Abstract 15630: Novel Mitochondrial Protein D-glutamate Cyclase Decreases During the Progression of Heart Failure
Shuhei TateishiMakoto AriyoshiMasumi KataneKenji HamaseYurika MiyoshiMaiko NakaneAtsushi HoshinoYoshifumi OkawaYuichiro MitaSatoshi KaimotoMotoki UchihashiKuniyoshi FukaiKazunori OnoDaichi HatoRyoetsu YamanakaSakiko HondaYohei FushimuraEri Kwai-KanaiNaotada IshiharaMasashi MitaHiroshi HommaSatoaki Matoba
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Introduction: Recently, importance of D-amino acids in mammals has been drawing attention. D-Amino acids are enantiomers of L-amino acids and have been recognized as biomarkers and bioactive substa...Cite
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.
Mitochondrial respiratory chain
Pressure overload
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The burden of heart failure (HF) in terms of health care expenditures, hospitalizations, and mortality is substantial and growing. The failing heart has been described as "energy-deprived" and mitochondrial dysfunction is a driving force associated with this energy supply-demand imbalance. Existing HF therapies provide symptomatic and longevity benefit by reducing cardiac workload through heart rate reduction and reduction of preload and afterload but do not address the underlying causes of abnormal myocardial energetic nor directly target mitochondrial abnormalities. Numerous studies in animal models of HF as well as myocardial tissue from explanted failed human hearts have shown that the failing heart manifests abnormalities of mitochondrial structure, dynamics, and function that lead to a marked increase in the formation of damaging reactive oxygen species and a marked reduction in on demand adenosine triphosphate synthesis. Correcting mitochondrial dysfunction therefore offers considerable potential as a new therapeutic approach to improve overall cardiac function, quality of life, and survival for patients with HF.
Afterload
Preload
Adenosine triphosphate
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Mitochondrial dysfunction in heart failure includes greater susceptibility to mitochondrial permeability transition (MPT), which may worsen cardiac function and decrease survival. Treatment with a mixture of the n3 polyunsaturated fatty acids (n3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is beneficial in heart failure patients and increases resistance to MPT in animal models. We assessed whether DHA and EPA have similar effects when given individually, and whether they prolong survival in heart failure. Male δ-sarcoglycan null cardiomyopathic hamsters were untreated or given either DHA, EPA, or a 1:1 mixture of DHA + EPA at 2.1% of energy intake. Treatment did not prolong survival: mean survival was 298 ± 15 days in untreated hamsters and 335 ± 17, 328 ± 14, and 311 ± 15 days with DHA, EPA, and DHA + EPA, respectively (n = 27-32/group). A subgroup of cardiomyopathic hamsters treated for 26 wk had impaired left ventricular function and increased cardiomyocyte apoptosis compared with normal hamsters, which was unaffected by n3 PUFA treatment. Evaluation of oxidative phosphorylation in isolated subsarcolemmal and interfibrillar mitochondria with substrates for complex I or II showed no effect of n3 PUFA treatment. On the other hand, interfibrillar mitochondria from cardiomyopathic hamsters were significantly more sensitive to Ca(2+)-induced MPT, which was completely normalized by treatment with DHA and partially corrected by EPA. In conclusion, treatment with DHA or EPA normalizes Ca(2+)-induced MPT in cardiomyopathic hamsters but does not prolong survival or improve cardiac function. This suggest that greater susceptibility to MPT is not a contributor to cardiac pathology and poor survival in heart failure.
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Pyruvate is an important metabolic substrate for the heart that is formed in the cytosol by glycolysis or conversion of lactate, and then must be transported into the mitochondrial matrix for further metabolism. The mitochondrial pyruvate carrier (MPC) is composed of MPC1 and MPC2 proteins that are each required for complex stability and transport activity. Indeed, mice with cardiac-specific knockout of MPC2 (CS-MPC2-/- mice) exhibited concomitant MPC1 degradation and marked reduction in pyruvate-stimulated mitochondrial respiration. While cardiac function and heart size was normal in 6 week old CS-MPC2-/- mice, serial echocardiograms demonstrated drastic increases in heart size, chamber dilation, and loss of contractile function at 10 and 16 weeks of age. Gene markers of heart failure, hypoxia, and fibrosis were markedly increased in CS-MPC2-/- hearts. Mitochondria isolated from 16 week old failing CS-MPC2-/- hearts exhibited normal respiration on glutamate/malate, succinate, palmitoylcarnitine, and 3-hydroxybutyrate/malate, indicating preservation of mitochondrial energetics with anaplerotic malate, or substrates to produce acetyl-CoA independent of pyruvate. Expression of genes encoding fat and ketone oxidation enzymes was not down-regulated in failing CS-MPC2-/- hearts as is typically observed in heart failure, suggesting these hearts may rely on fat or ketone body oxidation for ATP production. However, targeted metabolomics of hearts from 6 week old CS-MPC2-/- chow-fed mice suggested TCA cycle dysfunction due to decreased acetyl-CoA levels that are insufficient to condense with oxaloacetate, causing an accumulation of oxaloacetate/aspartate, malate, and fumarate. To determine whether increasing the availability of usable substrates (fatty acids and ketones) would rescue the cardiac dysfunction, CS-MPC2-/- mice were fed a high fat, low carbohydrate (ketogenic) diet. Ketogenic diet strikingly decreased hypertrophy and improved functional parameters in 10 week old mice. In conclusion, loss of mitochondrial pyruvate utilization leads to altered cardiac substrate metabolism and inability to maintain TCA cycle flux, resulting in dilated cardiomyopathy that can be corrected by administration of a ketogenic diet.
Acetyl-CoA
Palmitoylcarnitine
ATP citrate lyase
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Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood.To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo.Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro.Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.
Lipotoxicity
Mitochondrial ROS
DNAJA3
DNM1L
FIS1
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Cardiomyocyte redox dysregulation contributes to oxidative and metabolic stress during heart failure. We previously showed in endothelial and vascular smooth muscle cells, cytochrome b5 reductase 3...
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Introduction: Despite the growing clinical burden of diabetic cardiomyopathy, there is no specific treatment for the disorder. We previously demonstrated that increased activity of AMP deaminase (AMPD) contributes to diastolic dysfunction in diabetic myocardium through ATP depletion and augmentation of xanthine oxidase-mediated ROS production. Additionally, the threshold for mitochondrial permeability transition (mPT), a major mechanism of cell necrosis, was found to be reduced by diabetes. Hypothesis: Increased localization of AMPD in mitochondria-associated ER membranes (MAMs) lowers the threshold for mPT, via accelerated Ca 2+ transport from the ER, by interaction with IP3 receptor (IP3R), VDAC and/or mitochondrial Ca 2+ uniporter (MCU). Methods and Results: The 90-kDa full-length and 70-kDa N-terminus-truncated form of AMPD3, a main isoform in rat hearts, were detected in the myocardium. The 90-kDa full-length AMPD3 was exclusively observed in outer mitochondrial membranes (OMMs), and both 90-kDa AMPD3 and 70-kDa AMPD3 were observed in MAMs. The 90-kDa AMPD3 levels in OMMs and MAMs were significantly higher in OLETF, type 2 diabetic rats, than in LETO, non-diabetic control rats, while 70-kDa AMPD3 levels were comparable in OLETF and LETO. The area of the MAM quantified by using electron micrographs was 57% larger in OLETF than in LETO (p<0.05). Immunoprecipitation experiments showed that 90-kDa AMPD3 was associated with VDAC, whereas 70-kDa AMPD3 did not interact with IP3R, VDAC or MCU. Ca 2+ retention capacity (CRC), an index of the threshold for mPT, analyzed in MAM-containing crude mitochondria was 21% lower in OLETF than in LETO (p<0.05). Although an inhibitor of AMPD (cpd3) had no effect on CRC, knockdown of 90-kDa AMPD3 significantly ameliorated H 2 O 2 -induced loss of mitochondrial membrane potential that was determined by TMRE in cultured isolated cardiomyocytes. Conclusions: The results suggest that 90-kDa AMPD3 in MAMs plays a role in regulation of the mPT threshold in a deaminase activity-independent manner and that increased AMPD3 in MAMs contributes to mitochondrial respiratory dysfunction in diabetic myocardium.
Voltage-dependent anion channel
Diabetic Cardiomyopathy
Uniporter
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During experimental hypertensive cardiac hypertrophy, the heart energy metabolism reverts from the normal adult type that obtains the majority of its requirement for adenosine triphosphate (ATP) from metabolism of fatty acids and oxidative phosphorylation (OXPHOS), to the fetal form, which metabolizes glucose and lactate. Mitochondrial synthesis and function require an estimated 1000 polypeptides, 37 of which are encoded by mitochondrial (mt) DNA, the rest by nuclear (n) DNA. Inherited or acquired aberrations of either mtDNA or nDNA mitochondrial genes cause mitochondrial dysfunction. Tissue expression of OXPHOS enzyme defects is often heterogeneous. As a result, cardiomyopathy and cardiac failure are frequent but unpredictable complications of mitochondrial encephalopathy, neuropathy, and myopathy. Several nuclear genes that encode mitochondrial proteins have been sequenced and specific defects associated with nuclear genes that affect mitochondrial structure and function have been linked to hypertrophic and dilated cardiomyopathies and to cardiac conduction defects. Thyroid hormone and exercise stimulate expression of a nuclear respiratory factor (NRF) that induces the nuclear gene TFAM, which encodes the mitochondrial transcription factor A that controls mitochondrial replication and transcription. TFAM-null mouse embryos lack mitochondria and fail to develop a heart. Mitochondrial dysfunction enhances the generation of radical oxygen species (ROS), which damage mtDNA, nDNA, proteins, and lipid membranes. Mice lacking the mitochondrial antioxidant enzyme manganese-superoxide dismutase (SOD) develop dilated cardiomyopathy. Palliative mitochondrial therapy with L-acetyl-carnitine and coenzyme Q10 improves cardiac function in patients with cardiomyopathy. Cure is only achievable by mitochondrial gene therapy. Experimental direct gene therapy uses vectors or targeting signal sequences to insert genes into mtDNA; indirect gene therapy employs viral or non-viral vectors to introduce genes into nDNA. Clinical repair of damaged somatic and germline genes that encode mitochondrial proteins may soon be within reach.
TFAM
Mitochondrial disease
DNAJA3
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Mitochondrial dysfunction has been implicated in several cardiovascular diseases; however, the roles of mitochondrial oxidative stress and DNA damage in hypertensive cardiomyopathy are not well understood.We evaluated the contribution of mitochondrial reactive oxygen species (ROS) to cardiac hypertrophy and failure by using genetic mouse models overexpressing catalase targeted to mitochondria and to peroxisomes.Angiotensin II increases mitochondrial ROS in cardiomyocytes, concomitant with increased mitochondrial protein carbonyls, mitochondrial DNA deletions, increased autophagy and signaling for mitochondrial biogenesis in hearts of angiotensin II-treated mice. The causal role of mitochondrial ROS in angiotensin II-induced cardiomyopathy is shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by angiotensin II, as well as heart failure induced by overexpression of Gαq. Furthermore, primary damage to mitochondrial DNA, induced by zidovudine administration or homozygous mutation of mitochondrial polymerase γ, is also shown to contribute directly to the development of cardiac hypertrophy, fibrosis and failure.These data indicate the critical role of mitochondrial ROS in cardiac hypertrophy and failure and support the potential use of mitochondrial-targeted antioxidants for prevention and treatment of hypertensive cardiomyopathy.
Mitochondrial ROS
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