Rumi Maruyama

Preventive Effect of Erythropoietin on Cardiac Dysfunction in Doxorubicin-Induced Cardiomyopathy

Background— Doxorubicin is a highly effective antineoplastic drug, but its clinical use is limited by its adverse side effects on the heart. We investigated possible protective effects of erythropoietin against doxorubicin-induced cardiomyopathy. Methods and Results— Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, human recombinant erythropoietin (5000 U/kg) was started simultaneously. Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by erythropoietin treatment. Erythropoietin also protected hearts against doxorubicin-induced cardiomyocyte atrophy and degeneration, myocardial fibrosis, inflammatory cell infiltration, and downregulation of expression of GATA-4 and 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin. Cyclooxygenase-2 expression was upregulated in doxorubicin-treated hearts, and that, too, was attenuated by erythropoietin. No doxorubicin-induced apoptotic effects were seen, nor were any changes seen in the expression of tumor necrosis factor-&agr; or transforming growth factor-&bgr;1. Antiatrophic and GATA-4 restoring effects of erythropoietin were demonstrated in the in vitro experiments with cultured cardiomyocytes exposed to doxorubicin, which indicated the direct cardioprotective effects of erythropoietin beyond erythropoiesis. Cardiac erythropoietin receptor expression was downregulated in doxorubicin-induced cardiomyopathy but was restored by erythropoietin. Among the downstream mediators of erythropoietin receptor signaling, activation of extracellular signal-regulated kinase was reduced by doxorubicin but restored by erythropoietin. By contrast, erythropoietin was ineffective when administered after cardiac dysfunction was established in the chronic stage. Conclusions— The present study indicates a protective effect of erythropoietin against doxorubicin-induced cardiomyopathy.

Postinfarction Gene Therapy Against Transforming Growth Factor-β Signal Modulates Infarct Tissue Dynamics and Attenuates Left Ventricular Remodeling and Heart Failure

Background—Fibrosis and progressive failure are prominent pathophysiological features of hearts after myocardial infarction (MI). We examined the effects of inhibiting transforming growth factor-β (TGF-β) signaling on post-MI cardiac fibrosis and ventricular remodeling and function. Methods and Results—MI was induced in mice by left coronary artery ligation. An adenovirus harboring soluble TGF-β type II receptor (Ad.CAG-sTβRII), a competitive inhibitor of TGF-β, was then injected into the hindlimb muscles on day 3 after MI (control, Ad.CAG-LacZ). Post-MI survival was significantly improved among sTβRII-treated mice (96% versus control at 71%), which also showed a significant attenuation of ventricular dilatation and improved function 4 weeks after MI. At the same time, histological analysis showed reduced fibrous tissue formation. Although MI size did not differ in the 2 groups, MI thickness was greater and circumference was smaller in the sTβRII-treated group; within the infarcted area, α-smooth muscle actin–positive cells were abundant, which might have contributed to infarct contraction. Apoptosis among myofibroblasts in granulation tissue during the subacute stage (10 days after MI) was less frequent in the sTβRII-treated group, and sTβRII directly inhibited Fas-induced apoptosis in cultured myofibroblasts. Finally, treatment of MI-bearing mice with sTβRII was ineffective if started during the chronic stage (4 weeks after MI). Conclusions—Postinfarction gene therapy aimed at suppressing TGF-β signaling mitigates cardiac remodeling by affecting cardiac fibrosis and infarct tissue dynamics (apoptosis inhibition and infarct contraction). This suggests that such therapy may represent a new approach to the treatment of post-MI heart failure, applicable during the subacute stage.

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.

The role of autophagy emerging in postinfarction cardiac remodelling

AIMS Autophagy is activated in cardiomyocytes in ischaemic heart disease, but its dynamics and functional roles remain unclear after myocardial infarction. We observed the dynamics of cardiomyocyte autophagy and examined its role during postinfarction cardiac remodelling. METHODS AND RESULTS Myocardial infarction was induced in mice by ligating the left coronary artery. During both the subacute and chronic stages (1 and 3 weeks postinfarction, respectively), autophagy was found to be activated in surviving cardiomyocytes, as demonstrated by the up-regulated expression of microtubule-associated protein-1 light chain 3-II (LC3-II), p62 and cathepsin D, and by electron microscopic findings. Activation of autophagy, specifically the digestion step, was prominent in cardiomyocytes 1 week postinfarction, especially in those bordering the infarct area, while the formation of autophagosomes was prominent 3 weeks postinfarction. Bafilomycin A1 (an autophagy inhibitor) significantly aggravated postinfarction cardiac dysfunction and remodelling. Cardiac hypertrophy was exacerbated in this group and was accompanied by augmented ventricular expression of atrial natriuretic peptide. In these hearts, autophagic findings (i.e. expression of LC3-II and the presence of autophagosomes) were diminished, and activation of AMP-activated protein kinase was enhanced. Treatment with rapamycin (an autophagy enhancer) brought about opposite outcomes, including mitigation of cardiac dysfunction and adverse remodelling. A combined treatment with bafilomycin A1 and rapamycin offset each effect on cardiomyocyte autophagy and cardiac remodelling in the postinfarction heart. CONCLUSION These findings suggest that cardiomyocyte autophagy is an innate mechanism that protects against progression of postinfarction cardiac remodelling, implying that augmenting autophagy could be a therapeutic strategy.

Critical Roles for the Fas/Fas Ligand System in Postinfarction Ventricular Remodeling and Heart Failure

In myocardial infarction (MI), granulation tissue cells disappear via apoptosis to complete a final scarring with scanty cells. Blockade of this apoptosis was reported to improve post-MI ventricular remodeling and heart failure. However, the molecular biological mechanisms for the apoptosis are unknown. Fas and Fas ligand were overexpressed in the granulation tissue at the subacute stage of MI (1 week after MI) in mice, where apoptosis frequently occurred. In mice lacking functioning Fas (lpr strain) and in those lacking Fas ligand (gld strain), apoptotic rate of granulation tissue cells was significantly fewer compared with that of genetically controlled mice, and post-MI ventricular remodeling and dysfunction were greatly attenuated. Mice were transfected with adenovirus encoding soluble Fas (sFas), a competitive inhibitor of Fas ligand, on the third day of MI. The treatment resulted in suppression of granulation tissue cell apoptosis and produced a thick, cell-rich infarct scar containing rich vessels and bundles of smooth muscle cells with a contractile phenotype at the chronic stage (4 weeks after MI). This accompanied not only alleviation of heart failure but also survival improvement. However, the sFas gene delivery during scar tissue phase was ineffective, suggesting that beneficial effects of the sFas gene therapy owes to inhibition of granulation tissue cell apoptosis. The Fas/Fas ligand interaction plays a critical role for granulation tissue cell apoptosis after MI. Blockade of this apoptosis by interfering with the Fas/Fas ligand interaction may become one of the therapeutic strategies against chronic heart failure after large MI.

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 μmol/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 &agr;‐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. (Circulation. 2003;108:104‐109.)

Autophagy limits acute myocardial infarction induced by permanent coronary artery occlusion

Ischemia is known to potently stimulate autophagy in the heart, which may contribute to cardiomyocyte survival. In vitro, transfection with small interfering RNAs targeting Atg5 or Lamp-2 (an autophagy-related gene necessary, respectively, for the initiation and digestion step of autophagy), which specifically inhibited autophagy, diminished survival among cultured cardiomyocytes subjected to anoxia and significantly reduced their ATP content, confirming an autophagy-mediated protective effect against anoxia. We next examined the dynamics of cardiomyocyte autophagy and the effects of manipulating autophagy during acute myocardial infarction in vivo. Myocardial infarction was induced by permanent ligation of the left coronary artery in green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) transgenic mice in which GFP-LC3 aggregates to be visible in the cytoplasm when autophagy is activated. Autophagy was rapidly (within 30 min after coronary ligation) activated in cardiomyocytes, and autophagic activity was particularly strong in salvaged cardiomyocytes bordering the infarcted area. Treatment with bafilomycin A1, an autophagy inhibitor, significantly increased infarct size (31% expansion) 24 h postinfarction. Interestingly, acute infarct size was significantly reduced (23% reduction) in starved mice showing prominent autophagy before infarction. Treatment with bafilomycin A1 reduced postinfarction myocardial ATP content, whereas starvation increased myocardial levels of amino acids and ATP, and the combined effects of bafilomycin A1 and starvation on acute infarct size offset one another. The present findings suggest that autophagy is an innate and potent process that protects cardiomyocytes from ischemic death during acute myocardial infarction.

Autophagic Degeneration and Death of Cardiomyocytes in Heart Failure

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 andcell 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 autophagic findings, an increase in cardiomyocyte size, and a reduction in myocardial fibrosis. G-CSF inducedchanges in molecular signaling included activation of Akt and 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. By contrast, neither cardiomyocyte apoptosis nor regeneration of cardiomyocytes from bone marrowderived cells was significant. It thus appears that autophagic death is an importantcontributor to cardiomyocyte loss in the cardiomyopathic hamster and that G-CSF exerts a beneficial effect, mainly via an antiautophagic mechanism. Addendum to: Autophagic Cardiomyocyte Death in Cardiomyopathic Hamsters and Its Prevention by Granulocyte Colony-Stimulating Factor S. Miyata, G. Takemura, Y. Kawase, Y. Li, H. Okada, R. Maruyama, H. Ushikoshi, M. Esaki, H. Kanamori, L. Li, Y. Misao, A. Tezuka, T. Toyo-Oka, S. Minatoguchi, T. Fujiwara and H. Fujiwara Am J Pathol 2006; 168:386-97

Induction of apoptosis by gallic acid in lung cancer cells.

The apoptosis-inducing effect of gallic acid (3,4,5-trihydroxybenzoic acid) was investigated in four human lung cancer cell lines, SBC-3 (small cell carcinoma), EBC-1 (squamous cell carcinoma), A549 (adenocarcinoma) and SBC-3/CDDP (cisplatin-resistant subclone of SBC-3). Gallic acid induced apoptosis in a dose-dependent manner as evidenced by analyses of DNA fragmentation, changes in cell morphology and loss of viability. Fifty percent inhibitory concentration (IC50) values of gallic acid on the cell viability of SBC-3, EBC-1 and A549 were around 10, 20 and 60 microg/ml, respectively. The IC50 value for SBC-3/CDDP cells was almost the same as that of SBC-3, suggesting that susceptibility of cells to gallic acid-induced apoptosis is not altered by the acquisition of cisplatin resistance. The apoptotic process was effectively triggered by 30 min exposure to gallic acid. A caspase inhibitor and alpha-tocopherol effectively prevented the gallic acid-induced apoptosis, indicating the involvememt of caspase activation and oxidative processes during the course of apoptosis in gallic acid-treated cancer cells. These findings suggest the possible applicability of gallic acid in lung cancer therapy, especially to circumvent resistance to anti-cancer drugs.

Prior starvation mitigates acute doxorubicin cardiotoxicity through restoration of autophagy in affected cardiomyocytes

AIMS Active autophagy has recently been reported in doxorubicin-induced cardiotoxicity; here we investigated its pathophysiological role. METHODS AND RESULTS Acute cardiotoxicity was induced in green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) transgenic mice by administering two intraperitoneal injections of 10 mg/kg doxorubicin with a 3 day interval. A starvation group was deprived of food for 48 h before each injection to induce autophagy in advance. Doxorubicin treatment caused left ventricular dilatation and dysfunction within 6 days. Cardiomyocyte autophagy appeared to be activated in the doxorubicin group, based on LC3, p62, and cathepsin D expression, while it seemed somewhat diminished by starvation prior to doxorubicin treatment. Unexpectedly, however, myocardial ATP levels were reduced in the doxorubicin group, and this reduction was prevented by earlier starvation. Electron microscopy revealed that the autophagic process was indeed initiated in the doxorubicin group, as shown by the increased lysosomes, but was not completed, i.e. autophagolysosome formation was rare. Starvation prior to doxorubicin treatment partly restored autophagosome formation towards control levels. Autophagic flux assays in both in vivo and in vitro models confirmed that doxorubicin impairs completion of the autophagic process in cardiomyocytes. The activities of both AMP-activated protein kinase and the autophagy-initiating kinase unc-51-like kinase 1 (ULK1) were found to be decreased by doxorubicin, and these were restored by prior starvation. CONCLUSION Prior starvation mitigates acute doxorubicin cardiotoxicity; the underlying mechanism may be, at least in part, restoration and further augmentation of myocardial autophagy, which is impaired by doxorubicin, probably through inactivation of AMP-activated protein kinase and ULK1.