Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The Mitochondrial Calcium Uniporter controls skeletal muscle mass in vivo

ABSTRACT: Muscle atrophy contributes to the poor prognosis of many physiopathological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca2+] which control aerobic metabolism, cell death and survival pathways. We have investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function and trophism, by overexpressing or silencing the Mitochondrial Calcium Uniporter (MCU). The results coherently demonstrate that both in developing and in adult muscles MCU-dependent mitochondrial Ca2+ uptake has a marked trophic effect that does not depend on autophagy or aerobic control, but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1M-NM-14 and Igf1-Akt/PKB. In adult mice, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca2+-dependent organelle-to-nucleus signaling route, which links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in sarcopenia. Experiments were performed on biological replicates of single skeletal muscle fibres. Seven fibres were chosen for their Mutochondrial Calcium Uniporter (MCU) overexpression and other seven fibres because MCU was silenced. Overexpression and silencing were performed injecting skeletal muscle with AAV containing MCU gene or short interfering oligos specific for MCU. As control was profiled eigth fibres transfected with AAV and eigth wild type fibres. Analyses were performed 7 days and 14 days after the AAV injection (3 fibers after 7 days and 4 fibers after 14 days for MCU overexpression and silencing, four fibres after 7 days and four after 14 days for control).

ORGANISM(S): Mus musculus

SUBMITTER: Gerolamo Lanfranchi

PROVIDER: E-GEOD-60931 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

The mitochondrial calcium uniporter controls skeletal muscle trophism in vivo.

Mammucari Cristina C Gherardi Gaia G Zamparo Ilaria I Raffaello Anna A Boncompagni Simona S Chemello Francesco F Cagnin Stefano S Braga Alessandra A Zanin Sofia S Pallafacchina Giorgia G Zentilin Lorena L Sandri Marco M De Stefani Diego D Protasi Feliciano F Lanfranchi Gerolamo G Rizzuto Rosario R

Cell reports 20150226 8

Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca(2+)], which control aerobic metabolism, cell death, and survival pathways. We investigated in vivo the effects of mitochondrial Ca(2+) homeostasis in skeletal muscle function and trophism by overexpressing or silencing the mitochondrial calcium uniporter (MCU). The results demonstrate that in both devel ...[more]

PMID: 25732818

Similar Datasets

Project description:This experiment was conducted to identify the mitochondrial protein changes in the presence and absence of LONP1 in skeletal muscle. The following abstract from the submitted manuscript describes the major findings of this work.Disuse-associated loss of muscle LONP1 impairs mitochondrial quality and causes reduced skeletal muscle mass and strength. Zhisheng Xu, Tingting Fu, Qiqi Guo, Danxia Zhou, Wanping Sun, Zheng Zhou, Lin Liu, Liwei Xiao, Yujing Yin, Yuhuan Jia, Xin Pan, Lei Fang, Min-sheng Zhu, Wenyong Fei, Bin Lu and Zhenji Gan. Mitochondrial proteolysis is an evolutionarily conserved quality control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a critical role in controlling mitochondrial quality as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a pivotal role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel an intriguing link between mitochondrial protein quality and muscle mass maintenance during muscle disuse.

Project description:Background: skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. Methodology/Principal Findings: we have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. Conclusions/Significance: as gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. EDL and soleus muscles were incubated with type I collagenase to dissociate intact myofibres that were separated under stereo microscope from the bulk of hyper contracted fibres. Isolated myofibres were divided in two parts: one was immersed in Laemmli buffer for fibre typing; the other was placed in RNA extraction buffer for RNA amplification. We analyzed the transcription profiles of 10 biological replicas of type 2B single muscle fibres from EDL and 10 biological replicas of type 1 single muscle fibres from soleus. Microarray competitive hybridizations were carried out against an artificial control with a balanced composition of type 1 and type 2B fibres (20 hybridizations). Each oligonucleotide is spotted in two replicates on the glass slide, so for every data two intra-slide replicas are present. The control was created as follows: three couples of soleus and EDL muscles were removed from 3 different mice and treated with collagenase. Total RNA was extracted separately from EDL and soleus muscles. By mixing about 1/3 RNA from EDL and 2/3 RNA from soleus muscles the control had approximately the same contributions of type 1 and type 2B fibres. Purified RNA samples from single fibres and from control were amplified twice using the Amino Allyl MessageAmpM-bM-^DM-" II aRNA Amplification Kit (Ambion).

Project description:Transgenic overexpression of Galgt2 in the skeletal muscles of mdx mice inhibits the development of disease pathology associated with muscular dystrophy. This is the case both in transgenic mice, where Galgt2 overexpression occurs from embryonic timepoints onward and in mdx mice where Galgt2 is overexpressed in the early postnatal period using Adeno-associated virus (AAV). Here, we use gene expression profiling to compare transcriptional changes resulting from embryonic and postnatal Galgt2 overexpression in mdx skeletal muscle. A surprising number of changes were in genes known to ameliorate muscular dystrophy when overexpressed (agrin, integrin alpha 7, ADAM12, Bcl2) or to cause muscular dystrophy when mutated (collagen VI (alpha1,alpha2), plectin 1, dystroglycan, selenoprotein N1, integrin alpha7, biglycan, dysferlin). Several genes involved in calcium homeostasis were also changed. In Galgt2 transgenic mice, where embryonic overexpression of Galgt2 in skeletal muscles alters neuromuscular development and muscle growth, the number of gene expression changes was vastly greater, however, 14% of genes altered in postnatal AAV-Galgt2 infected mdx muscles were also changed with embryonic overexpression. These experiments suggest that postnatal overexpression of Galgt2 inhibits muscular dystrophy in mdx mice via induction of a group of genes that, in aggregate, can govern membrane stability, membrane repair, calcium homeostasis, and apoptosis. Transgenic overexpression of Galgt2 in the skeletal muscles of mdx mice inhibits the development of disease pathology associated with muscular dystrophy. This is the case both in transgenic mice, where Galgt2 overexpression occurs from embryonic timepoints onward and in mdx mice where Galgt2 is overexpressed in the early postnatal period using Adeno-associated virus (AAV). Here, we use gene expression profiling to compare transcriptional changes resulting from embryonic and postnatal Galgt2 overexpression in mdx skeletal muscle. A surprising number of changes were in genes known to ameliorate muscular dystrophy when overexpressed (agrin, integrin alpha7, ADAM12, Bcl2) or to cause muscular dystrophy when mutated (collagen VI (alpha1,alpha2), plectin 1, dystroglycan, selenoprotein N1, integrin alpha7, biglycan, dysferlin). Several genes involved in calcium homeostasis were also changed. In Galgt2 transgenic mice, where embryonic overexpression of Galgt2 in skeletal muscles alters neuromuscular development and muscle growth, the number of gene expression changes was vastly greater, however, 14% of genes altered in postnatal AAV-Galgt2 infected mdx muscles were also changed with embryonic overexpression. These experiments suggest that postnatal overexpression of Galgt2 inhibits muscular dystrophy in mdx mice via induction of a group of genes that, in aggregate, can govern membrane stability, membrane repair, calcium homeostasis, and apoptosis. Experiment Overall Design: Microarrays were done in three groups at a time. 1) Transgenic Overexpression of Galgt2 - comprised to Wild Type (WT), Galgt2 Transgenic (CT), Dystrophin-Deficient (mdx), and Galgt2 Transgenic and Dystrophin-Deficient (CTmdx) each in duplicate 2) AAV-Mediated Galgt2 Gene Delivery in to the mdx gastrocnemius muscle in the postnatal period - comprised of AAVGalgt2 and PBS each in duplicate, and 3) Myoblasts and myotubes - comptised of one sample each of C1C12 myoblasts and myotubes, C2C12 myoblasts and myotubes stably transfected with Galgt2

Project description:Methods for studying mitochondrial adaptations in skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II skeletal muscle fibres. In this project, we utilised the latest Mass spectrometry (MS) techniques to provide new insights into mitochondrial adaptations in type I and II fibres in response to two different types of training – moderate-intensity-continuous training (MICT) and sprint-interval training (SIT). An 8-week training intervention was undertaken by 23 men who performed either MICT or SIT. Single muscle fibres from skeletal muscle biopsies were collected at rest, before and after the 8 weeks of training, and pooled for subsequent MS analysis. A proteomic workflow was applied that permitted a three-tiered comparison and quantification of mitochondrial proteins in different fibre types. Our protocol includes tandem mass tag labelling for increased identification of low-abundant proteins. We quantified more than 45% of known mitochondrial proteins in skeletal muscle. When comparing type I to type II fibres,24 mitochondrial proteins were differentially expressed following MICT and 10 following SIT. These altered proteins were associated with known cellular pathways within the mitochondria, including oxidative phosphorylation, the TCA cycle and fatty acid oxidation. There were distinct trends for fibre-type-specific protein responses to different types of exercise training. Following MICT proteins mostly increased in abundance in type I fibres, without analogous changes observed following SIT. This greater upregulation of mitochondrial proteins observed following MICT, suggests exercise volume is a powerful stimulus for mitochondrial adaptations. This upregulation mostly seen in type I fibres is consistent with the predominate recruitment of this fibre type with MICT. When normalised to mitochondrial content further evidence to fibre-specific non-stoichiometrically induced increases in the expression of fatty acid mitochondrial proteins is presented, highlighting mitochondria as a pivotal subcellular site in facilitating substrate utilisation to increase ATP production in type I fibres. Conversely, the research suggests very high-intensity exercise training altered the systematic biogenesis of oxidative phosphorylation (OXPHOS) components, in particular complex IV subunits of the OXPHOS pathway. These results question the existing knowledge of fibre-type-specific changes to mitochondrial proteins in response to exercise training and provide a valuable contribution to understanding the mechanisms by which exercise helps to improve health and prevent disease.

Project description:Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)8–13 expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets. A17.1 transgenic mice have previously been described. Male A17.1 mice and WT controls were generated by crossing the heterozygous carrier strain A17.1 obtained from Rubinsztein's group with the FvB background mice. The mice were genotyped by PCR 3–4 weeks after birth. Wild type FvB and A17.1 mice were housed in minimal disease facilities (Royal Holloway, University of London) with food and water ad libitum. Total RNA was extracted from skeletal muscles using RNA Bee (Amsbio) according to the manufacturer's instructions. RNA integration number (RIN) was determined with RNA 6000 Nano (Agilent Technologies). RNA with RIN >7 were used for subsequent steps. RNA labelling was performed with the Illumina® TotalPrep RNA Amplification kit (Ambion) according to the manufacturer's protocol, and subsequently was hybridized to Illumina Mouse v1.1 Bead arrays.

Dataset Information

The Mitochondrial Calcium Uniporter controls skeletal muscle mass in vivo

Publications

The mitochondrial calcium uniporter controls skeletal muscle trophism in vivo.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets