Maki Katamura

p53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia

Inhibition of tumor suppressor p53 is cardioprotective against ischemic injury and provides resistance to subsequent cardiac remodeling. We investigated p53-mediated expansion of ischemic damage with a focus on mitochondrial integrity in association with autophagy and apoptosis. p53(-/-) heart showed that autophagic flux was promoted under ischemia without a change in cardiac tissue ATP content. Electron micrographs revealed that ischemic border zone in p53(-/-) mice had 5-fold greater numbers of autophagic vacuoles containing mitochondria, indicating the occurrence of mitophagy, with an apparent reduction of abnormal mitochondria compared with those in WT mice. Analysis of autophagic mediators acting downstream of p53 revealed that TIGAR (TP53-induced glycolysis and apoptosis regulator) was exclusively up-regulated in ischemic myocardium. TIGAR(-/-) mice exhibited the promotion of mitophagy followed by decrease of abnormal mitochondria and resistance to ischemic injury, consistent with the phenotype of p53(-/-) mice. In p53(-/-) and TIGAR(-/-) ischemic myocardium, ROS production was elevated and followed by Bnip3 activation which is an initiator of mitophagy. Furthermore, the activation of Bnip3 and mitophagy due to p53/TIGAR inhibition were reversed with antioxidant N-acetyl-cysteine, indicating that this adaptive response requires ROS signal. Inhibition of mitophagy using chloroquine in p53(-/-) or TIGAR(-/-) mice exacerbated accumulation of damaged mitochondria to the level of wild-type mice and attenuated cardioprotective action. These findings indicate that p53/TIGAR-mediated inhibition of myocyte mitophagy is responsible for impairment of mitochondrial integrity and subsequent apoptosis, the process of which is closely involved in p53-mediated ventricular remodeling after myocardial infarction.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts

OBJECTIVES The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

p53 Promotes Cardiac Dysfunction in Diabetic Mellitus Caused by Excessive Mitochondrial Respiration-Mediated Reactive Oxygen Species Generation and Lipid Accumulation

Background— Diabetic cardiomyopathy is characterized by energetic dysregulation caused by glucotoxicity, lipotoxicity, and mitochondrial alterations. p53 and its downstream mitochondrial assembly protein, synthesis of cytochrome c oxidase 2 (SCO2), are important regulators of mitochondrial respiration, whereas the involvement in diabetic cardiomyopathy remains to be determined. Methods and Results— The role of p53 and SCO2 in energy metabolism was examined in both type I (streptozotocin [STZ] administration) and type II diabetic (db/db) mice. Cardiac expressions of p53 and SCO2 in 4-week STZ diabetic mice were upregulated (185% and 152% versus controls, respectively, P<0.01), with a marked decrease in cardiac performance. Mitochondrial oxygen consumption was increased (136% versus control, P<0.01) in parallel with augmentation of mitochondrial cytochrome c oxidase (complex IV) activity. Reactive oxygen species (ROS)-damaged myocytes and lipid accumulation were increased in association with membrane-localization of fatty acid translocase protein FAT/CD36. Antioxidant tempol reduced the increased expressions of p53 and SCO2 in STZ-diabetic hearts and normalized alterations in mitochondrial oxygen consumption, lipid accumulation, and cardiac dysfunction. Similar results were observed in db/db mice, whereas in p53-deficient or SCO2-deficient diabetic mice, the cardiac and metabolic abnormalities were prevented. Overexpression of SCO2 in cardiac myocytes increased mitochondrial ROS and fatty acid accumulation, whereas knockdown of SCO2 ameliorated them. Conclusions— Myocardial p53/SCO2 signal is activated by diabetes-mediated ROS generation to increase mitochondrial oxygen consumption, resulting in excessive generation of mitochondria-derived ROS and lipid accumulation in association with cardiac dysfunction.

p53 and TIGAR regulate cardiac myocyte energy homeostasis under hypoxic stress

Bioenergetic homeostasis is altered in heart failure and may play an important role in pathogenesis. p53 has been implicated in heart failure, and although its role in regulating tumorigenesis is well characterized, its activities on cellular metabolism are just beginning to be understood. We investigated the role of p53 and its transcriptional target gene TP53-induced glycolysis and apoptosis regulator (TIGAR) in myocardial energy metabolism under conditions simulating ischemia that can lead to heart failure. Expression of p53 and TIGAR was markedly upregulated after myocardial infarction, and apoptotic myocytes were decreased by 42% in p53-deficient mouse hearts compared with those in wild-type mice. To examine the effect of p53 on energy metabolism, cardiac myocytes were exposed to hypoxia. Hypoxia induced p53 and TIGAR expression in a p53-dependent manner. Knockdown of p53 or TIGAR increased glycolysis with elevated fructose-2,6-bisphosphate levels and reduced myocyte apoptosis. Hypoxic stress decreased phosphocreatine content and the mitochondrial membrane potential of myocytes without changes in ATP content, the effects of which were prevented by the knockdown of TIGAR. Inhibition of glycolysis by 2-deoxyglucose blocked these bioenergetic effects and TIGAR siRNA-mediated prevention of apoptosis, and, in contrast, overexpression of TIGAR reduced glucose utilization and increased apoptosis. Our data demonstrate that p53 and TIGAR inhibit glycolysis in hypoxic myocytes and that inhibition of glycolysis is closely involved in apoptosis, suggesting that p53 and TIGAR are significant mediators of cellular energy homeostasis and cell death under ischemic stress.

p53 Promotes Cardiac Dysfunction in Diabetic Mellitus Due to Excessive Mitochondrial Respiration-Mediated ROS Generation and Lipid Accumulation

Background— Diabetic cardiomyopathy is characterized by energetic dysregulation caused by glucotoxicity, lipotoxicity, and mitochondrial alterations. p53 and its downstream mitochondrial assembly protein, synthesis of cytochrome c oxidase 2 (SCO2), are important regulators of mitochondrial respiration, whereas the involvement in diabetic cardiomyopathy remains to be determined. Methods and Results— The role of p53 and SCO2 in energy metabolism was examined in both type I (streptozotocin [STZ] administration) and type II diabetic (db/db) mice. Cardiac expressions of p53 and SCO2 in 4-week STZ diabetic mice were upregulated (185% and 152% versus controls, respectively, P<0.01), with a marked decrease in cardiac performance. Mitochondrial oxygen consumption was increased (136% versus control, P<0.01) in parallel with augmentation of mitochondrial cytochrome c oxidase (complex IV) activity. Reactive oxygen species (ROS)-damaged myocytes and lipid accumulation were increased in association with membrane-localization of fatty acid translocase protein FAT/CD36. Antioxidant tempol reduced the increased expressions of p53 and SCO2 in STZ-diabetic hearts and normalized alterations in mitochondrial oxygen consumption, lipid accumulation, and cardiac dysfunction. Similar results were observed in db/db mice, whereas in p53-deficient or SCO2-deficient diabetic mice, the cardiac and metabolic abnormalities were prevented. Overexpression of SCO2 in cardiac myocytes increased mitochondrial ROS and fatty acid accumulation, whereas knockdown of SCO2 ameliorated them. Conclusions— Myocardial p53/SCO2 signal is activated by diabetes-mediated ROS generation to increase mitochondrial oxygen consumption, resulting in excessive generation of mitochondria-derived ROS and lipid accumulation in association with cardiac dysfunction.

Curcumin Attenuates Doxorubicin-Induced Cardiotoxicity by Inducing Autophagy via the Regulation of JNK Phosphorylation

Background and Objective: Doxorubicin (DOX) has been used in cancer therapy for several decades. However, cardiac complications induced by DOX dose-dependently limit the clinical implication at optimal antitumor efficacy. Curcumin (Cur), a natural compound, has been effective as an anticancer agent in various types of cancers. It also protects against cardiac hypertrophy and heart failure, though its effect on cardiomyopathy caused by DOX treatment is unclear. To elucidate the role of curcumin on heart failure, we used the DOX induced cardiomyopathy model and primary cultured cardiac myocytes. Method and Results: Male C57/BL6 mice were randomized to 4 courses of treatment administered for 4 weeks: Phosphate-Buffered Saline (PBS), Cur, DOX, and DOX+Cur. DOX-treated mice exhibited severe cardiac dysfunction, and the mortality was higher than that in PBS-treated mice. In DOX-treated mice, the number of apoptotic cardiac myocytes was higher and the fibrotic areas were larger than in PBS-treated mice. The cardiotoxic effects of DOX were ameliorated by treatment with Cur. In DOX-treated GFP-LC3 transgenic mice, Cur induced autophagy and decreased apoptosis in the heart. Cur also induced autophagy and suppressed DOX-induced apoptosis in neonatal rat cardiac myocytes. Inhibition of autophagy by 3-methyladenine decreased the cardioprotective effect of Cur. Furthermore, Cur decreased c-Jun N-terminal kinase (JNK) phosphorylation, resulting in reduction of apoptosis. The JNK inhibitor SP600125 abolished these effects. Conclusion: Cur protects the heart from DOX-induced cardiotoxicity by inducing autophagy and decreasing cardiomyocyte apoptosis. The mechanism involves JNK-mediated modification of apoptosis. Induction of cardiac autophagy may be a novel therapeutic approach for preventing DOX-induced cardiotoxicity.

Myocardial stretch induced by increased left ventricular diastolic pressure preconditions isolated perfused hearts of normotensive and spontaneously hypertensive rats

Objective: The aim of our study was to determine whether myocardial stretch (non-ischemic stress) could precondition isolated perfused hearts of both normotensive Wister-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).Methods: The perfused hearts in Langendorff mode were subjected to 30 min of global no-flow ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) were measured. In the control group, LVEDP was set at 10 mmHg. In the stretch group, LVEDP was increased to 30 or 60 mmHg for 5 min before 30 min of ischemia. In the ischemic preconditioning group, the hearts were exposed to two cycles of a 5-min period of ischemia before 30 min of ischemia. Myocardial lactate contents were measured at the baseline and at the end of the 60 mmHg stretch.Results: Hemodynamic parameters of LVDP and LVEDP at 30 min of reperfusion improved in the stretch group (LVEDP at 60 mmHg) and the ischemic preconditioning group. Coronary flow did not decrease during the stretch. Recovery of the coronary flow during reperfusion was better in the stretch and ischemic preconditioning groups. Postischemic contractile function was better in WKY rats than in SHR. Myocardial lactate contents at the end of 60 mmHg stretch were negligible. Conclusions: Myocardial stretch induced by increasing LVEDP preconditioned isolated perfused hearts of both WKY rats and SHR, via mechanisms not involving myocardial ischemia during stretch.

An alpha-adrenergic agonist protects hearts by inducing Akt1-mediated autophagy

Alpha-adrenergic agonists is known to be protective in cardiac myocytes from apoptosis induced by beta-adrenergic stimulation. Although there has been a recent focus on the role of cardiac autophagy in heart failure, its role in heart failure with adrenergic overload has not yet been elucidated. In the present study, we investigated the contribution of autophagy to cardiac failure during adrenergic overload both in vitro and in vivo. Neonatal rat cardiac myocytes overexpressing GFP-tagged LC3 were prepared and stimulated with the alpha1-adrenergic agonist, phenylephrine (PE), the beta-adrenergic agonist, isoproterenol (ISO), or norepinephrine (NE) in order to track changes in the formation of autophagosomes in vitro. All adrenergic stimulators increased cardiac autophagy by stimulating autophagic flux. Blocking autophagy by the knockdown of autophagy-related 5 (ATG5) exacerbated ISO-induced apoptosis and negated the anti-apoptotic effects of PE, which indicated the cardioprotective role of autophagy during adrenergic overload. PE-induced cardiac autophagy was mediated by the PI3-kinase/Akt pathway, but not by MEK/ERK, whereas both pathways mediated the anti-apoptotic effects of PE. Knock down of Akt1 was the most essential among the three Akt family members examined for the induction of cardiac autophagy. The four-week administration of PE kept the high level of cardiac autophagy without heart failure in vivo, whereas autophagy levels in a myocardium impaired by four-week persistent administration of ISO or NE were the same with the control state. These present study indicated that cardiac autophagy played a protective role during adrenergic overload and also that the Akt pathway could mediate cardiac autophagy for the anti-apoptotic effects of the alpha-adrenergic pathway.

Effects of glibenclamide and nicorandil in post-ischaemic contractile dysfunction of perfused hearts in normotensive and spontaneously hypertensive rats

Objective We have demonstrated previously that nicorandil, an ATP-sensitive potassium channel opener, improved postischaemic contractile dysfunction of perfused hearts in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats dose-dependently. This study aimed to characterize the effect of glibenclamide, an ATPsensitive potassium channel blocker, and nicorandil in postischaemic contractile dysfunction of SHR and WKY rats Methods The perfused hearts were subjected to 30 min of global ischaemia and then 30 min of reperfusion. Administration of 10 or 50µmol/l glibenclamide or of a combination of glibenclamide and 300 µmol/l nicorandil was performed for 10 min before the ischaemia. The left ventricular developed pressure and end-diastolic pressure were measured Results Postischaemic contractile function was better in WKY rats than it was in SHR. Neither glibenclamide nor a combination of glibenclamide and nicorandil influenced the postischaemic contractile function or increased the incidence of reperfusion arrhythmias. The recoveries of coronary flow and heart rate after reperfusion were poor and the incidence of reperfusion arrhythmias was low in SHR Conclusions These results suggest that nicorandil improves postischaemic contractile dysfunction via a mechanism involving ATP-sensitive potassium channel opening both in SHR and in WKY rats. The hypertensive hearts were more susceptible to cardiac reperfusion dysfunction, compared with normal hearts