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Masahiko Koda

Publications and source records attributed to Masahiko Koda.

At least 19 recordsLinked to original sources
PubMed2006

Preventive and therapeutic effects of angiotensin II type 1 receptor blocker on hepatic fibrosis induced by bile duct ligation in rats.

BACKGROUND: The aim of this study was to examine the preventive and therapeutic effects of an angiotensin II type 1 receptor blocker, candesartan, on cholestasis-induced liver fibrosis. METHODS: Candesartan was administered orally for 21 days immediately after bile duct ligation to evaluate its preventive effect, and for 21 days starting 3 weeks after bile duct ligation to evaluate its therapeutic effect. Fibrosis was assessed by measuring hepatic hydroxyproline (Hyp) content. The activated hepatic stellate cells (HSCs) were assessed by alpha-smooth muscle actin (alpha-SMA) immunostaining. The gene expression of collagen I, transforming growth factor-beta1 (TGF-beta1), and connective tissue growth factor (CTGF) in the liver was examined by real-time reverse transcriptase-polymerase chain reaction. RESULTS: As a preventive effect, candesartan reduced the hepatic Hyp content by 36%, alpha-SMA-positive cells by 65%, hepatic TGF-beta1 content by 35%, and the expression of collagen I by 72%, TGF-beta1 by 67%, and CTGF mRNA by 69%. As a therapeutic effect, candesartan reduced the hepatic Hyp content by 48%, TGF-beta1 content by 54%, and the expression of collagen I by 47%, TGF-beta1 by 43%, and CTGF mRNA by 53%. Significant decreases in lipid peroxidation markers, hepatic thiobarbituric acid-reactive substance, and 4-hydroxy-2-nonenal were observed in candesartan-treated rats. CONCLUSIONS: Candesartan attenuated liver fibrosis via suppression of collagen I and TGF-beta1 expression, HSC activation, and lipid peroxidation protein, showing its preventive and therapeutic effects on cholestasis-induced liver fibrosis.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2006

Endothelin-1 enhances fibrogenic gene expression, but does not promote DNA synthesis or apoptosis in hepatic stellate cells.

BACKGROUND: In liver injury, the pool of hepatic stellate cell (HSC) increases and produces extracellular matrix proteins, decreasing during the resolution of fibrosis. The profibrogenic role of endothelin-1 (ET-1) in liver fibrosis remains disputed. We therefore studied the effect of ET-1 on proliferation, apoptosis and profibrogenic gene expression of HSCs. RESULTS: First passage HSC predominantly expressed endothelin A receptor (ETAR) mRNA and 4th passage HSC predominantly expressed the endothelin B receptor (ETBR) mRNA. ET-1 had no effect on DNA synthesis in 1st passage HSC, but reduced DNA synthesis in 4th passage HSC by more than 50%. Inhibition of proliferation by endothelin-1 was abrogated by ETBR specific antagonist BQ788, indicating a prominent role of ETBR in growth inhibition. ET-1 did not prevent apoptosis induced by serum deprivation or Fas ligand in 1st or 4th passage HSC. However, ET-1 increased procollagen alpha1(I), transforming growth factor beta-1 and matrix metalloproteinase (MMP)-2 mRNA transcripts in a concentration-dependent manner in 1st, but not in 4th passage HSC. Profibrogenic gene expression was abrogated by ETAR antagonist BQ123. Both BQ123 and BQ788 attenuated the increase of MMP-2 expression by ET-1. CONCLUSION: We show that ET-1 stimulates fibrogenic gene expression for 1st passage HSC and it inhibits HSC proliferation for 4th passage HSC. These data indicate the profibrogenic and antifibrogenic action of ET-1 for HSC are involved in the process of liver fibrosis.

Journal Article↗
PubMed2006

Sonographic subcutaneous and visceral fat indices represent the distribution of body fat volume.

BACKGROUND: A reliable method for direct measurement of both subcutaneous and visceral fat volume is the measurement of fat tissue area from tomographic pictures by CT or by MR imaging. However, these are not widely usable because of high cost and/or exposure to radiation. METHODS: We compared sonographic subcutaneous and visceral fat indices with fat distribution by serial-slice MR imaging in 17 subjects. Sonographic subcutaneous or visceral fat index is standardized thickness of subcutaneous fat tissue or the intra-abdominal depth at the level of umbilicus by height. RESULTS: Sonographic visceral fat index and intra-abdominal depth were significantly correlated with visceral fat volume by serial-slice MR imaging (r = 0.746, r = 0.726, respectively). Similarly, sonographic subcutaneous fat index and subcutaneous fat thickness were significantly correlated with subcutaneous fat volume by serial-slice MR imaging (r = 0.825, r = 0.816, respectively). The ratio of sonographic visceral fat index and sonographic subcutaneous fat index was closely correlated with the ratio of the visceral fat volume and the subcutaneous fat volume by single-slice MR imaging, which proves to be related to cardiovascular disease risk (r = 0.722). CONCLUSION: Sonographic subcutaneous or visceral fat index could be an easily measured and inexpensive indicator for the assessment of fat distribution instead of CT or MR imaging.

Adult↗
PubMed2006

Nuclear hypertrophy reflects increased biosynthetic activities in myocytes of human hypertrophic hearts.

BACKGROUND: The nucleus of the myocytes in human hypertrophic hearts is characterized by its bizarre shape and widespread clumping of chromatin. The functional significance has not been determined. METHODS AND RESULTS: Left ventricular (LV) endomyocardial biopsies obtained from patients with dilated cardiomyopathy (DCM, n=23), postmyocarditis (n=13), hypertrophic cardiomyopathy (HCM, n=21), apical hypertrophic cardiomyopathy (APH, n=11) and hypertensive heart disease (HHD, n=11), and from nonhypertrophic hearts (controls, n=14) were examined. Myocyte size and LV mass index were similar among the hypertrophic hearts, but the nuclear hypertrophy score (grade 0-3) was highest in hearts with systolic failure (DCM and postmyocarditis) and higher in those without it (HCM, APH, and HHD), compared with controls. So were biosynthetic activities such as DNA repair/synthesis, immunohistochemically assessed by proliferating cell nuclear antigen, transcription activity by spliceosome component of 35 kDa, and translation efficiency by 70 kDa S6 protein kinase. There were significant correlations between nuclear hypertrophy and each biosynthetic activity. Additionally, most of the proliferating cell nuclear antigen-positive nuclei co-expressed oxidative DNA damage markers. CONCLUSION: A link is suggested between structural alteration and molecular biological events in the nuclei of myocytes from human hypertrophic hearts; the nuclear hypertrophy reflects increased biosynthetic activities of DNA repair/synthesis, transcription, and translation efficiency.

Aged↗
PubMed2005

Myocardial apoptotic index based on in situ DNA nick end-labeling of endomyocardial biopsies does not predict prognosis of dilated cardiomyopathy.

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.

Adult↗
PubMed2004

Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment.

BACKGROUND: We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony-stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. METHODS AND RESULTS: In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 microg x kg(-1) x d(-1) of human recombinant G-CSF (G) was injected subcutaneously from 1 to 5 days after MI. Smaller left ventricular (LV) dimension, increased LV ejection fraction, and thicker infarct-LV wall were seen in G at 3 months after MI. At 2, 7, and 14 days and 3 months after MI, necrotic tissue areas were 14.2+/-1.5/13.4+/-1.1, 0.4+/-0.1/1.8+/-0.5*, 0/0, and 0/0 mm2 x slice(-1) x kg(-1), granulation areas 0/0, 4.0+/-0.7/8.5+/-1.0*, 3.9+/-0.8/5.7+/-0.7,* and 0/0 mm2 x slice(-1) x kg(-1), and scar areas 0/0, 0/0, 0/0, and 4.2+/-0.5/7.9+/-0.9* mm2 x slice(-1) x kg(-1) in G and S, respectively (*P<0.05, G versus S). Clear increases of macrophages and of matrix metalloproteinases (MMP) 1 and 9 were seen in G at 7 days after MI. This suggests that G accelerates absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues via expression of MMPs. Meanwhile, surviving myocardial tissue areas within the risk areas were significantly increased in G despite there being no difference in LV weight, LV wall area, or cardiomyocyte size between G and S. Confocal microscopy revealed significant increases of cardiomyocytes with positive 3,3,3',3'-tetramethylindocarbocyanine perchlorate and positive troponin I in G, suggesting enhanced myocardial regeneration by G. CONCLUSIONS: The acceleration of the healing process and myocardial regeneration may play an important role for the beneficial effect of post-MI G-CSF treatment.

Animals↗
PubMed2004

The influence on liver parenchymal function and complications of radiofrequency ablation or the combination with transcatheter arterial embolization for hepatocellular carcinoma.

Objectives: We investigated how radiofrequency ablation (RFA) alone or the combination with transcatheter arterial embolization (TAE-RFA) for hepatocellular carcinoma (HCC) affects the liver parenchymal function of underlying chronic liver disease to find a predictive factor for selecting appropriate candidates for RFA. Methods: In 53 HCC patients (RFA alone, 25; TAE-RFA, 28), liver laboratory tests and development of complications were monitored until 6 months after treatments. Serum albumin deteriorated within 1 month and recovered after 6 months in both groups, whereas the elevation of the Pugh score continued in RFA alone group and that of serum bilirubin continued in TAE-RFA group until 6 months. The elevation of Pugh score 6 months after RFA was significantly more frequent in patients with a high pre-treatment Pugh score (>/=8 points) than in those with a low pre-treatment Pugh score (<8 points). Complications after RFA were observed in 11 of the 53 patients: temporary complications in 5, refractory ascites in 6. Refractory ascites developed in only patients with a high pre-treatment Pugh score. Conclusion: Liver parenchymal functions in patients with a low pre-treatment Pugh score were transiently deteriorated by RFA or TAE-RFA. However, in patients with a high Pugh score, RFA or TAE-RFA induces long-term deterioration of the liver parenchymal functions and causes serious complications. Therefore, patients with a Pugh score >/=8 points would not be good candidates for RFA or TAE-RFA.

Journal Article↗
PubMed2004

Cardiomyopathy with prominent autophagic degeneration, accompanied by an elevated plasma brain natriuretic peptide level despite the lack of overt heart failure.

A 75-year-old man without overt heart failure showed an abnormally high level of brain natriuretic peptide (BNP) in plasma: 600 pg/ml. The left ventricular endomyocardial biopsy revealed prominent vacuolar degeneration in the myocytes, most of which were positive for PAS stain and BNP immunoreaction. Ultrastructurally, degenerative changes of myocytes were marked, such as deposits of glycogen and lipofuscin granules in the cytoplasm, but the most prominent finding was giant vacuoles containing degraded mitochondria, glycogen granules, myofibrils, and myelin-like structures (autophagosomes). This case may belong to one of the unclassified cardiomyopathies characterized by prominent autophagic vacuoles.

Aged↗
PubMed2004

Percutaneous sonographically guided radiofrequency ablation with artificial pleural effusion for hepatocellular carcinoma located under the diaphragm.

OBJECTIVE: Sonographically guided radiofrequency ablation is usually one of the most effective treatments for hepatocellular carcinoma. However, the localization of the tumor is a major limiting factor in the use of a sonographically guided procedure. In our experience, sonographic examination with artificial pleural effusion has improved the visualization of hepatocellular carcinomas under the diaphragm. We investigated the safety, benefits, and local efficacy of radiofrequency ablation with artificial pleural effusion. SUBJECTS AND METHODS: Twenty-five lesions in 23 patients were treated using radiofrequency ablation with artificial pleural effusion, for which 5% glucose solution was injected into the pleural cavity. RESULTS: Artificial pleural effusion allowed us to visualize the whole tumor on gray-scale sonography in 22 lesions that were not detectable or were poorly visible and to obtain a safer and easier puncture line in 14 lesions. In 23 (92%) of the 25 lesions, artificial pleural effusion was helpful in performing percutaneous radiofrequency ablation. Complete necrosis after radiofrequency ablation was obtained in 22 (88%) of the 25 lesions. During a mean (+/- SD) follow-up period of 10.6 +/- 6.5 months, local recurrence at the ablation site was diagnosed in only one (4.5%) of the 22 lesions. Mild cough in three patients and mild dyspnea in two patients were observed as adverse effects of artificial pleural effusion, but these effects were temporary. Oxygen saturation of the blood during artificial pleural effusion was slightly decreased. CONCLUSION: Radiofrequency ablation with artificial pleural effusion is a safe and beneficial treatment option that offers excellent local control through visualization of hepato-cellular carcinomas under the diaphragm.

Aged↗
PubMed2003

Qualitative assessment of tumor vascularity in hepatocellular carcinoma by contrast-enhanced coded ultrasound: comparison with arterial phase of dynamic CT and conventional color/power Doppler ultrasound.

The purpose of this study was to evaluate the detection rate of tumor vessels and vascularity in hepatocellular carcinoma (HCC) by contrast-enhanced coded US using Levovist, and to compare with conventional color/power Doppler US (CDUS) and dynamic CT. Ninety nodules (72 hypo/isoechoic nodules, 18 hyperechoic nodules) in 61 patients were studied. We observed tumor vessels by continuous transmission at the early vascular phase (40 s following administration of Levovist) and vascularity by intermittent transmission (intervals of 2-3 s) at the late vascular phase (40 to approximately 120 s). The detection rate of tumor vessels at the early vascular phase was 97% in hypo/isoechoic nodules and 70% in hyperechoic nodules with high density in dynamic CT being higher than that by CDUS. Tumor vascularity at the late vascular phase in hypo/isoechoic and hyperechoic nodules was hyper-enhancement in 78 and 40%, iso-enhancement in 19 and 40%, and hypo-enhancement in 3 and 0%, respectively. The detection rates of tumor vessels and vascularity in hyperechoic nodules were similar to those by CDUS. The detection rates of tumor vessels and vascularity were not affected by the tumor size in HCC tumors with high density in dynamic CT. Contrast-enhanced US with Levovist was superior to CDUS and equal to dynamic CT to assess tumor vessels in hypo/isoechoic nodules. Although it was equal to CDUS for hyperechoic nodules, this modality is useful in evaluating tumor hemodynamics.

Adult↗
PubMed2003

Inhibition of granulation tissue cell apoptosis during the subacute stage of myocardial infarction improves cardiac remodeling and dysfunction at the chronic stage.

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.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Postinfarction treatment with an adenoviral vector expressing hepatocyte growth factor relieves chronic left ventricular remodeling and dysfunction in mice.

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.

Adenoviridae↗
PubMed2003

Myocytes positive for in situ markers for DNA breaks in human hearts which are hypertrophic, but neither failed nor dilated: a manifestation of cardiac hypertrophy rather than failure.

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.

Adult↗
PubMed2003

Hepatocellular carcinoma with sarcomatous change arising after radiofrequency ablation for well-differentiated hepatocellular carcinoma.

We report a case involving well-differentiated hepatocellular carcinoma (HCC) developing to HCC with sarcomatous changes after radiofrequency ablation (RFA). In a cirrhotic patient with both hepatitis B surface antigen and hepatitis C virus RNA, a well-differentiated HCC with a diameter of 2 cm was detected in segment IV of the liver. A combination of transcatheter arterial embolization and percutaneous ethanol injection (PEI) was performed and, after 8 months, PEI was performed for recurrent tumors. Fifteen months after the first treatment, a recurrent tumor measuring 3.5 cm in diameter was detected in segment IV, which was demonstrated as well-differentiated HCC by tumor biopsy, and treated by RFA. Although the treated lesion was reduced to 2.5 cm in diameter 6 months after RFA, the tumor rapidly enlarged to 6 cm in diameter 2 months later and progressed to lymph node metastasis. Aspiration biopsy showed spindle-shaped sarcomatoid cells with positive staining for both vimentin and keratin. The patient died of HCC progression 10 months after RFA. Autopsy findings showed both sarcomatoid cells and trabecular HCC cells. The sarcomatoid cells had metastasized to the lymph nodes and distant organs. This is the first case illustrating a sarcomatous HCC after RFA. Of interest, RFA may be related to the development of sarcomatous HCC.

Journal Article↗
PubMed2002

Sensitivity to apoptosis signal, clearance rate, and ultrastructure of fas ligand-induced apoptosis in in vivo adult cardiac cells.

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.

Animals↗
PubMed2002

Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes.

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.

Animals↗