RESUMEN
Background Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. Purpose To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 (13C) MRI with hyperpolarized (HP) [1-13C]-pyruvate. Materials and Methods For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-13C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired t test was used for comparing 13C data among the states. Results At rest, HP [1-13C]-lactate and [1-13C]-alanine were detected in calf muscle, but [13C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP 13C signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-13C]-lactate-to-tC ratio increased 1.7-fold (P = .04). HP [13C]-bicarbonate-to-tC ratio increased 8.4-fold (P = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting (P = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {13C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-13C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021 Online supplemental material is available for this article.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Ejercicio Físico , Músculo Esquelético/metabolismo , Ácido Pirúvico/metabolismo , Adulto , Bicarbonatos/metabolismo , Estudios de Factibilidad , Humanos , Ácido Láctico/metabolismo , Masculino , Estudios ProspectivosRESUMEN
McArdle disease and mitochondrial myopathy impair muscle oxidative phosphorylation (OXPHOS) by distinct mechanisms: the former by restricting oxidative substrate availability caused by blocked glycogen breakdown, the latter because of intrinsic respiratory chain defects. We applied metabolic profiling to systematically interrogate these disorders at rest, when muscle symptoms are typically minimal, and with exercise, when symptoms of premature fatigue and potential muscle injury are unmasked. At rest, patients with mitochondrial disease exhibit elevated lactate and reduced uridine; in McArdle disease purine nucleotide metabolites, including xanthine, hypoxanthine, and inosine are elevated. During exercise, glycolytic intermediates, TCA cycle intermediates, and pantothenate expand dramatically in both mitochondrial disease and control subjects. In contrast, in McArdle disease, these metabolites remain unchanged from rest; but urea cycle intermediates are increased, likely attributable to increased ammonia production as a result of exaggerated purine degradation. Our results establish skeletal muscle glycogen as the source of TCA cycle expansion that normally accompanies exercise and imply that impaired TCA cycle flux is a central mechanism of restricted oxidative capacity in this disorder. Finally, we report that resting levels of long-chain triacylglycerols in mitochondrial myopathy correlate with the severity of OXPHOS dysfunction, as indicated by the level of impaired O2 extraction from arterial blood during peak exercise. Our integrated analysis of exercise and metabolism provides unique insights into the biochemical basis of these muscle oxidative defects, with potential implications for their clinical management.
Asunto(s)
Ciclo del Ácido Cítrico/fisiología , Metabolismo Energético/fisiología , Ejercicio Físico/fisiología , Enfermedad del Almacenamiento de Glucógeno Tipo V/patología , Miopatías Mitocondriales/patología , Músculo Esquelético/patología , Adolescente , Adulto , Anciano , Ciclo del Ácido Cítrico/genética , Transporte de Electrón/fisiología , Femenino , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo V/genética , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Metaboloma/fisiología , Persona de Mediana Edad , Mitocondrias/metabolismo , Miopatías Mitocondriales/genética , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno/fisiología , Triglicéridos/metabolismo , Adulto JovenRESUMEN
Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a â¼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.
Asunto(s)
Reprogramación Celular , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Gotas Lipídicas/metabolismo , Mitocondrias/metabolismo , Azufre/metabolismo , Aconitato Hidratasa/metabolismo , Metabolismo Energético , Células HEK293 , Humanos , Redes y Vías MetabólicasRESUMEN
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
Asunto(s)
Alelos , Complejo I de Transporte de Electrón/deficiencia , Proteínas de la Membrana/genética , Enfermedades Mitocondriales/genética , Mutación/genética , Fenotipo , Adolescente , Adulto , Edad de Inicio , Secuencia de Aminoácidos , Niño , Complejo I de Transporte de Electrón/genética , Femenino , Humanos , Lactante , Masculino , Proteínas de la Membrana/química , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
Long-term rehabilitative strategies are important for individuals with well-healed burn injuries. Such information is particularly critical because patients are routinely surviving severe burn injuries given medical advances in the acute care setting. The purpose of this study was to test the hypothesis that a 6-mo community-based exercise training program will increase maximal aerobic capacity (VÌo2max) in subjects with prior burn injuries, with the extent of that increase influenced by the severity of the burn injury (i.e., percent body surface area burned). Maximal aerobic capacity (indirect calorimetry) and skeletal muscle oxidative enzyme activity (biopsy of the vastus lateralis muscle) were measured pre- and postexercise training in noninjured control subjects (n = 11) and in individuals with well-healed burn injuries (n = 13, moderate body surface area burned; n = 20, high body surface area burned). Exercise training increased VÌo2max in all groups (control: 15 ± 5%; moderate body surface area: 11 ± 3%; high body surface area: 11 ± 2%; P < 0.05), though the magnitude of this improvement did not differ between groups (P = 0.7). Exercise training also increased the activity of the skeletal muscle oxidative enzymes citrate synthase (P < 0.05) and cytochrome c oxidase (P < 0.05), an effect that did not differ between groups (P = 0.2). These data suggest that 6 mo of progressive exercise training improves VÌo2max in individuals with burn injuries and that the magnitude of body surface area burned does not lessen this adaptive response.
Asunto(s)
Quemaduras , Terapia por Ejercicio , Ejercicio Físico , Consumo de Oxígeno/fisiología , Adulto , Tolerancia al Ejercicio , Femenino , Humanos , MasculinoRESUMEN
OBJECTIVE: Single, large-scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large-scale mtDNA deletions in skeletal muscle. METHODS: We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large-scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. RESULTS: We have defined 3 "classes" of single, large-scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. INTERPRETATION: Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex-specific protein-encoding genes. Furthermore, removal of mt-tRNA genes impacts specific complexes only at high deletion levels, when complex-specific protein-encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115-130.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Eliminación de Secuencia/genética , Adulto , Anciano , Biopsia , Estudios de Cohortes , Complejo I de Transporte de Electrón/genética , Complejo IV de Transporte de Electrones/genética , Femenino , Eliminación de Gen , Dosificación de Gen , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/patología , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Fosforilación Oxidativa , Adulto JovenRESUMEN
ISCU myopathy is an inherited disease that primarily affects individuals of northern Swedish descent who share a single point mutation in the fourth intron of the ISCU gene. The current study shows correction of specific phenotypes associated with disease following treatment with an antisense oligonucleotide (ASO) targeted to the site of the mutation. We have shown that ASO treatment diminished aberrant splicing and increased ISCU protein levels in both patient fibroblasts and patient myotubes in a concentration dependent fashion. Upon ASO treatment, levels of SDHB in patient myotubular cell lines increased to levels observed in control myotubular cell lines. Additionally, we have shown that both patient fibroblast and myotubular cell lines displayed an increase in complex II activity with a concomitant decrease in succinate levels in patient myotubular cell lines after ASO treatment. Mitochondrial and cytosolic aconitase activities increased significantly following ASO treatment in patient myotubes. The current study suggests that ASO treatment may serve as a viable approach to correcting ISCU myopathy in patients.
Asunto(s)
Acidosis Láctica/congénito , Proteínas Hierro-Azufre/genética , Enfermedades Musculares/congénito , Oligonucleótidos Antisentido/genética , Succinato Deshidrogenasa/genética , Acidosis Láctica/genética , Acidosis Láctica/patología , Acidosis Láctica/terapia , Femenino , Humanos , Intrones/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Enfermedades Musculares/terapia , Oligonucleótidos Antisentido/uso terapéutico , Fenotipo , Mutación Puntual , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Succinato Deshidrogenasa/biosíntesisRESUMEN
Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function.
Asunto(s)
Enfermedades Mitocondriales/genética , Modelos Moleculares , Procesamiento Postranscripcional del ARN/genética , ARN de Transferencia/genética , ARNt Metiltransferasas/genética , Secuencia de Aminoácidos , Emparejamiento Base , Secuencia de Bases , Exoma/genética , Mutación del Sistema de Lectura/genética , Humanos , Enfermedades Mitocondriales/patología , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , ARNt Metiltransferasas/químicaRESUMEN
Iron-sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.
Asunto(s)
Acidosis Láctica/congénito , Factores de Crecimiento de Fibroblastos/metabolismo , Hidroximetilglutaril-CoA Sintasa/metabolismo , Proteínas Hierro-Azufre/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Musculares/congénito , Factores de Transcripción/genética , Acidosis Láctica/genética , Acidosis Láctica/metabolismo , Acidosis Láctica/patología , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Metabolismo Energético , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Humanos , Hidroximetilglutaril-CoA Sintasa/genética , Proteínas Hierro-Azufre/genética , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/metabolismoRESUMEN
Glycogen storage diseases (GSD) are inborn errors of glycogen or glucose metabolism. In the GSDs that affect muscle, the consequence of a block in skeletal muscle glycogen breakdown or glucose use, is an impairment of muscular performance and exercise intolerance, owing to 1) an increase in glycogen storage that disrupts contractile function and/or 2) a reduced substrate turnover below the block, which inhibits skeletal muscle ATP production. Immobility is associated with metabolic alterations in muscle leading to an increased dependence on glycogen use and a reduced capacity for fatty acid oxidation. Such changes may be detrimental for persons with GSD from a metabolic perspective. However, exercise may alter skeletal muscle substrate metabolism in ways that are beneficial for patients with GSD, such as improving exercise tolerance and increasing fatty acid oxidation. In addition, a regular exercise program has the potential to improve general health and fitness and improve quality of life, if executed properly. In this review, we describe skeletal muscle substrate use during exercise in GSDs, and how blocks in metabolic pathways affect exercise tolerance in GSDs. We review the studies that have examined the effect of regular exercise training in different types of GSD. Finally, we consider how oral substrate supplementation can improve exercise tolerance and we discuss the precautions that apply to persons with GSD that engage in exercise.
Asunto(s)
Tolerancia al Ejercicio , Ejercicio Físico , Enfermedad del Almacenamiento de Glucógeno/clasificación , Enfermedad del Almacenamiento de Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Metabolismo de los Hidratos de Carbono , Glucosa/metabolismo , Glucógeno/metabolismo , Humanos , Metabolismo de los Lípidos , Calidad de VidaRESUMEN
BACKGROUND: ISCU myopathy is a disease caused by muscle-specific deficiency of the Fe-S cluster scaffold protein ISCU. RESULTS: MyoD expression enhanced ISCU mRNA mis-splicing, and oxidative stress exacerbated ISCU depletion in patient cells. CONCLUSION: ISCU protein deficiency in patients results from muscle-specific mis-splicing as well as oxidative stress. SIGNIFICANCE: Oxidative stress negatively influences the mammalian Fe-S cluster assembly machinery by destabilization of ISCU. Iron-sulfur (Fe-S) cluster cofactors are formed on the scaffold protein ISCU. ISCU myopathy is a disease caused by an intronic mutation that leads to abnormally spliced ISCU mRNA. We found that two predominant mis-spliced ISCU mRNAs produce a truncated and short-lived ISCU protein product in multiple patient cell types. Expression of the muscle-specific transcription factor MyoD further diminished normal splicing of ISCU mRNA in patient myoblasts, demonstrating that the process of muscle differentiation enhances the loss of normal ISCU mRNA splicing. ISCU protein was nearly undetectable in patient skeletal muscle, but was higher in patient myoblasts, fibroblasts, and lymphoblasts. We next treated patient cells with pro-oxidants to mimic the oxidative stress associated with muscle activity. Brief hydrogen peroxide treatment or incubation in an enriched oxygen atmosphere led to a marked further reduction of ISCU protein levels, which could be prevented by pretreatment with the antioxidant ascorbate. Thus, we conclude that skeletal muscle differentiation of patient cells causes a higher degree of abnormal ISCU splicing and that oxidative stress resulting from skeletal muscle work destabilizes the small amounts of normal ISCU protein generated in patient skeletal muscles.
Asunto(s)
Diferenciación Celular , Proteínas Hierro-Azufre/genética , Enfermedades Mitocondriales/metabolismo , Músculo Esquelético/citología , Estrés Oxidativo , Empalme del ARN , Adulto , Anciano , Animales , Femenino , Humanos , Proteínas Hierro-Azufre/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/fisiopatología , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Especificidad de Órganos , Adulto JovenRESUMEN
A trimethylamine (TMA) moiety is present in carnitine and acetylcarnitine, and both molecules play critical roles in muscle metabolism. At 7 T, the chemical shift dispersion was sufficient to routinely resolve the TMA signals from carnitine at 3.20 and from acetylcarnitine at 3.17 ppm in the (1)H-MRS (Magnetic Resonance Spectroscopy) of human soleus muscle with a temporal resolution of about 2 min. In healthy, sedentary adults, the concentration of acetylcarnitine increased nearly 10-fold, to 4.1 ± 1.0 mmol/kg, in soleus muscle after 5 min of calf-raise exercise and recovered to a baseline concentration of 0.5 ± 0.3 mmol/kg. While the half-time for decay of acetylcarnitine was the same whether measured from the TMA signal (18.8 ± 5.6 min) or measured from the methyl signal (19.4 ± 6.1 min), the detection of acetylcarnitine by its TMA signal in soleus has the advantage of higher sensitivity and without overlapping from lipid signals. Although the activity of carnitine acetyltransferase is sufficient to allow equilibrium between carnitine and coenzyme-A pools, the exchange in TMA signal between carnitine and acetylcarnitine is slow in soleus following exercise on 7T (1)H-NMR time scale. The TMA signal provides a simple and direct measure of the relative amounts of carnitine and acetylcarnitine.
Asunto(s)
Acetilcarnitina/metabolismo , Carnitina/metabolismo , Ejercicio Físico , Espectroscopía de Resonancia Magnética , Metilaminas/metabolismo , Músculo Esquelético/metabolismo , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Human skeletal muscle respiratory chain defects restrict the ability of working muscle to extract oxygen from blood, and result in a hyperkinetic circulation during exercise in which oxygen delivery is excessive relative to oxygen uptake and oxygen levels within contracting muscle are abnormally high. To investigate the role of the muscle microcirculation in this anomalous circulatory response and possible implications for the regulation of muscle angiogenesis, we assessed muscle oxidative capacity during cycle exercise and determined capillary levels and distribution and vascular endothelial growth factor expression in quadriceps muscle biopsies in patients with mitochondrial myopathy attributable to heteroplasmic mitochondrial DNA mutations. We found that in patients with mitochondrial myopathy, muscle capillary levels were twice that of sedentary healthy subjects (3.0 ± 0.9% compared with 1.4 ± 0.3%, P < 0.001) despite the fact that oxygen utilization during peak cycle exercise was half that of control subjects (11.1 ± 4.0 ml/kg/min compared with 20.7 ± 7.9 ml/kg/min, P < 0.01); that capillary area was greatest in patients with the most severe muscle oxidative defects and was more than two times higher around muscle fibre segments with defective (i.e. cytochrome oxidase negative/succinic dehydrogenase-positive or 'ragged-red' fibres) compared with more preserved respiratory chain function; and that vascular endothelial growth factor expression paralleled capillary distribution. The increased muscle capillary levels in patients correlated directly (r(2) = 0.68, P < 0.05) with the severity of the mismatch between systemic oxygen delivery (cardiac output) and oxygen utilization during cycle exercise. Our results suggest that capillary growth is increased as a result of impaired muscle oxidative phosphorylation in mitochondrial myopathy, thus promoting increased blood flow to respiration-incompetent muscle fibres and a mismatch between oxygen delivery and utilization during exercise. Furthermore, the finding of high capillary levels despite elevated tissue oxygen levels during exercise in respiration-deficient muscle fibres implies that mitochondrial metabolism activates angiogenesis in skeletal muscle by a mechanism that is independent of hypoxia.
Asunto(s)
Capilares/fisiopatología , Miopatías Mitocondriales/fisiopatología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/fisiopatología , Consumo de Oxígeno/fisiología , Adulto , Capilares/metabolismo , Ejercicio Físico/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miopatías Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Oxígeno/metabolismoRESUMEN
Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases.
Asunto(s)
Enfermedades Mitocondriales , Fosforilación Oxidativa , Humanos , Longevidad , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismoRESUMEN
Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C-->T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.
Asunto(s)
Proteínas de Ciclo Celular/genética , ADN Mitocondrial/genética , Eliminación de Gen , Mutación , Oftalmoplejía Externa Progresiva Crónica/genética , Ribonucleótido Reductasas/genética , Biopsia , Análisis Mutacional de ADN , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Dosificación de Gen , Ligamiento Genético , Genotipo , Heterocigoto , Humanos , Inmunohistoquímica , Escala de Lod , Masculino , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Músculo Esquelético/cirugía , Oftalmoplejía Externa Progresiva Crónica/fisiopatología , Linaje , Mutación Puntual , Análisis de Secuencia de ADN , Succinato Deshidrogenasa/metabolismoRESUMEN
Mammalian ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, possesses an iron-sulfur [2Fe-2S] cluster that does not participate in catalysis. We investigated ferrochelatase expression in iron-deficient erythropoietic tissues of mice lacking iron regulatory protein 2, in iron-deficient murine erythroleukemia cells, and in human patients with ISCU myopathy. Ferrochelatase activity and protein levels were dramatically decreased in Irp2(-/-) spleens, whereas ferrochelatase mRNA levels were increased, demonstrating posttranscriptional regulation of ferrochelatase in vivo. Translation of ferrochelatase mRNA was unchanged in iron-depleted murine erythroleukemia cells, and the stability of mature ferrochelatase protein was also unaffected. However, the stability of newly formed ferrochelatase protein was dramatically decreased during iron deficiency. Ferrochelatase was also severely depleted in muscle biopsies and cultured myoblasts from patients with ISCU myopathy, a disease caused by deficiency of a scaffold protein required for Fe-S cluster assembly. Together, these data suggest that decreased Fe-S cluster availability because of cellular iron depletion or impaired Fe-S cluster assembly causes reduced maturation and stabilization of apo-ferrochelatase, providing a direct link between Fe-S biogenesis and completion of heme biosynthesis. We propose that decreased heme biosynthesis resulting from impaired Fe-S cluster assembly can contribute to the pathogenesis of diseases caused by defective Fe-S cluster biogenesis.
Asunto(s)
Anemia Ferropénica/metabolismo , Ferroquelatasa/metabolismo , Hemo/biosíntesis , Hierro/metabolismo , Miopatías Mitocondriales/metabolismo , Azufre/metabolismo , Anemia Ferropénica/genética , Anemia Ferropénica/patología , Animales , Biopsia , Línea Celular Tumoral , Eritrocitos/citología , Eritrocitos/enzimología , Ferroquelatasa/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Proteína 2 Reguladora de Hierro/genética , Leucemia Eritroblástica Aguda/patología , Ratones , Ratones Mutantes , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Estrés Oxidativo/fisiología , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismoRESUMEN
Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles, including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by (31)P magnetic resonance spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in beta-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from the loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the beta-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.
Asunto(s)
Calpaína/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/anomalías , Músculo Esquelético/metabolismo , Estrés Oxidativo , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calpaína/genética , Ácidos Grasos/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/enzimología , Mitocondrias/genética , Proteínas Musculares/genética , Músculo Esquelético/enzimología , Oxidación-ReducciónRESUMEN
A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.
Asunto(s)
Tolerancia al Ejercicio/genética , Proteínas Hierro-Azufre/genética , Miopatías Mitocondriales/genética , Sitios de Empalme de ARN/genética , Aconitato Hidratasa/deficiencia , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Homocigoto , Humanos , Mitocondrias/enzimología , Miopatías Mitocondriales/enzimología , Datos de Secuencia Molecular , Mutación , Linaje , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Succinato Deshidrogenasa/deficiencia , SueciaRESUMEN
Exertional dyspnea limits exercise in some mitochondrial myopathy (MM) patients, but the clinical features of this syndrome are poorly defined, and its underlying mechanism is unknown. We evaluated ventilation and arterial blood gases during cycle exercise and recovery in five MM patients with exertional dyspnea and genetically defined mitochondrial defects, and in four control subjects (C). Patient ventilation was normal at rest. During exercise, MM patients had low Vo(2peak) (28 ± 9% of predicted) and exaggerated systemic O(2) delivery relative to O(2) utilization (i.e., a hyperkinetic circulation). High perceived breathing effort in patients was associated with exaggerated ventilation relative to metabolic rate with high VE/VO(2peak), (MM = 104 ± 18; C = 42 ± 8, P ≤ 0.001), and Ve/VCO(2peak)(,) (MM = 54 ± 9; C = 34 ± 7, P ≤ 0.01); a steeper slope of increase in ΔVE/ΔVCO(2) (MM = 50.0 ± 6.9; C = 32.2 ± 6.6, P ≤ 0.01); and elevated peak respiratory exchange ratio (RER), (MM = 1.95 ± 0.31, C = 1.25 ± 0.03, P ≤ 0.01). Arterial lactate was higher in MM patients, and evidence for ventilatory compensation to metabolic acidosis included lower Pa(CO(2)) and standard bicarbonate. However, during 5 min of recovery, despite a further fall in arterial pH and lactate elevation, ventilation in MM rapidly normalized. These data indicate that exertional dyspnea in MM is attributable to mitochondrial defects that severely impair muscle oxidative phosphorylation and result in a hyperkinetic circulation in exercise. Exaggerated exercise ventilation is indicated by markedly elevated VE/VO(2), VE/VCO(2), and RER. While lactic acidosis likely contributes to exercise hyperventilation, the fact that ventilation normalizes during recovery from exercise despite increasing metabolic acidosis strongly indicates that additional, exercise-specific mechanisms are responsible for this distinctive pattern of exercise ventilation.
Asunto(s)
Disnea/etiología , Disnea/fisiopatología , Mitocondrias Musculares/fisiología , Miopatías Mitocondriales/complicaciones , Miopatías Mitocondriales/fisiopatología , Esfuerzo Físico/fisiología , Acidosis/fisiopatología , Adulto , Análisis de los Gases de la Sangre , Dióxido de Carbono/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Lactatos/sangre , Masculino , Persona de Mediana Edad , Consumo de Oxígeno/fisiología , Ventilación Pulmonar/fisiología , Pruebas de Función RespiratoriaRESUMEN
Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, ß-hydroxy acylcarnitines, and ß-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.