Takatomo Watanabe

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.

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.

Left atrial function assessed by speckle tracking echocardiography as a predictor of new-onset non-valvular atrial fibrillation: results from a prospective study in 580 adults

AIMS The aim of this prospective study was to evaluate left atrial (LA) function for the prediction of increased risk of new-onset non-valvular atrial fibrillation (AF). Risk stratification for new-onset AF based on LA remodelling may have a major public health impact. Although left atrial volume (LAV) or LA dimension have been proposed as predictors of AF, other predictive parameters of LA function have not yet been fully examined. METHODS AND RESULTS LA emptying function (EF), strain rate (SR), and LAV were evaluated in the apical four-chamber view by speckle tracking echocardiography in 580 consecutive adults (age 64 ± 17, 303 men) without a history of atrial arrhythmias. During a follow-up period of 28 months, 32 subjects (age 73 ± 9, 18 men) developed electrocardiographically confirmed AF. Subjects with new-onset AF had lower LA active EF (16 ± 5 vs. 28 ± 8%, P< 0.001) and lower LA SR at atrial contraction (-0.9 ± 0.2 vs. -1.4 ± 0.5 S(-1), P< 0.001), but larger maximum LAV index (59 ± 12 vs. 46 ± 16 mL/m(2), P< 0.001) compared with non-AF subjects at baseline. In multivariate logistic regression analysis, LA active EF was the only independent predictor of new-onset AF. Using an LA active EF cut-off of ≤20%, the sensitivity and specificity for new-onset AF based on receiver operator characteristic curve analysis were 88 and 81%, respectively (area under the curve: 0.92). CONCLUSION Reduced LA active EF (booster pump function) assessed by speckle tracking echocardiography independently predicts the risk of new-onset AF, suggesting a stronger association between LA functional remodelling and AF than between LA size and AF.

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.

Left atrial global and regional function in patients with paroxysmal atrial fibrillation has already been impaired before enlargement of left atrium: velocity vector imaging echocardiography study.

AIMS Left atrial volume (LAV) has been proposed as a predictor of atrial fibrillation (AF) and LA function has been investigated by velocity vector imaging (VVI) echocardiography. The aim of this study was to determine whether LA function was associated with the existence of AF. METHODS AND RESULTS We examined emptying function (EF) as a global function and strain rate (SR) as a regional function of LA function during three phases of the cardiac cycle (reservoir, conduit, and booster pump phase). The parameters were measured (apical four-chamber view) by VVI in 302 subjects [126 sinus rhythm, 91 paroxysmal AF (PAF), and 85 chronic AF]. Global and regional LA function were significantly lower in PAF patients during sinus rhythm (LA total EF: 35 ± 8%; SR at atrial contraction: -0.8 ± 0.3s(-1)) and much lower in chronic AF patients (LA total EF 22 ± 8%) than in subjects with sinus rhythm (LA total EF: 47 ± 7%; SR at atrial contraction: -1.4 ± 0.4s(-1)). In multivariate logistic regression analysis, LA active EF and SR at atrial contraction were independent features of PAF. CONCLUSION LA function, particularly LA active relaxation and contraction, was lower in PAF patients than in subjects with sinus rhythm, regardless of LA size and age. LA functional impairment was observed regardless of hypertension before LA enlargement in patients with PAF. Reduced LA function, as assessed by VVI, is an important feature of AF as well as LA structure.

Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes

Little is known about the association between autophagy and diabetic cardiomyopathy. Also unknown are possible distinguishing features of cardiac autophagy in type 1 and type 2 diabetes. In hearts from streptozotocin-induced type 1 diabetic mice, diastolic function was impaired, though autophagic activity was significantly increased, as evidenced by increases in microtubule-associated protein 1 light chain 3/LC3 and LC3-II/-I ratios, SQSTM1/p62 (sequestosome 1) and CTSD (cathepsin D), and by the abundance of autophagic vacuoles and lysosomes detected electron-microscopically. AMP-activated protein kinase (AMPK) was activated and ATP content was reduced in type 1 diabetic hearts. Treatment with chloroquine, an autophagy inhibitor, worsened cardiac performance in type 1 diabetes. In addition, hearts from db/db type 2 diabetic model mice exhibited poorer diastolic function than control hearts from db/+ mice. However, levels of LC3-II, SQSTM1 and phosphorylated MTOR (mechanistic target of rapamycin) were increased, but CTSD was decreased and very few lysosomes were detected ultrastructurally, despite the abundance of autophagic vacuoles. AMPK activity was suppressed and ATP content was reduced in type 2 diabetic hearts. These findings suggest the autophagic process is suppressed at the final digestion step in type 2 diabetic hearts. Resveratrol, an autophagy enhancer, mitigated diastolic dysfunction, while chloroquine had the opposite effects in type 2 diabetic hearts. Autophagy in the heart is enhanced in type 1 diabetes, but is suppressed in type 2 diabetes. This difference provides important insight into the pathophysiology of diabetic cardiomyopathy, which is essential for the development of new treatment strategies.

Quantitative validation of left atrial structure and function by two-dimensional and three-dimensional speckle tracking echocardiography: a comparative study with three-dimensional computed tomography.

BACKGROUND The aim of this study was to validate the accuracy of three-dimensional (3D) speckle tracking echocardiography (STE) and two-dimensional (2D)-STE for the assessment of left atrial (LA) volume and function by comparison with 3D-computed tomography (CT) performed on the same day as STE. METHODS LA phasic volume and emptying function (EF) were measured in 28 patients with paroxysmal atrial fibrillation undergoing catheter ablation (62±11 years old) using both 3D-STE and 2D-STE during sinus rhythm. LA phasic volume and function measured by 3D-STE and 2D-STE were validated using 3D-CT as a gold standard. RESULTS The intraobserver correlation coefficient and variability in maximum LA volume assessed by 3D-STE were 0.99 and 1.4±6.0%, respectively. The interobserver correlation coefficient and variability in maximum LA volume assessed by 3D-STE were 0.99 and 0.2±4.5%, respectively. There were strong correlations between LA phasic volume measured by 3D-CT and those measured by 3D-STE (r=0.98, p<0.001). There were correlations between LA phasic function measured by 3D-CT and those measured by 3D-STE (r=0.85-0.88, p<0.001). There was a better agreement between 3D-CT and 3D-STE in the assessment of LA phasic volumes and function than between 3D-CT and 2D-STE in apical 2- and 4-chamber view. CONCLUSIONS 3D-STE allows more accurate measurement of LA volume and function than 2D-STE and has high reproducibility.

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.

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.