Tomonori Kawaguchi

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.

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.

Functional Significance and Morphological Characterization of Starvation-Induced Autophagy in the Adult Heart

To examine the functional significance and morphological characteristics of starvation-induced autophagy in the adult heart, we made green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) transgenic mice starve for up to 3 days. Electron microscopy revealed round, homogenous, electron-dense lipid droplet-like vacuoles that initially appeared in cardiomyocytes as early as 12 hours after starvation; these vacuoles were identified as lysosomes based on cathepsin D-immunopositive reactivity and acid phosphatase activity. The increase in the number of lysosomes depended on the starvation interval; typical autophagolysosomes with intracellular organelles also appeared, and their numbers increased at the later phases of starvation. Myocardial expression of autophagy-related proteins, LC3-II, cathepsin D, and ubiquitin, increased, whereas both myocardial ATP content and starvation integral decreased. Treatment with bafilomycin A1, an autophagy inhibitor, did not affect cardiac function in normally fed mice but significantly depressed cardiac function and caused significant left ventricular dilatation in mice starved for 3 days. The cardiomyocytes were occupied with markedly accumulated lysosomes in starved mice treated with bafilomycin A1, and both the myocardial amino acid content, which was increased during starvation, and the myocardial ATP content were severely decreased, potentially contributing to cardiac dysfunction. The present findings suggest a critical role of autophagy in the maintenance of cardiac function during starvation in the adult.

Resveratrol Reverses Remodeling in Hearts with Large, Old Myocardial Infarctions through Enhanced Autophagy-Activating AMP Kinase Pathway

We investigated the effect of resveratrol, a popular natural polyphenolic compound with antioxidant and proautophagic actions, on postinfarction heart failure. Myocardial infarction was induced in mice by left coronary artery ligation. Four weeks postinfarction, when heart failure was established, the surviving mice were started on 2-week treatments with one of the following: vehicle, low- or high-dose resveratrol (5 or 50 mg/kg/day, respectively), chloroquine (an autophagy inhibitor), or high-dose resveratrol plus chloroquine. High-dose resveratrol partially reversed left ventricular dilation (reverse remodeling) and significantly improved cardiac function. Autophagy was augmented in those hearts, as indicated by up-regulation of myocardial microtubule-associated protein-1 light chain 3-II, ATP content, and autophagic vacuoles. The activities of AMP-activated protein kinase and silent information regulator-1 were enhanced in hearts treated with resveratrol, whereas Akt activity and manganese superoxide dismutase expression were unchanged, and the activities of mammalian target of rapamycin and p70 S6 kinase were suppressed. Chloroquine elicited opposite results, including exacerbation of cardiac remodeling associated with a reduction in autophagic activity. When resveratrol and chloroquine were administered together, the effects offset one another. In vitro, compound C (AMP-activated protein kinase inhibitor) suppressed resveratrol-induced autophagy in cardiomyocytes, but did not affect the events evoked by chloroquine. In conclusion, resveratrol is a beneficial pharmacological tool that augments autophagy to bring about reverse remodeling in the postinfarction heart.

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.

Anti-Fas Gene Therapy Prevents Doxorubicin-Induced Acute Cardiotoxicity through Mechanisms Independent of Apoptosis

Activation of Fas signaling is a key mediator of doxorubicin cardiotoxicity, which involves both cardiomyocyte apoptosis and myocardial inflammation. In this study, acute cardiotoxicity was induced in mice by doxorubicin, and some mice simultaneously received an intramuscular injection of adenoviral vector encoding mouse soluble Fas (sFas) gene (Ad.CAG-sFas), an inhibitor of Fas/Fas ligand interaction. Two weeks later, left ventricular dilatation and dysfunction were apparent in the LacZ-treated control group, but both were significantly mitigated in the sFas-treated group. The in situ nick-end labeling-positive rate were similar in the two groups, and although electron microscopy revealed cardiomyocyte degeneration, no apoptotic structural features and no activation of caspases were detected, suggesting an insignificant role of apoptosis in this model. Instead, sFas treatment reversed doxorubicin-induced down-regulation of GATA-4 and attenuated ubiquitination of myosin heavy chain and troponin I to preserve these sarcomeric proteins. In addition, doxorubicin-induced significant leukocyte infiltration, fibrosis, and oxidative damage to the myocardium, all of which were largely reversed by sFas treatment. sFas treatment also suppressed doxorubicin-induced p53 overexpression, phosphorylation of c-Jun N-terminal kinase, c-Jun, and inhibitor of nuclear factor-kappaB, as well as production of cyclooxygenase-2 and monocyte chemoattractant protein-1, and it restored extracellular signal-regulated kinase activation. Therefore, sFas gene therapy prevents the progression of doxorubicin-induced acute cardiotoxicity, with accompanying attenuation of the cardiomyocyte degeneration, inflammation, fibrosis, and oxidative damage caused by Fas signaling.

Erythropoietin Receptor Signaling Mitigates Renal Dysfunction-Associated Heart Failure by Mechanisms Unrelated to Relief of Anemia

OBJECTIVES We examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure. BACKGROUND Although EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects. METHODS Mice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks. RESULTS Although only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice. CONCLUSIONS EPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia.

Postinfarction gene therapy with adenoviral vector expressing decorin mitigates cardiac remodeling and dysfunction

The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-beta (TGF-beta) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorins chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-beta1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-beta signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.

Restriction of food intake prevents postinfarction heart failure by enhancing autophagy in the surviving cardiomyocytes.

We investigated the effect of restriction of food intake, a potent inducer of autophagy, on postinfarction cardiac remodeling and dysfunction. Myocardial infarction was induced in mice by left coronary artery ligation. At 1 week after infarction, mice were randomly divided into four groups: the control group was fed ad libitum (100%); the food restriction (FR) groups were fed 80%, 60%, or 40% of the mean amount of food consumed by the control mice. After 2 weeks on the respective diets, left ventricular dilatation and hypofunction were apparent in the control group, but both parameters were significantly mitigated in the FR groups, with the 60% FR group showing the strongest therapeutic effect. Cardiomyocyte autophagy was strongly activated in the FR groups, as indicated by up-regulation of microtubule-associated protein 1 light chain 3-II, autophagosome formation, and myocardial ATP content. Chloroquine, an autophagy inhibitor, completely canceled the therapeutic effect of FR. This negative effect was associated with reduced activation of AMP-activated protein kinase and of ULK1 (a homolog of yeast Atg1), both of which were enhanced in hearts from the FR group. In vitro, the AMP-activated protein kinase inhibitor compound C suppressed glucose depletion-induced autophagy in cardiomyocytes, but did not influence activity of chloroquine. Our findings imply that a dietary protocol with FR could be a preventive strategy against postinfarction heart failure.

Asialoerythropoietin, a Nonerythropoietic Derivative of Erythropoietin, Displays Broad Anti-Heart Failure Activity

Background—We investigated the effects of asialoerythropoietin (asialoEPO), a nonerythrogenic erythropoietin derivative, on 3 murine models of heart failure with different etiologies. Methods and Results—Doxorubicin (15 mg/kg) induced heart failure within 2 weeks (toxic cardiomyopathy). Treatment with asialoEPO (6.9 &mgr;g/kg) for 2 weeks thereafter attenuated the associated left ventricular dysfunction and dilatation. In addition, the asialoEPO-treated heart showed less myocardial fibrosis, inflammation, and oxidative damage, and diminished atrophic cardiomyocyte degeneration, which was accompanied by restored expression of GATA-4 and sarcomeric proteins. Mice with large 6-week-old myocardial infarctions exhibited marked left ventricular dysfunction with adverse remodeling (ischemic cardiomyopathy). AsialoEPO treatment for 4 weeks significantly mitigated progression of the dysfunction and remodeling and reduced myocardial fibrosis, inflammation, and oxidative damage. Finally, 25-week-old &dgr;-sarcoglycan-deficient mice (genetic cardiomyopathy) were treated with asialoEPO for 5 weeks. AsialoEPO mitigated the progressive cardiac remodeling and dysfunction through cardiomyocyte hypertrophy, and upregulated expression of GATA-4 and sarcomeric proteins. AsialoEPO appears to act by altering the activity of the downstream erythropoietin receptor signals extracellular signal-regulated protein kinase, Akt, signal transducer, and activator of transcription 3 and 5 in a model-specific manner. Conclusions—The findings suggest that asialoEPO exerts broad cardioprotective effects through distinct mechanisms depending on the model, which are independent of the erythrogenic action. This compound may be promising for the treatment of heart failure of various etiologies.