sobrevida , Disfunção Ventricular/tratamento farmacológico , Remodelação Ventricular/efeitos dos fármacosRESUMO
BACKGROUND: Sensitivity to apoptotic signals, the clearance rate of apoptosis, and the apoptotic ultrastructure have not been studied in cells of the in vivo adult heart. METHODS AND RESULTS: To minimize the systemic influence, soluble Fas ligand was injected directly into in vivo rat hearts and livers (as the control) at concentrations of 0, 0.5, 2, and 5 microg/mL (groups C, F0.5, F2, and F5). Apoptotic cardiomyocytes and apoptotic noncardiomyocytes of the heart were identified with similar incidences only in F5. Their incidences peaked at 12 hours after injection (2.0+/-0.09% in cardiomyocytes) and diminished markedly 24 hours later. Caspase-3 was activated only in F5. Boc-Asp-fmk, a pancaspase inhibitor, inhibited apoptosis, suggesting that the apoptosis sensitivity was regulated upstream of caspase-3. Apoptotic noncardiomyocytes showed typical ultrastructure. In addition to the typical ultrastructure, such as cellular shrinkage, chromatin condensation, and apoptotic bodies, however, apoptotic cardiomyocytes showed unique features: doughnut-like, but not half-moon- or crescent-like, chromatin condensation; frequent plasma membrane rupture even during the early stage; condensed mitochondria with wrinkled cristae inside; the appearance of cytoplasmic lipid-like droplets; and myofibrillar derangement. In the livers, typical apoptosis was induced in hepatocytes and nonhepatocytes of the liver even in the F0.5 group, which were cleared 24 hours later. CONCLUSIONS: Compared with liver cells, cardiomyocytes as well as noncardiomyocytes of the heart are more resistant against the apoptotic signal, but the clearance is similarly rapid (within 24 hours). The ultrastructure of apoptotic cardiomyocytes is unique. These findings provide new insights into the dynamics of cell death in the heart.
Assuntos
Apoptose , Glicoproteínas de Membrana/toxicidade , Miocárdio/citologia , Miocárdio/ultraestrutura , Animais , Caspase 3 , Caspases/metabolismo , Fragmentação do DNA , Proteína Ligante Fas , Coração/efeitos dos fármacos , Insuficiência Cardíaca/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Cinética , Masculino , Miocárdio/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) is implicated in tissue regeneration, angiogenesis, and antiapoptosis. However, its chronic effects are undetermined on postinfarction left ventricular (LV) remodeling and heart failure. METHODS AND RESULTS: In mice, on day 3 after myocardial infarction (MI), adenovirus encoding human HGF (Ad.CAG-HGF) was injected into the hindlimb muscles (n=13). As a control (n=15), LacZ gene was used. A persistent increase in plasma human HGF was confirmed in the treated mice: 1.0+/-0.2 ng/mL 4 weeks later. At 4 weeks after MI, the HGF-treated mice showed improved LV remodeling and dysfunction compared with controls, as indicated by the smaller LV cavity and heart/body weight ratio, greater % fractional shortening and LV +/-dP/dt, and lower LV end-diastolic pressure. The cardiomyocytes near MI, including the papillary muscles and trabeculae, were greatly hypertrophied in the treated mice. The old infarct size was similar between the groups, but the infarct wall was thicker in the treated mice, where the density of noncardiomyocyte cells, including vessels, was greater. Fibrosis of the ventricular wall was significantly reduced in them. Examination of 10-day-old MI revealed no proliferation or apoptosis but showed augmented expression of c-Met/HGF receptor in cardiomyocytes near MI, whereas a greater proliferating activity and smaller apoptotic rate of granulation tissue cells in the HGF-treated hearts was observed compared with controls. CONCLUSIONS: Postinfarction HGF gene therapy improved LV remodeling and dysfunction through hypertrophy of cardiomyocytes, infarct wall thickening, preservation of vessels, and antifibrosis. These findings imply a novel therapeutic approach against postinfarction heart failure.
Assuntos
Adenoviridae , Vetores Genéticos/uso terapêutico , Fator de Crescimento de Hepatócito/biossíntese , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Adenoviridae/genética , Animais , Bromodesoxiuridina , Divisão Celular/efeitos dos fármacos , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Vetores Genéticos/genética , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia , Taxa de Sobrevida , Fatores de Tempo , Transdução Genética/métodos , Resultado do Tratamento , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/genética , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/genéticaRESUMO
BACKGROUND: DNA breaks detected largely by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate in situ nick end-labeling (TUNEL) are observed in the hearts of patients with diseases such as dilated cardiomyopathy (DCM). STUDY OBJECTIVES: To determine the prognostic value of TUNEL assays in cases of DCM. DESIGN, SETTING, AND PARTICIPANTS: DCM patients were selected from among patients who had undergone left ventricular (LV) biopsy during the period from 1994 to 2001 in our hospital. Of those, 46 (35 men and 11 women; mean [+/- SD] age, 58 +/- 11 years) who were followed up for > 3 years after the undergoing the biopsy (mean follow-up period, 4.9 +/- 2.0 years) or died during the follow-up period were entered into the present study. The myocardial apoptotic index was assessed in deparaffinized biopsy specimens that were stained using a conventional TUNEL assay. In addition, all surviving patients received a follow-up echocardiographic examination. RESULTS: Ten of the 46 biopsy specimens (22%) contained TUNEL-positive myocytes; their mean apoptotic index was 0.44 +/- 1.05%. The apoptotic index showed no relation to cardiac functional parameters determined at the time of biopsy, however. Seven patients died during the follow-up period, and 19 of the surviving patients were readmitted to the hospital because of a worsening of their heart failure. There was no significant difference in the apoptotic indexes of biopsy specimens from the dead and surviving patients, or between the surviving patients who were readmitted to the hospital and those who were not. There was also no significant correlation between the apoptotic index and changes in the LV ejection fraction, LV end-diastolic diameter, or LV posterior wall thickness during follow-up. CONCLUSION: The apoptotic index derived from TUNEL assays is not predictive of the prognosis of patients with DCM-induced heart failure.
Assuntos
Apoptose , Cardiomiopatia Dilatada/patologia , Miocárdio/patologia , Adulto , Idoso , Apoptose/genética , Biópsia , Cardiomiopatia Dilatada/genética , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , PrognósticoRESUMO
OBJECTIVE: In cultured cardiomyocytes, apoptosis is induced not by Fas stimulation, a popular inducer of apoptosis, but by an additional treatment with actinomycin D (AD), a transcription inhibitor, although the mechanism is unknown. Our hypothesis is that Fas stimulation not only activates pro-apoptotic signals but also may inhibit some of them, and this inhibition is blocked by AD. METHODS: Cultured neonatal mouse cardiomyocytes were treated with agonistic anti-Fas antibody (FA), AD, or both (FA+AD). In this system, apoptotic signals related to Fas-induced apoptotic pathways were examined by RT-PCR and immunoblotting. In addition, antisense oligonucleotide (AS) studies were carried out. RESULTS: The treatment with FA+AD induced up-regulation of Fas, activation of c-Jun N-terminal kinase (JNK), which is one of the key molecules of the alternate pathway of Fas-induced apoptosis, up-regulation of Bax, up-regulation and activation of caspase-3, activation of caspase-3-dependent DNase (CAD), and final DNA fragmentation and apoptotic morphologies in cardiomyocytes. FA alone or AD alone did not affect any part of the above pathway. However, mRNA of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inactivator of JNK, was up-regulated by FA alone, but not by FA+AD or AD alone. Pretreatment with AS against MKP-1 induced apoptosis in FA alone-treated cardiomyocytes, whereas AS against JNK1 prevented apoptosis induced by FA+AD. On the other hand, FA+AD did not result in the activation of either caspase-8, one of the key molecules of the classic pathway in Fas-induced apoptosis, p38 MAPK, or extracellular signal-regulated kinase (ERK). CONCLUSIONS: Cardiomyocyte apoptosis by FA+AD depends on the alternate pathway through the JNK, Bax and caspase-3, and CAD-dependent pathways including a positive feedback mechanism of Fas up-regulation. The molecular mechanism that prevents Fas stimulation alone from inducing apoptosis involves up-regulation of MKP-1, an inhibitor of JNK; this up-regulation is inhibited by AD.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Dactinomicina/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Miocárdio/metabolismo , Fosfoproteínas Fosfatases , Receptor fas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Proteínas Reguladoras de Apoptose , Northern Blotting/métodos , Western Blotting/métodos , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/genética , Células Cultivadas , DNA Antissenso , Desoxirribonucleases/genética , Fosfatase 1 de Especificidade Dupla , Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Proteínas Imediatamente Precoces/genética , MAP Quinase Quinase 4 , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Miocárdio/ultraestrutura , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção/métodos , Receptor fas/genética , Receptor fas/imunologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Chronic congestive heart failure (CHF) causes structural remodeling of the liver, generally leading to nutmeg liver. Male UM-X7.1 hamsters, a strain developing cardiomyopathy, had no CHF and decompensated CHF (n = 6 each) at the age of 10 and 30 weeks, respectively. We used age-matched, male Syrian hamsters without CHF (n = 6 each) as controls. All the 30-week-old UM-X7.1 hamsters had a typical nutmeg liver in which the population of hepatocytes was decreased. Positive in situ nick end labeling (TUNEL) was found in 2.2 +/- 0.74% of hepatocytes in congestive livers, being significantly higher compared with the other groups without CHF (< 0.5%). DNA ladder pattern was also evident in the congestive livers. Electron microscopy revealed a typical apoptotic ultrastructure in the hepatocytes of the 30-week-old UM-X7.1 hamsters. However, many showed secondary necrotic changes. Although hepatocytes undergoing oncosis (primary necrosis) are rare, they were also found. The level of soluble Fas ligand in the plasma was increased, and Fas receptor in the liver was overexpressed in the CHF animals. In addition, both the Bax/Bcl-2 ratio and the Bad/Bcl-xL ratio were increased, and caspase-3 was activated in them. Our findings suggest that hepatocyte apoptosis contributes to hepatic remodeling under conditions of CHF.
Assuntos
Apoptose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Hepatócitos/ultraestrutura , Hepatopatias/metabolismo , Hepatopatias/patologia , Animais , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/congênito , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Caspase 3 , Caspases/biossíntese , Cricetinae , Insuficiência Cardíaca/etiologia , Hepatócitos/metabolismo , Marcação In Situ das Extremidades Cortadas , Hepatopatias/etiologia , Masculino , Mesocricetus , Microscopia Eletrônica , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína bcl-X , Receptor fas/biossínteseRESUMO
Numerous cardiomyocytes were found to show autophagic vacuolar degeneration in the UM-X7.1 hamster model of human dilated cardiomyopathy, and autophagy-related proteins--i.e., ubiquitin, cathepsin D and Rab7--were upregulated in those hearts. Importantly, Evans blue-positive cardiomyocytes with leaky plasma membranes were also positive for cathepsin D, suggesting a link between autophagic degeneration and cell death. Treatment with granulocyte colony-stimulating factor (G-CSF) significantly improved survival, cardiac function and remodeling in these animals, and such beneficial effects were accompanied by a reduction in autophagy, an increase in cardiomyocyte size, and a reduction in myocardial fibrosis. G-CSF-induced changes in molecular signaling included activation of Akt and Stat3 (signal transducer and activator of transcription-3), a reduction in the level of myocardial tumor necrosis factor-alpha, and an increase in those of matrix metalloproteinases. In contrast, neither cardiomyocyte apoptosis nor regeneration of cardiomyocytes from bone marrow-derived cells was significant. It thus appears that autophagic death and autophagy-dependent degeneration are important contributors to loss of cardiomyocyte function in the cardiomyopathic hamster and that G-CSF exerts a beneficial effect, mainly via an anti-autophagic mechanism.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/patologia , Animais , Cardiomiopatia Dilatada/patologia , Cricetinae , Fator Estimulador de Colônias de Granulócitos/fisiologia , Insuficiência Cardíaca/etiologia , Humanos , Modelos BiológicosRESUMO
In UM-X7.1 hamster model of human dilated cardiomyopathy, heart failure progressively develops and causes 50% mortality by 30 weeks of age. Through ultrastructural analysis, we found that many cardiomyocytes of this model contain typical autophagic vacuoles including degraded mitochondria, glycogen granules, and myelin-like figures. In addition, ubiquitin, cathepsin D, and Rab7 were overexpressed as determined by immunoassays. Importantly, most cardiomyocytes with leaky plasma membranes were positive for cathepsin D, suggesting a direct link between autophagic degeneration and cell death. Meanwhile, cardiomyocyte apoptosis appeared insignificant. Granulocyte colony-stimulating factor (10 microg/kg/day), injected 5 days/week from 15 to 30 weeks of age, improved survival among 30-week-old hamsters (100% versus 53% in the untreated hamsters, P < 0.0001); ventricular function and remodeling, increased cardiomyocyte size, and reduced myocardial fibrosis followed by a dramatic reduction in the autophagic findings were also seen. Granulocyte colony-stimulating factor also down-regulated tumor necrosis factor-alpha and increased activities of Akt signal transducer and activator of transcription-3, and matrix metalloproteinases. However, there was no clear evidence of transdifferentiation from bone marrow cells into cardiomyocytes. In conclusion, autophagic death is important for cardiomyocyte loss in the cardiomyopathic hamster, and the beneficial effect of granulocyte colony-stimulating factor acts mainly via an anti-autophagic mechanism rather than anti-apo-ptosis or regeneration.
Assuntos
Apoptose , Autofagia , Cardiomiopatia Dilatada/prevenção & controle , Fibrose/prevenção & controle , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Miócitos Cardíacos/patologia , Animais , Cardiomiopatia Dilatada/metabolismo , Catepsina D/metabolismo , Cricetinae , Fibrose/metabolismo , Masculino , Mesocricetus , Mitocôndrias/patologia , Bainha de Mielina , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes , Fator de Transcrição STAT3/metabolismo , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Vacúolos , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The significance of DNA breaks reported in failing hearts is controversial, although they may suggest myocyte apoptosis and may thus be responsible for the progression of heart failure. This study attempted to check the validity of the in situ markers for DNA breaks for detecting myocyte death and to evaluate separately two factors, failure or hypertrophy, crucial for DNA breaks in pathological human hearts. In the autopsy study, myocytes showed positivity for in situ nick end-labelling (TUNEL) and of Taq and Pfu polymerase-based in situ ligation assays not only in dilated cardiomyopathy (DCM, n = 9) with failure, but also in hypertrophic cardiomyopathy (HCM, n = 8) and hypertensive heart disease (HHD, n = 4) without failure. There was a significant correlation between each in situ marker and heart weight. The incidence of TUNEL-positive myocytes always exceeded that seen in in situ ligation assays. In addition, there were significant correlations between the in situ markers and the expression of the proliferating cell nuclear antigen (PCNA) and of the spliceosome component of 35 kD (SC-35). Similarly, in the left ventricular biopsy study using 23 DCM, 21 HCM, 11 HHD, and 13 non-hypertrophic hearts, the incidence of the in situ markers showed significant correlations with the left ventricular mass index and myocyte size, but not with cardiac function and dilatation. Positivity of myocytes for in situ markers for DNA breaks, such as TUNEL and in situ ligation assays, may be an epiphenomenon accompanying cardiac hypertrophy, but not myocyte death in pathological human hearts.