Atsushi Hoshino

Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart

Cumulative evidence indicates that mitochondrial dysfunction has a role in heart failure progression, but whether mitochondrial quality control mechanisms are involved in the development of cardiac dysfunction remains unclear. Here we show that cytosolic p53 impairs autophagic degradation of damaged mitochondria and facilitates mitochondrial dysfunction and heart failure in mice. Prevalence and induction of mitochondrial autophagy is attenuated by senescence or doxorubicin treatment in vitro and in vivo. We show that cytosolic p53 binds to Parkin and disturbs its translocation to damaged mitochondria and their subsequent clearance by mitophagy. p53-deficient mice show less decline of mitochondrial integrity and cardiac functional reserve with increasing age or after treatment with doxorubicin. Furthermore, overexpression of Parkin ameliorates the functional decline in aged hearts, and is accompanied by decreased senescence-associated β-galactosidase activity and proinflammatory phenotypes. Thus, p53-mediated inhibition of mitophagy modulates cardiac dysfunction, raising the possibility that therapeutic activation of mitophagy by inhibiting cytosolic p53 may ameliorate heart failure and symptoms of cardiac ageing.

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.

Inhibition of p53 preserves Parkin-mediated mitophagy and pancreatic β-cell function in diabetes

Significance Tumor suppressor p53 has been known to have a broader role that extends to the regulation of energy metabolism. We investigated the role of islet p53 and found that genetic and pharmacological inhibition of p53 preserves insulin secretion and glucose tolerance in both streptozotocin-induced type 1 and db/db mouse models of type 2 diabetes. Glucolipotoxicy induces accumulation of p53 in the cytosol via oxidative stress and endoplasmic reticulum stress. Cytosolic p53 inhibits the autophagic clearance of damaged mitochondria by an inhibitory protein–protein interaction with Parkin, leading to the impairment of mitochondrial energetics and subsequent insulin secretion signals in islet β-cells. Mitochondrial compromise is a fundamental contributor to pancreatic β-cell failure in diabetes. Previous studies have demonstrated a broader role for tumor suppressor p53 that extends to the modulation of mitochondrial homeostasis. However, the role of islet p53 in glucose homeostasis has not yet been evaluated. Here we show that p53 deficiency protects against the development of diabetes in streptozotocin (STZ)-induced type 1 and db/db mouse models of type 2 diabetes. Glucolipotoxicity stimulates NADPH oxidase via receptor for advanced-glycation end products and Toll-like receptor 4. This oxidative stress induces the accumulation of p53 in the cytosolic compartment of pancreatic β-cells in concert with endoplasmic reticulum stress. Cytosolic p53 disturbs the process of mitophagy through an inhibitory interaction with Parkin and induces mitochondrial dysfunction. The occurrence of mitophagy is maintained in STZ-treated p53−/− mice that exhibit preserved glucose oxidation capacity and subsequent insulin secretion signaling, leading to better glucose tolerance. These protective effects are not observed when Parkin is deleted. Furthermore, pifithrin-α, a specific inhibitor of p53, ameliorates mitochondrial dysfunction and glucose intolerance in both STZ-treated and db/db mice. Thus, an intervention with cytosolic p53 for a mitophagy deficiency may be a therapeutic strategy for the prevention and treatment of diabetes.

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.

The Bedtime Administration Ameliorates Blood Pressure Variability and Reduces Urinary Albumin Excretion in Amlodipine-Olmesartan Combination Therapy

Chronotherapy has the potential to improve blood pressure (BP) variability and to decrease stroke and cardiovascular events. The present study examined the efficacy and safety of the bedtime administration in amlodipine-olmesartan combination therapy, as compared with the morning administration. The present study was an open-label, randomized crossover study of the effects of the morning vs. bedtime administration of amlodipine-olmesartan combination. The subject was 31 essential hypertensive patients. Morning BP surge (MBPS) and nocturnal BP pattern were analyzed from ambulatory BP data. Glucose and lipid profiles and cardiovascular-renal data were also collected. The bedtime administration reduced MBPS significantly (24.2 ± 13.5 mmHg vs. 32.3 ± 14.2 mmHg, p < 0.001) with no excessive nocturnal BP fall. In nondipper, the bedtime administration significantly improved nocturnal BP. On the other hand, it did not reduce nocturnal BP in dipper. Urinary albumin/creatinine ratio was lower in the bedtime administration than in the morning administration (42.5 ± 59.9 mg/g vs. 75.3 ± 26.4 mg/g, p = 0.044). In amlodipine-olmesartan combination therapy, the bedtime administration reduced better MBPS with correcting nocturnal BP fall and improved urinary albumin excretion. The bedtime dosing of amlodipine and olmesartan seems more apt than the morning dose to obtain the therapeutic goal.

Oxidative Post-Translational Modifications Develop LONP1 Dysfunction in Pressure Overload Heart Failure

Background—Mitochondrial compromise is a fundamental contributor to heart failure. Recent studies have revealed that several surveillance systems maintain mitochondrial integrity. The present study evaluated the role of mitochondrial AAA+ protease in a mouse model of pressure overload heart failure. Methods and Results—The fluorescein isothiocyanate casein assay and immunoblotting for endogenous mitochondrial proteins revealed a marked reduction in ATP-dependent proteolytic activity in failing heart mitochondria. The level of reduced cysteine was decreased, and tyrosine nitration and protein carbonylation were promoted in Lon protease homolog (LONP1), the most abundant mitochondrial AAA+ protease, in heart failure. Comprehensive analysis revealed that electron transport chain protein levels were increased even with a reduction in the expression of their corresponding mRNAs in heart failure, which indicated decreased protein turnover and resulted in the accumulation of oxidative damage in the electron transport chain. The induction of mitochondria-targeted human catalase ameliorated proteolytic activity and protein homeostasis in the electron transport chain, leading to improvements in mitochondrial energetics and cardiac contractility even during the late stage of pressure overload. Moreover, the infusion of mitoTEMPO, a mitochondria-targeted superoxide dismutase mimetic, recovered oxidative modifications of LONP1 and improved mitochondrial respiration capacity and cardiac function. The in vivo small interfering RNA repression of LONP1 partially canceled the protective effects of mitochondria-targeted human catalase induction and mitoTEMPO infusion. Conclusions—Oxidative post-translational modifications attenuate mitochondrial AAA+ protease activity, which is involved in impaired electron transport chain protein homeostasis, mitochondrial respiration deficiency, and left ventricular contractile dysfunction. Oxidatively inactivated proteases may be an endogenous target for mitoTEMPO treatment in pressure overload heart failure.

Activation of PPAR-α in the early stage of heart failure maintained myocardial function and energetics in pressure-overload heart failure

Failing heart loses its metabolic flexibility, relying increasingly on glucose as its preferential substrate and decreasing fatty acid oxidation (FAO). Peroxisome proliferator-activated receptor α (PPAR-α) is a key regulator of this substrate shift. However, its role during heart failure is complex and remains unclear. Recent studies reported that heart failure develops in the heart of myosin heavy chain-PPAR-α transgenic mice in a manner similar to that of diabetic cardiomyopathy, whereas cardiac dysfunction is enhanced in PPAR-α knockout mice in response to chronic pressure overload. We created a pressure-overload heart failure model in mice through transverse aortic constriction (TAC) and activated PPAR-α during heart failure using an inducible transgenic model. After 8 wk of TAC, left ventricular (LV) function had decreased with the reduction of PPAR-α expression in wild-type mice. We examined the effect of PPAR-α induction during heart failure using the Tet-Off system. Eight weeks after the TAC operation, LV construction was preserved significantly by PPAR-α induction with an increase in PPAR-α-targeted genes related to fatty acid metabolism. The increase of expression of fibrosis-related genes was significantly attenuated by PPAR-α induction. Metabolic rates measured by isolated heart perfusions showed a reduction in FAO and glucose oxidation in TAC hearts, but the rate of FAO preserved significantly owing to the induction of PPAR-α. Myocardial high-energy phosphates were significantly preserved by PPAR-α induction. These results suggest that PPAR-α activation during pressure-overloaded heart failure improved myocardial function and energetics. Thus activating PPAR-α and modulation of FAO could be a promising therapeutic strategy for heart failure.NEW & NOTEWORTHY The present study demonstrates the role of PPAR-α activation in the early stage of heart failure using an inducible transgenic mouse model. Induction of PPAR-α preserved heart function, and myocardial energetics. Activating PPAR-α and modulation of fatty acid oxidation could be a promising therapeutic strategy for heart failure.

D -Glutamate is metabolized in the heart mitochondria

D-Amino acids are enantiomers of L-amino acids and have recently been recognized as biomarkers and bioactive substances in mammals, including humans. In the present study, we investigated functions of the novel mammalian mitochondrial protein 9030617O03Rik and showed decreased expression under conditions of heart failure. Genomic sequence analyses showed partial homology with a bacterial aspartate/glutamate/hydantoin racemase. Subsequent determinations of all free amino acid concentrations in 9030617O03Rik-deficient mice showed high accumulations of D-glutamate in heart tissues. This is the first time that a significant amount of D-glutamate was detected in mammalian tissue. Further analysis of D-glutamate metabolism indicated that 9030617O03Rik is a D-glutamate cyclase that converts D-glutamate to 5-oxo-D-proline. Hence, this protein is the first identified enzyme responsible for mammalian D-glutamate metabolism, as confirmed in cloning analyses. These findings suggest that D-glutamate and 5-oxo-D-proline have bioactivities in mammals through the metabolism by D-glutamate cyclase.