Mitochondrial Matrix Enzyme Research Articles

Proteomic and phosphoproteomic remodeling in a rabbit model of electrical storm

Abstract Background Electrical storm (ES), characterized by repetitive ventricular tachycardia/fibrillation (VT/VF) over a short time-interval, is a major problem with substantial mortality in patients with implantable cardioverter-defibrillators (ICDs) and structural heart diseases, but therapeutic strategy in the survivors has not been established, likely due to incomplete understanding of the pathophysiology. Objective To explore molecular mechanisms of ICD-shocked VT/VF-related death in experimental ES. Methods The model was created by inducing chronic complete atrioventricular-block in ICD-implanted rabbits, causing cardiac hypertrophy, QT-prolongation, and VT/VF-episodes. Relative protein/phosphoprotein abundances in hearts from 5 rabbits with ES (defined as ≥3 VF-episodes/24-hr period) experiencing 81±28 VF-episodes and 5 controls (CTL) were assessed by iTRAQ LC-MS/MS and key regulatory pathways were examined by Ingenuity-Pathway-Analysis. Results Proteomic analysis identified 1938 proteins, 302 of which were differentially expressed between ES and CTL (P<0.05). ES was profoundly associated with mitochondrial dysfunction and oxidative phosphorylation inactivation along with decreased measurement and predicted inhibition of electron transport chain-proteins. Reductions in mitochondrial matrix-enzymes inferred downregulation of fatty acid β-oxidation and TCA-cycle. Transcriptional factors including nuclear respiratory factor 1 and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha involved in mitochondrial biogenesis were predicted downregulated and TP53 linked to mitochondrial depolarization upregulated. Phosphoproteome detected 641 phosphorylated proteins, 168 of which have changes in phosphorylation state in ES versus CTL (P<0.05). Aberrant phosphorylation-patterns occurred in Z-disc proteins of sarcomeres and Ca2+-handling proteins. Protein-kinase A and G, calcium, Ca2+/calmodulin protein-kinase, AMP-activated protein-kinase, and extracellular signal-regulated kinases/mitogen-activated protein-kinase signalings were activated, all coupling to nuclear integrators and mitochondrial transcription, which showed prediction opposite to proteomic data. Conclusions ES may cause mitochondrial damage, which can explain mechanical dysfunction and associated mortality. Direct effects of ICD-shocked VT/VF merit further investigations.

Abstract 144: Pyruvate Dehydrogenase Kinase 4 Contributes To Abdominal Aortic Aneurysm Through Accelerating Smooth Muscle Cell Metabolic Reprogramming And Nlrp3 Inflammasome-dependent Pyroptosis

Background: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate upon rupture. Understanding its molecular mechanisms of progression is crucial for therapeutic strategery. Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular metabolism. This study explored the role of PDK4 in AAA development, focusing on its impact on vascular smooth muscle cells (VSMCs). Methods: The expression of PDK4 in human and mice AAA tissues was measured. The role of PDK4 in AAA development was evaluated in VSMC-specific PDK4 knockout mice using a PCSK9/Ang II-induced AAA model. Seahorse analysis was used to assess oxidative phosphorylation and glycolysis levels. RNA-sequencing analysis and mitochondrial functional analysis were used to explore the molecular mechanisms. Results: We observed an increased expression of VSMC-PDK4 in human AAA tissues and AAA tissues from a PCSK9/AngII-induced AAA mouse model. PDK4 deficiency in VSMCs significantly inhibited AAA formation, whereas PDK4 overexpression aggravated AAA development. PDK4 promoted the metabolic reprogramming of VSMCs and VSMC-specific deletion of PDK4 ameliorated the contractile-to-synthetic phenotype switch in VSMCs. PDK4 activated NLRP3 inflammasome by impairing mitochondrial function, which contributed to the pyroptosis of VSMCs and instigated an inflammatory response. Mitochondria-targeted ROS scavenging and NLRP3 inhibitor MCC950 effectively attenuated NLRP3 inflammasome activation and pyroptosis mediated by PDK4. Supplementation with MCC950 was beneficial to ameliorating AAA in a VSMC PDK4-dependent manner in mouse models. Conclusion: PDK4 promotes smooth muscle cell metabolic reprogramming and NLRP3 inflammasome-dependent pyroptosis in AAA development, suggesting that targeting PDK4 may be a promising therapeutic strategy for the prevention and treatment of AAA.

PPAR-gamma agonist pioglitazone recovers mitochondrial quality control in fibroblasts from PITRM1-deficient patients.

Introduction: Biallelic variants in PITRM1 are associated with a slowly progressive syndrome characterized by intellectual disability, spinocerebellar ataxia, cognitive decline and psychosis. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests diverse oligopeptides, including the mitochondrial targeting sequences (MTS) that are cleaved from proteins imported across the inner mitochondrial membrane by the mitochondrial processing peptidase (MPP). Mitochondrial peptidases also play a role in the maturation of Frataxin, the protein affected in Friedreich's ataxia. Recent studies in yeast indicated that the mitochondrial matrix protease Ste23, which is a homologue of the human insulin-degrading enzyme (IDE), cooperates with Cym1 (homologue of PITRM1) to ensure the proper functioning of the preprotein processing machinery. In humans, IDE could be upregulated by Peroxisome Proliferator-Activated Receptor Gamma (PPARG) agonists. Methods: We investigated preprotein processing, mitochondrial membrane potential and MTS degradation in control and patients' fibroblasts, and we evaluated the pharmacological effect of the PPARG agonist Pioglitazone on mitochondrial proteostasis. Results: We discovered that PITRM1 dysfunction results in the accumulation of MTS, leading to the disruption and dissipation of the mitochondrial membrane potential. This triggers a feedback inhibition of MPP activity, consequently impairing the processing and maturation of Frataxin. Furthermore, we found that the pharmacological stimulation of PPARG by Pioglitazone upregulates IDE and also PITRM1 protein levels restoring the presequence processing machinery and improving Frataxin maturation and mitochondrial function. Discussion: Our findings provide mechanistic insights and suggest a potential pharmacological strategy for this rare neurodegenerative mitochondrial disease.

Goldilocks calcium concentrations and the regulation of oxidative phosphorylation: Too much, too little, or just right

Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca2+ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra- or intramitochondrial Ca2+ modulates oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supraphysiological increases in cytosolic and mitochondrial Ca2+ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4± 0.6μM) and medium (22.0± 2.4μM) Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and α-ketoglutarate, respectively, but not complex II-linked succinate. In intact cells, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

Pharmacokinetics of some newly synthesized 1, 5- benzothiazepine scaffolds: A molecular docking and molecular dynamics simulation approach

A highly efficient in silico pharmacokinetics was studied for some newly synthesized 1, 5- benzothiazepine derivatives. A total of fourteen title moieties were prepared by the condensation of substituted chalcones and 2- aminothiophenol that led to ring expansion in the presence of CuO nanocatalytic framework. The reported synthetic route is advantageous due to shorter reaction time and enhanced yield. The drug-target binding analysis revealed the potential therapeutic targets against 1,5- benzothiazepine derivatives using auto in silico consensus inverse docking (ACID) server. Among the seven selected 1,5- benzothiazepine derivatives, consensus inverse docking (CID) against PDB-binding database identified potential therapeutic targets using structure-based screening. The comparative analysis predicted 4-hydroxyphenylpyruvate dioxygenase (HPPD) (Uniprot ID: P32754), involved in the regulation of blood tyrosine levels by catalyzing the reaction of HPPD to homogentisic acid in tyrosine catabolism pathway. Dimethylglycine dehydrogenase (DMGDH) (Uniprot ID: Q9AGP8), a flavin adenine dinucleotide (FAD)‐ and tetrahydrofolate (THF)‐dependent, mitochondrial matrix enzyme that degrades choline and transfers electrons to the respiratory chain, one‐carbon metabolism, and folate receptor beta (Uniprot ID: P14207), which is a glycosylphosphatidylinositol-linked protein capturing ligandsfrom the extracellular matrix and passages them inside the cell throughendosomalpathway and recognized as an emerging biomarker for cancer and chronic inflammatory diseases, as potential targets for the selected derivatives. These findings warrant for in vitro cell-based experimental validation of the currently identified actives.

“CEREBRAL PALSY MIMIC OF A 5YEAR OLD GIRL CHILD”

Summary: Glutaric aciduria type I (GA I) is an autosomal recessive disorder caused by deficiency of Glutaryl CoA dehydrogenase, a mitochondrial matrix enzyme involved in the degradation of lysine, hydroxyl lysine and trypophan[1] resulting in accumulation of glutaric acid,3-hydroxy glutaric acid in central nervous system. The estimated prevalence of GA-I is approximately 1 in 1,00,000 live births.[2] The classic symptom of glutaric aciduria type I (GAI) is irreversible focal striatal necrosis during an acute illness, most often between the ages of 3 and 18 months termed as “Encephalopathic crisis” results in dystonic-dyskinetic disorder.

Alterations in Inflammatory Cytokines and Redox Homeostasis in LPS-Induced Pancreatic Beta-Cell Toxicity and Mitochondrial Stress: Protection by Azadirachtin

Inflammation and redox imbalance are hallmarks of cancer, diabetes, and other degenerative disorders. Pathophysiological response to these disorders leads to oxidative stress and mitochondrial dysfunction by alterations and reprogramming in cellular signaling and metabolism. Pancreatic beta cells are very sensitive to the inflammatory and altered nutrient signals and hence play a crucial role in diabetes and cancer. In this study, we treated insulin-secreting pancreatic beta cells, Rin-5F, with the bacterial endotoxin, LPS (1 μg/ml) to induce an inflammatory response in vitro and then treated the cells with a known anti-inflammatory, anticancer and antioxidant phytochemical, azadirachtin (AZD, 25 µM for 24 h). Our results demonstrated lipid peroxidation and nitric oxide production causing increased nitro/oxidative stress and alterations in the activities of anti-oxidant enzymes, superoxide dismutase and catalase after LPS treatment. Pro-inflammatory responses caused by translocation of nuclear factor kappa B and release of inflammatory cytokines were also observed. These changes were accompanied by GSH-dependent redox imbalance and alterations in mitochondrial membrane potential and respiratory complexes enzyme activities leading to mitochondrial respiratory dysfunction, reduced ATP synthesis, and intrinsic caspase-9 mediated apoptosis. Caspase-9 was activated due to alterations in Bcl-2 and Bax proteins and release of cytochrome c into the cytosol. The activities of oxidative stress-sensitive mitochondrial matrix enzymes, aconitase, and glutamate dehydrogenase were also inhibited. Treatment with AZD showed beneficial effects on the recovery of antioxidant enzymes, inflammatory responses, and mitochondrial functions. GSH-dependent redox homeostasis also recovered after the treatment with AZD. This study may help in better understanding the etiology and pathogenesis of inflammation-induced disorders in pancreatic beta cells to better manage therapeutic strategies.

Mitochondrial transport and metabolism of the vitamin B-derived cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ , and related diseases: A review.

Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD+, which are all structurally similar to nucleotides and derived from different B‐vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease‐causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor‐dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ are the focus of this review.

Photobiomodulation and Oxidative Stress: 980 nm Diode Laser Light Regulates Mitochondrial Activity and Reactive Oxygen Species Production.

Photobiomodulation with 808 nm laser light electively stimulates Complexes III and IV of the mitochondrial respiratory chain, while Complexes I and II are not affected. At the wavelength of 1064 nm, Complexes I, III, and IV are excited, while Complex II and some mitochondrial matrix enzymes seem to be not receptive to photons at that wavelength. Complex IV was also activated by 633 nm. The mechanism of action of wavelengths in the range 900–1000 nm on mitochondria is less understood or not described. Oxidative stress from reactive oxygen species (ROS) generated by mitochondrial activity is an inescapable consequence of aerobic metabolism. The antioxidant enzyme system for ROS scavenging can keep them under control. However, alterations in mitochondrial activity can cause an increment of ROS production. ROS and ATP can play a role in cell death, cell proliferation, and cell cycle arrest. In our work, bovine liver isolated mitochondria were irradiated for 60 sec, in continuous wave mode with 980 nm and powers from 0.1 to 1.4 W (0.1 W increment at every step) to generate energies from 6 to 84 J, fluences from 7.7 to 107.7 J/cm2, power densities from 0.13 to 1.79 W/cm2, and spot size 0.78 cm2. The control was equal to 0 W. The activity of the mitochondria's complexes, Krebs cycle enzymes, ATP production, oxygen consumption, generation of ROS, and oxidative stress were detected. Lower powers (0.1–0.2 W) showed an inhibitory effect; those that were intermediate (0.3–0.7 W) did not display an effect, and the higher powers (0.8–1.1 W) induced an increment of ATP synthesis. Increasing the power (1.2–1.4 W) recovered the ATP production to the control level. The interaction occurred on Complexes III and IV, as well as ATP production and oxygen consumption. Results showed that 0.1 W uncoupled the respiratory chain and induced higher oxidative stress and drastic inhibition of ATP production. Conversely, 0.8 W kept mitochondria coupled and induced an increase of ATP production by increments of Complex III and IV activities. An augmentation of oxidative stress was also observed, probably as a consequence of the increased oxygen consumption and mitochondrial isolation experimental conditions. No effect was observed using 0.5 W, and no effect was observed on the enzymes of the Krebs cycle.

PDK4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming

Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy regulation. Previous studies suggested that PDK4 is increased in the calcified vessels of patients with atherosclerosis and is closely associated with mitochondrial function, but the precise regulatory mechanisms remain largely unknown. This study aims to investigate the role of PDK4 in vascular calcification and the molecular mechanisms involved. Using a variety of complementary techniques, we found impaired autophagic activity in the process of vascular smooth muscle cells (VSMCs) calcification, whereas knocking down PDK4 had the opposite effect. PDK4 drives the metabolic reprogramming of VSMCs towards a Warburg effect, and the inhibition of PDK4 abrogates VSMCs calcification. Mechanistically, PDK4 disturbs the integrity of the mitochondria-associated endoplasmic reticulum membrane, concomitantly impairing mitochondrial respiratory capacity, which contributes to a decrease in lysosomal degradation by inhibiting the V-ATPase and lactate dehydrogenase B interaction. PDK4 also inhibits the nuclear translocation of the transcription factor EB, thus inhibiting lysosomal function. These changes result in the interruption of autophagic flux, which accelerates calcium deposition in VSMCs. In addition, glycolysis serves as a metabolic adaptation to improve VSMCs oxidative stress resistance, whereas inhibition of glycolysis by 2-deoxy-D-glucose induces the apoptosis of VSMCs and increases the calcium deposition in VSMCs. Our results suggest that PDK4 plays a key role in vascular calcification through autophagy inhibition and metabolic reprogramming.

Two novel ECHS1 variants, affecting splicing and reducing enzyme activity, is associated with mitochondrial encephalopathy in infant: a case report

BackgroundShort-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme involved in the second step of mitochondrial fatty acid β-oxidation. Mitochondrial diseases resulting from ECHS1 mutations are often characterised by encephalopathy, deafness, epilepsy, optic atrophy, cardiomyopathy, dystonia, and lactic acidosis. In this study, we report two novel heterogeneous variants, c.414 + 5G > A (in intron 3) and c.310C > G (in CDS), of ECHS1 in an infant with mitochondrial encephalopathy.Case presentationThe two novel variants, c.414 + 5G > A (Chr10:135183403) in intron 3 and c.310C > G (Chr10:135183512) in CDS, were identified by next generation sequencing (NGS). A minigene assay was used to analyse the function of the c.414 + 5G > A variant. ECHS1 enzyme activity was measured by spectrophotometry in the patient-derived myoblasts. The 2-year old patient presented with mitochondrial encephalopathy since birth. Clinical features were encephalopathy, epilepsy, and hindered psychomotor and language development. Serum lactate and blood ammonia levels were elevated, and brain magnetic resonance imaging showed abnormal signals in the bilateral frontal, parietal, and occipital cortices and brainstem and basal ganglia. We found two novel heterogeneous variants in ECHS1 in this patient. Minigene assay revealed the c.414 + 5G > A variant as the cause of intronic cryptic splice site activation and 39 bp deletion in mature mRNA. In silico analysis predicted that c.310C > G might change glutamine (Q) to glutamic acid (E) in the 104th amino acid sequence (p.Q104E). To investigate the impact of these two variants on protein function, we constructed a 3D model of human ECHS1 and showed that the variants might alter the highly conserved region in close proximity to the active site, which might hinder, or even halt, enzymatic activity. The experimental assay showed that ECHS1 enzyme activity in the patient-derived myoblasts decreased compared to that in control.ConclusionsOur findings are the first to report a mitochondrial encephalopathy infant carrying two novel ECHS1 variants, c.414 + 5G > A and c.310C > G, which might be deleterious variants, function as pathogenicity markers for mitochondrial encephalopathy, and facilitate disease diagnosis.

Aldehyde dehydrogenase 2 protects cardiomyocytes against lipotoxicity via the AKT/glycogen synthase kinase 3 beta pathways

Aldehyde dehydrogenase 2, a mitochondrial matrix enzyme, plays a crucial role in protecting the heart against stress, such as ischemia reperfusion and alcohol injury. The present study aimed to investigate the effect of aldehyde dehydrogenase 2 on lipotoxic cardiomyopathy and to explore the possible mechanisms in vitro. Primary cardiomyocytes in the lipotoxic group were treated with oxidatively modified low-density lipoprotein (50 mg/L) for 24 h. Overexpression of aldehyde dehydrogenase 2 was achieved using the aldehyde dehydrogenase 2 activator, Alda-1 (20 μM). We found that cardiomyocyte apoptosis was attenuated by aldehyde dehydrogenase 2 overexpression. In addition, aldehyde dehydrogenase 2 overexpression inhibited the expression of BCL2 associated X, apoptosis regulator (BAX) and caspase 3, while it enhanced protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK-3β) phosphorylation. The results suggested that aldehyde dehydrogenase 2 is cardioprotective against lipotoxic cardiomyopathy, probably by reducing apoptosis through the AKT/glycogen synthase kinase 3 beta (GSK-3β) pathway. Our findings partially revealed the molecular mechanism of aldehyde dehydrogenase 2’s cardioprotective effect against lipotoxic injury, and suggest a new therapeutic strategy to treat lipotoxic cardiomyopathy.

Assessment of mitochondrial respiratory chain enzymes in cells and tissues.

Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.

Advanced glycation end products accelerate calcification in VSMCs through HIF-1\u03b1/PDK4 activation and suppress glucose metabolism

Arterial media calcification is associated with diabetes mellitus. Previous studies have shown that advanced glycation end products (AGEs) are responsible for vascular smooth muscle cell (VSMC) calcification, but the underlying mechanisms remain unclear. Hypoxia-inducible factor-1α (HIF-1α), one of the major factors during hypoxia, and pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular metabolism shift, have been reported in VSMC calcification. The potential link among HIF-1α, PDK4, and AGEs-induced vascular calcification was investigated in this study. We observed that AGEs elevated HIF-1α and PDK4 expression levels in a dose-dependent manner and that maximal stimulation was attained at 24 h. Two important HIF-1α-regulated genes, vascular endothelial growth factor A (VEGFA) and glucose transporter 1 (GLUT-1), were significantly increased after AGEs exposure. Stabilization or nuclear translocation of HIF-1α increased PDK4 expression. PDK4 inhibition attenuated AGEs-induced VSMC calcification, which was evaluated by measuring the calcium content, alkaline phosphatase (ALP) activity and runt-related transcription factor 2 (RUNX2) expression levels and by Alizarin red S staining. In addition, the glucose consumption, lactate production, key enzymes of glucose metabolism and oxygen consumption rate (OCR) were decreased during AGEs-induced VSMC calcification. In conclusion, this study suggests that AGEs accelerate vascular calcification partly through the HIF-1α/PDK4 pathway and suppress glucose metabolism.

Restoring mitochondrial biogenesis with metformin attenuates β-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of PDK4/oxidative stress-mediated apoptosis.

Mitochondrial abnormalities have long been observed in the development of vascular calcification. Metformin, a member of the biguanide class of antidiabetic drugs, has recently received attention owing to new findings regarding its protective role in cardiovascular disease. Since the precise control of mitochondrial quantity and quality is critical for the survival and function of vascular smooth muscle cells (VSMCs), maintaining mitochondrial homeostasis may be a potential protective factor for VSMCs against osteoblast-like phenotypic transition. However, limited studies have been reported in this area. Here, we investigated the role of metformin in the phenotypic transformation of VSMCs, as well as its intracellular signal transduction pathways. We demonstrated that supplementation with metformin restored the β-glycerophosphate (β-GP)-mediated impairment of mitochondrial biogenesis in VSMCs, as evidenced by an increased mitochondrial DNA copy number, a restored mitochondrial membrane potential (MMP), and upregulated mitochondrial biogenesis-related gene expression, whereas the AMP-activated protein kinase (AMPK) inhibitor compound C suppressed these effects. We also observed that overexpression of pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular energy metabolism, exacerbated β-GP-induced oxidative stress and subsequent apoptosis in VSMCs but that these effects were suppressed by dichloroacetate, a widely reported PDK4 inhibitor. More importantly, enhanced mitochondrial biogenesis attenuated the β-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype through inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, whereas disruption of mitochondrial biogenesis by zidovudine aggravated β-GP-induced apoptosis in VSMCs. In addition, inhibition of autophagy by small interfering RNA targeting Atg5 reduced mitochondrial biogenesis in VSMCs. In summary, we uncovered a novel mechanism by which metformin attenuates the phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, and mitochondrial homeostasis is involved in this process.

Mitochondrial PITRM1 peptidase loss-of-function in childhood cerebellar atrophy

ObjectiveTo identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy.MethodsIdentification of the underlying mutations by whole...

Intermediary metabolism and fatty acid oxidation: novel targets of electron transport chain-driven injury during ischemia and reperfusion.

Cardiac ischemia-reperfusion (I/R) damages the electron transport chain (ETC), causing mitochondrial and cardiomyocyte injury. Reversible blockade of the ETC at complex I during ischemia protects the ETC and decreases cardiac injury. In the present study, we used an unbiased proteomic approach to analyze the extent of ETC-driven mitochondrial injury during I/R. Isolated-perfused mouse (C57BL/6) hearts underwent 25-min global ischemia (37°C) and 30-min reperfusion. In treated hearts, amobarbital (2 mM) was given for 1 min before ischemia to rapidly and reversibly block the ETC at complex I. Mitochondria were isolated at the end of reperfusion and subjected to unbiased proteomic analysis using tryptic digestion followed by liquid chromatography-mass spectrometry with isotope tags for relative and absolute quantification. Amobarbital treatment decreased cardiac injury and protected respiration. I/R decreased the content ( P < 0.05) of multiple mitochondrial matrix enzymes involved in intermediary metabolism compared with the time control. The contents of several enzymes in fatty acid oxidation were decreased compared with the time control. Blockade of ETC during ischemia largely prevented the decreases. Thus, after I/R, not only the ETC but also multiple pathways of intermediary metabolism sustain damage initiated by the ETC. If these damaged mitochondria persist in the myocyte, they remain a potent stimulus for ongoing injury and the transition to cardiomyopathy during prolonged reperfusion. Modulation of ETC function during early reperfusion is a key strategy to preserve mitochondrial metabolism and to decrease persistent mitochondria-driven injury during longer periods of reperfusion that predispose to ventricular dysfunction and heart failure. NEW & NOTEWORTHY Ischemia-reperfusion (I/R) damages mitochondria, which could be protected by reversible blockade of the electron transport chain (ETC). Unbiased proteomics with isotope tags for relative and absolute quantification analyzed mitochondrial damage during I/R and found that multiple enzymes in the tricarboxylic acid cycle, fatty acid oxidation, and ETC decreased, which could be prevented by ETC blockade. Strategic ETC modulation can reduce mitochondrial damage and cardiac injury.

Nε-carboxymethyl-lysine promotes calcium deposition in VSMCs via intracellular oxidative stress-induced PDK4 activation and alters glucose metabolism.

Diabetes and vascular calcification are intrinsically linked. We previously reported that advanced glycation end products (AGEs) accelerate calcium deposition in vascular smooth muscle cells (VSMCs) via excessive oxidative stress. However, the underlying mechanism remains poorly understood. Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy metabolism. Since hyperactivation of PDK4 has been reported in calcified vessels and in patients with diabetes mellitus, inhibition of PDK4 expression may be a strategy for the prevention of diabetic vascular calcification. In this study, we used a rat VSMC model to investigate the role of PDK4 in diabetic vascular calcification and further explore the underlying mechanisms. We observed that Nε-carboxymethyl-lysine (CML), which is a major immunogen of AGEs, accelerated calcium deposition in VSMCs through PDK4 activation. An elevated level of reactive oxygen species (ROS) acted as a signal transduction intermediate to increase PDK4 expression. Either inhibition of PDK4 expression or RAGE (receptor for AGEs) blockade attenuated CML-induced VSMC calcification, as shown by decreased alkaline phosphatase (ALP) activity and runt-related transcription factor 2 (RUNX2) expression. Glucose consumption and lactate production were increased during CML-induced VSMC calcification. Importantly, CML accelerates glycolysis in VSMCs via a PDK4-dependent pathway. In conclusion, this study demonstrates a novel mechanism by which CML promotes VSMC calcification via PDK4 activation and alters glucose metabolism in VSMCs.

In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents

Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the F1FO complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader.

Glutaric Aciduria Type 1 and Acute Renal Failure: Case Report and Suggested Pathomechanisms.

Glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in tissues and body fluids. During catabolic crises, GA1 patients are prone to the development of striatal necrosis and a subsequent irreversible movement disorder during a time window of vulnerability in early infancy. Thus, GA1 had been considered a pure "cerebral organic aciduria" in the past. Single case reports have indicated the occurrence of acute renal dysfunction in children affected by GA1. In addition, growing evidence arises that GA1 patients may develop chronic renal failure during adulthood independent of the previous occurrence of encephalopathic crises. The underlying mechanisms are yet unknown. Here we report on a 3-year-old GA1 patient who died following the development of acute renal failure most likely due to haemolytic uraemic syndrome associated with a pneumococcal infection. We hypothesise that known GA1 pathomechanisms, namely the endothelial dysfunction mediated by 3OHGA, as well as the transporter mechanisms for the urinary excretion of GA and 3OHGA, are involved in the development of glomerular and tubular dysfunction, respectively, and may contribute to a pre-disposition of GA1 patients to renal disease. We recommend careful differential monitoring of glomerular and tubular renal function in GA1 patients.