Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype.

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.

Progressive exercise training improves maximal aerobic capacity in individuals with well-healed burn injuries.

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 (V̇o2max) 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 V̇o2max 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 V̇o2max in individuals with burn injuries and that the magnitude of body surface area burned does not lessen this adaptive response.

Pathological mechanisms underlying single large-scale mitochondrial DNA deletions.

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.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies.

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.

Impaired glycogen breakdown and synthesis in phosphoglucomutase 1 deficiency.

OBJECTIVE: We investigated metabolism and physiological responses to exercise in an 18-year-old woman with multiple congenital abnormalities and exertional muscle fatigue, tightness, and rhabdomyolysis. METHODS: We studied biochemistry in muscle and fibroblasts, performed mutation analysis, assessed physiological responses to forearm and cycle-ergometer exercise combined with stable-isotope techniques and indirect calorimetry, and evaluated the effect of IV glucose infusion and oral sucrose ingestion on the exercise response. RESULTS: Phosphoglucomutase type 1 (PGM1) activity in muscle and fibroblasts was severely deficient and PGM1 in muscle was undetectable by Western blot. The patient was compound heterozygous for missense (R422W) and nonsense (Q530X) mutations in PGM1. Forearm exercise elicited no increase in lactate, but an exaggerated increase in ammonia, and provoked a forearm contracture. Comparable to patients with McArdle disease, the patient developed a 'second wind' with a spontaneous fall in exercise heart rate and perceived exertion. Like in McArdle disease, this was attributable to an increase in muscle oxidative capacity. Carbohydrate oxidation was blocked during exercise, and the patient had exaggerated oxidation of fat to fuel exercise. Exercise heart rate and perceived exertion were lower after IV glucose and oral sucrose. Muscle glycogen level was low normal. CONCLUSIONS: The second wind phenomenon has been considered to be pathognomonic for McArdle disease, but we demonstrate that it can also be present in PGM1 deficiency. We show that severe loss of PGM1 activity causes blocked muscle glycogenolysis that mimics McArdle disease, but may also limit glycogen synthesis, which broadens the phenotypic spectrum of this disorder.

Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle.

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.

Reproducibility and absolute quantification of muscle glycogen in patients with glycogen storage disease by 13C NMR spectroscopy at 7 Tesla.

Carbon-13 magnetic resonance spectroscopy (13C MRS) offers a noninvasive method to assess glycogen levels in skeletal muscle and to identify excess glycogen accumulation in patients with glycogen storage disease (GSD). Despite the clinical potential of the method, it is currently not widely used for diagnosis or for follow-up of treatment. While it is possible to perform acceptable 13C MRS at lower fields, the low natural abundance of 13C and the inherently low signal-to-noise ratio of 13C MRS makes it desirable to utilize the advantage of increased signal strength offered by ultra-high fields for more accurate measurements. Concomitant with this advantage, however, ultra-high fields present unique technical challenges that need to be addressed when studying glycogen. In particular, the question of measurement reproducibility needs to be answered so as to give investigators insight into meaningful inter-subject glycogen differences. We measured muscle glycogen levels in vivo in the calf muscle in three patients with McArdle disease (MD), one patient with phosphofructokinase deficiency (PFKD) and four healthy controls by performing 13C MRS at 7T. Absolute quantification of the MRS signal was achieved by using a reference phantom with known concentration of metabolites. Muscle glycogen concentration was increased in GSD patients (31.5±2.9 g/kg w. w.) compared with controls (12.4±2.2 g/kg w. w.). In three GSD patients glycogen was also determined biochemically in muscle homogenates from needle biopsies and showed a similar 2.5-fold increase in muscle glycogen concentration in GSD patients compared with controls. Repeated inter-subject glycogen measurements yield a coefficient of variability of 5.18%, while repeated phantom measurements yield a lower 3.2% system variability. We conclude that noninvasive ultra-high field 13C MRS provides a valuable, highly reproducible tool for quantitative assessment of glycogen levels in health and disease.

The effect of rowing ergometry and resistive exercise on skeletal muscle structure and function during bed rest.

Exposure to microgravity causes functional and structural impairment of skeletal muscle. Current exercise regimens are time-consuming and insufficiently effective; an integrated countermeasure is needed that addresses musculoskeletal along with cardiovascular health. High-intensity, short-duration rowing ergometry and supplemental resistive strength exercise may achieve these goals. Twenty-seven healthy volunteers completed 5 wk of head-down-tilt bed rest (HDBR): 18 were randomized to exercise, 9 remained sedentary. Exercise consisted of rowing ergometry 6 days/wk, including interval training, and supplemental strength training 2 days/wk. Measurements before and after HDBR and following reambulation included assessment of strength, skeletal muscle volume (MRI), and muscle metabolism (magnetic resonance spectroscopy); quadriceps muscle biopsies were obtained to assess muscle fiber types, capillarization, and oxidative capacity. Sedentary bed rest (BR) led to decreased muscle volume (quadriceps: -9 ± 4%, P < 0.001; plantar flexors: -19 ± 6%, P < 0.001). Exercise (ExBR) reduced atrophy in the quadriceps (-5 ± 4%, interaction P = 0.018) and calf muscle, although to a lesser degree (-14 ± 6%, interaction P = 0.076). Knee extensor and plantar flexor strength was impaired by BR (-14 ± 15%, P = 0.014 and -22 ± 7%, P = 0.001) but preserved by ExBR (-4 ± 13%, P = 0.238 and +13 ± 28%, P = 0.011). Metabolic capacity, as assessed by maximal O2 consumption, (31)P-MRS, and oxidative chain enzyme activity, was impaired in BR but stable or improved in ExBR. Reambulation reversed the negative impact of BR. High-intensity, short-duration rowing and supplemental strength training effectively preserved skeletal muscle function and structure while partially preventing atrophy in key antigravity muscles. Due to its integrated cardiovascular benefits, rowing ergometry could be a primary component of exercise prescriptions for astronauts or patients suffering from severe deconditioning.

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.