Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca²⁺]_mit, whereas non-aggregate-prone mutations did not decrease [Ca²⁺]_mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca²⁺]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca²⁺]_mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.     

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.     

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila

Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age‐related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher‐order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos‐mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.     

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ⁰ cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.     

Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

Generation and bioenergetic analysis of cybrids containing mitochondrial DNA from mouse skeletal muscle during aging

Mitochondrial respiratory chain defects have been associated with various diseases and normal aging, particularly in tissues with high energy demands including skeletal muscle. Muscle-specific mitochondrial DNA (mtDNA) mutations have also been reported to accumulate with aging. Our understanding of the molecular processes mediating altered mitochondrial gene expression to dysfunction associated with mtDNA mutations in muscle would be greatly enhanced by our ability to transfer muscle mtDNA to established cell lines. Here, we report the successful generation of mouse cybrids carrying skeletal muscle mtDNA. Using this novel approach, we performed bioenergetic analysis of cells bearing mtDNA derived from young and old mouse skeletal muscles. A significant decrease in oxidative phosphorylation coupling and regulation capacity has been observed with cybrids carrying mtDNA from skeletal muscle of old mice. Our results also revealed decrease growth capacity and cell viability associated with the mtDNA derived from muscle of old mice. These findings indicate that a decline in mitochondrial function associated with compromised mtDNA quality during aging leads to a decrease in both the capacity and regulation of oxidative phosphorylation.     

Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer

Background

Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers.

Methods

Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed.

Results

We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells.

General Significance

Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.     

Mitochondrial neurogastrointestinal encephalomyopathy and thymidine metabolism: results and hypotheses

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease with mitochondrial DNA (mtDNA) alterations and is caused by mutations in the nuclear gene encoding thymidine phosphorylase (TP). The cardinal clinical manifestations are ptosis, ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Skeletal muscle shows mitochondrial abnormalities, including ragged-red fibers and cytochrome c oxidase deficiency, together with mtDNA depletion, multiple deletions or both. In MNGIE patients, TP mutations cause a loss-of-function of the cytosolic enzyme, TP. As a direct consequence of the TP defect, thymidine metabolism is altered. High blood levels of this nucleoside are likely to lead to mtDNA defects even in cells that do not express TP, such as skeletal muscle. We hypothesize that high concentrations of thymidine affect dNTP (deoxyribonucleoside triphosphate) metabolism in mitochondria more than in cytosol or nuclei, because mitochondrial dNTPs depend mainly on the thymidine salvage pathway, whereas nuclear dNTPs depend mostly on de novo pathway. The imbalance in the mitochondrial dNTP homeostasis affects mtDNA replication, leading to mitochondrial dysfunction.     

Mutations in FBXL4, Encoding a Mitochondrial Protein,Cause Early-Onset Mitochondrial Encephalomyopathy

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.     

Hyperactive Intracellular Calcium Signaling Associated with Localized Mitochondrial Defects in Skeletal Muscle of an Animal Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1^G93A mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca²⁺ release activity, which can include propagating Ca²⁺ waves. These Ca²⁺ waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca²⁺ uptake by Ru360 lead to cell-wide propagation of such Ca²⁺ release events. Our data reveal that mitochondria regulate Ca²⁺ signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Summary

Rat liver mitochondria have a specific Ca²⁺ release pathway which operates when NAD⁺ is hydrolysed to nicotinamide and ADPribose. NAD⁺ hydrolysis is Ca²⁺-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca²⁺ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD⁺ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD⁺ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca²⁺ release pathway activated by both tbh and GT. Ca²⁺ release increases with the mitochondrial Ca²⁺ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca²⁺ (‘Ca²⁺ cycling’) is prevented. These data support the notion that both tbh- and GT-induced Ca²⁺ release are not the consequence of an unspecific increase of the inner membrane permeability (‘pore’ formation). Tbh induces Ca²⁺ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca²⁺ loads and high tbh concentrations), Ca²⁺ release to about the same extent and rate as the Na⁺/Ca²⁺ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca²⁺ cycling and damage to mitochondria.     

Mitochondrial DNA diseases: Histological and cellular studies

Large-scale deletions and tRNA point mutations in mitochondrial DNA (mtDNA) are associated with a variety of different mitochondrial encephalomyopathies. Skeletal muscle in these patients shows a typical pathology, characterized by the focal accumulation of large numbers of morphologically and biochemically abnormal mitochondria (ragged-red fibers). Both mtDNA deletions and tRNA point mutations impair mitochondrial translation and produce deficiencies in oxidative phosphorylation. However, mutant and wild-type mtDNAs co-exist (mtDNA heteroplasmy) and the translation defect is not expressed until the ratio of mutant: wild-type mtDNAs exceeds a specific threshold. Below the threshold the phenotype can be rescued by intramitochondrial genetic complementation. The mosaic expression of the skeletal muscle pathology is thus determined by both the cellular and organellar distribution of mtDNA mutants.