MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Muscle fiber phenotype: a culprit of abnormal metabolism and function in skeletal muscle of humans with obesity.

The proportion of the different types of fibers in a given skeletal muscle contributes to its overall metabolic and functional characteristics. Greater proportion of type I muscle fibers is associated with favorable oxidative metabolism and function of the muscle. Humans with obesity have a lower proportion of type I muscle fibers. We discuss how lower proportion of type I fibers in skeletal muscle of humans with obesity may explain metabolic and functional abnormalities reported in these individuals. These include lower muscle glucose disposal rate, mitochondrial content, protein synthesis, and quality/contractile function, as well as increased risk for heart disease, lower levels of physical activity, and propensity for weight gain/resistance to weight loss. We delineate future research directions and the need to examine hybrid muscle fiber populations, which are indicative of a transitory state of fiber phenotype within skeletal muscle. We also describe methodologies for precisely characterizing muscle fibers and gene expression at the single muscle fiber level to enhance our understanding of the regulation of muscle fiber phenotype in obesity. By contextualizing research in the field of muscle fiber type in obesity, we lay a foundation for future advancements and pave the way for translation of this knowledge to address impaired metabolism and function in obesity.

Genes Whose Gain or Loss of Function Changes Type 1, 2A, 2X, or 2B Muscle Fibre Proportions in Mice-A Systematic Review.

Adult skeletal muscle fibres are classified as type 1, 2A, 2X, and 2B. These classifications are based on the expression of the dominant myosin heavy chain isoform. Muscle fibre-specific gene expression and proportions of muscle fibre types change during development and in response to exercise, chronic electrical stimulation, or inactivity. To identify genes whose gain or loss-of-function alters type 1, 2A, 2X, or 2B muscle fibre proportions in mice, we conducted a systematic review of transgenic mouse studies. The systematic review was conducted in accordance with the 2009 PRISMA guidelines and the PICO framework. We identified 25 "muscle fibre genes" (Akirin1, Bdkrb2, Bdnf, Camk4, Ccnd3, Cpt1a, Epas1, Esrrg, Foxj3, Foxo1, Il15, Mapk12, Mstn, Myod1, Ncor1, Nfatc1, Nol3, Ppargc1a, Ppargc1b, Sirt1, Sirt3, Thra, Thrb, Trib3, and Vgll2) whose gain or loss-of-function significantly changes type 1, 2A, 2X or 2B muscle fibre proportions in mice. The fact that 15 of the 25 muscle fibre genes are transcriptional regulators suggests that muscle fibre-specific gene expression is primarily regulated transcriptionally. A reanalysis of existing datasets revealed that the expression of Ppargc1a and Vgll2 increases and Mstn decreases after exercise, respectively. This suggests that these genes help to regulate the muscle fibre adaptation to exercise. Finally, there are many known DNA sequence variants of muscle fibre genes. It seems likely that such DNA sequence variants contribute to the large variation of muscle fibre type proportions in the human population.

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

KEY POINTS: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation-contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading. ABSTRACT: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)-positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C-terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross-sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading.

Multi-Omics Approach to Mitochondrial DNA Damage in Human Muscle Fibers.

Mitochondrial DNA deletions affect energy metabolism at tissue-specific and cell-specific threshold levels, but the pathophysiological mechanisms determining cell fate remain poorly understood. Chronic progressive external ophthalmoplegia (CPEO) is caused by mtDNA deletions and characterized by a mosaic distribution of muscle fibers with defective cytochrome oxidase (COX) activity, interspersed among fibers with retained functional respiratory chain. We used diagnostic histochemistry to distinguish COX-negative from COX-positive fibers in nine muscle biopsies from CPEO patients and performed laser capture microdissection (LCM) coupled to genome-wide gene expression analysis. To gain molecular insight into the pathogenesis, we applied network and pathway analysis to highlight molecular differences of the COX-positive and COX-negative fiber transcriptome. We then integrated our results with proteomics data that we previously obtained comparing COX-positive and COX-negative fiber sections from three other patients. By virtue of the combination of LCM and a multi-omics approach, we here provide a comprehensive resource to tackle the pathogenic changes leading to progressive respiratory chain deficiency and disease in mitochondrial deletion syndromes. Our data show that COX-negative fibers upregulate transcripts involved in translational elongation and protein synthesis. Furthermore, based on functional annotation analysis, we find that mitochondrial transcripts are the most enriched among those with significantly different expression between COX-positive and COX-negative fibers, indicating that our unbiased large-scale approach resolves the core of the pathogenic changes. Further enrichments include transcripts encoding LIM domain proteins, ubiquitin ligases, proteins involved in RNA turnover, and, interestingly, cell cycle arrest and cell death. These pathways may thus have a functional association to the molecular pathogenesis of the disease. Overall, the transcriptome and proteome show a low degree of correlation in CPEO patients, suggesting a relevant contribution of post-transcriptional mechanisms in shaping this disease phenotype.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function.

Single muscle fiber proteomics reveals unexpected mitochondrial specialization.

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

Calsequestrins in skeletal and cardiac muscle from adult Danio rerio.

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.

Spaceflight on the ISS changed the skeletal muscle proteome of two astronauts.

Skeletal muscle undergoes atrophy and loss of force during long space missions, when astronauts are persistently exposed to altered gravity and increased ionizing radiation. We previously carried out mass spectrometry-based proteomics from skeletal muscle biopsies of two astronauts, taken before and after a mission on the International Space Station. The experiments were part of an effort to find similarities between spaceflight and bed rest, a ground-based model of unloading, focused on proteins located at the costameres. We here extend the data analysis of the astronaut dataset and show compartment-resolved changes in the mitochondrial proteome, remodeling of the extracellular matrix and of the antioxidant response. The astronauts differed in their level of onboard physical exercise, which correlated with their respective preservation of muscle mass and force at landing in previous analyses. We show that the mitochondrial proteome downregulation during spaceflight, particularly the inner membrane and matrix, was dramatic for both astronauts. The expression of autophagy regulators and reactive oxygen species scavengers, however, showed partially opposite expression trends in the two subjects, possibly correlating with their level of onboard exercise. As mitochondria are primarily affected in many different tissues during spaceflight, we hypothesize that reactive oxygen species (ROS) rather than mechanical unloading per se could be the primary cause of skeletal muscle mitochondrial damage in space. Onboard physical exercise might have a strong direct effect on the prevention of muscle atrophy through mechanotransduction and a subsidiary effect on mitochondrial quality control, possibly through upregulation of autophagy and anti-oxidant responses.

Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition.

OBJECTIVE: Skeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively. METHODS: We analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice. RESULTS: We show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates. CONCLUSIONS: We show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions.

Acyl-CoA thioesterase-2 facilitates ß-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner.

Acyl-Coenzyme A (acyl-CoA) thioesters are compartmentalized intermediates that participate in in multiple metabolic reactions within the mitochondrial matrix. The limited availability of free CoA (CoASH) in the matrix raises the question of how the local acyl-CoA concentration is regulated to prevent trapping of CoASH from overload of any specific substrate. Acyl-CoA thioesterase-2 (ACOT2) hydrolyzes long-chain acyl-CoAs to their constituent fatty acids and CoASH, and is the only mitochondrial matrix ACOT refractory to inhibition by CoASH. Thus, we reasoned that ACOT2 may constitutively regulate matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up when lipid supply and energy demands were modest. When energy demand and pyruvate availability were elevated, lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2 , and overt inhibition of ß-oxidation was demonstrated in isolated mitochondria from Acot2 -depleted glycolytic SM. In mice fed a high fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. These observations suggest that ACOT2 supports CoASH availability to facilitate ß-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Plasma proteome profiling of healthy subjects undergoing bed rest reveals unloading-dependent changes linked to muscle atrophy.

BACKGROUND: Inactivity and unloading induce skeletal muscle atrophy, loss of strength and detrimental metabolic effects. Bed rest is a model to study the impact of inactivity on the musculoskeletal system. It not only provides information for bed-ridden patients care, but it is also a ground-based spaceflight analogue used to mimic the challenges of long space missions for the human body. In both cases, it would be desirable to develop a panel of biomarkers to monitor muscle atrophy in a minimally invasive way at point of care to limit the onset of muscle loss in a personalized fashion. METHODS: We applied mass spectrometry-based proteomics to measure plasma protein abundance changes in response to 10 days of bed rest in 10 young males. To validate the correlation between muscle atrophy and the significant hits emerging from our study, we analysed in parallel, with the same pipeline, a cohort of cancer patients with or without cachexia and age-matched controls. Our analysis resulted in the quantification of over 500 proteins. RESULTS: Unloading affected plasma concentration of proteins of the complement cascade, lipid carriers and proteins derived from tissue leakage. Among the latter, teneurin-4 increased 1.6-fold in plasma at bed rest day 10 (BR10) compared with BR0 (6.E9 vs. 4.3E9, P = 0.02) and decreased to 0.6-fold the initial abundance after 2 days of recovery at normal daily activity (R + 2, 2.7E9, P = 3.3E-4); the extracellular matrix protein lumican was decreased to 0.7-fold (1.2E9 vs. 8.5E8, P = 1.5E-4) at BR10 and remained as low at R + 2. We identified six proteins distinguishing subjects developing unloading-mediated muscle atrophy (decrease of >4% of quadriceps cross-sectional area) from those largely maintaining their initial muscle mass. Among them, transthyretin, a thyroid hormone-binding protein, was significantly less abundant at BR10 in the plasma of subjects with muscle atrophy compared with those with no atrophy (1.6E10 vs. 2.6E10, P = 0.001). Haptoglobin-related protein was also significantly reduced in the serum of cancer patients with cachexia compared with that of controls. CONCLUSIONS: Our findings highlight a combination or proteomic changes that can be explored as potential biomarkers of muscle atrophy occurring under different conditions. The panel of significant proteomic differences distinguishing atrophy-prone and atrophy-resistant subjects after 10 days of bed rest need to be tested in a larger cohort to validate their potential to predict inactivity-triggered muscle loss in humans.

NFAT isoforms control activity-dependent muscle fiber type specification.

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.

Signatures of muscle disuse in spaceflight and bed rest revealed by single muscle fiber proteomics.

Astronauts experience dramatic loss of muscle mass, decreased strength, and insulin resistance, despite performing daily intense physical exercise that would lead to muscle growth on Earth. Partially mimicking spaceflight, prolonged bed rest causes muscle atrophy, loss of force, and glucose intolerance. To unravel the underlying mechanisms, we employed highly sensitive single fiber proteomics to detail the molecular remodeling caused by unloading and inactivity during bed rest and changes of the muscle proteome of astronauts before and after a mission on the International Space Station. Muscle focal adhesions, involved in fiber-matrix interaction and insulin receptor stabilization, are prominently downregulated in both bed rest and spaceflight and restored upon reloading. Pathways of antioxidant response increased strongly in slow but not in fast muscle fibers. Unloading alone upregulated markers of neuromuscular damage and the pathway controlling EIF5A hypusination. These proteomic signatures of mechanical unloading in muscle fiber subtypes contribute to disentangle the effect of microgravity from the pleiotropic challenges of spaceflight.

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study.

BACKGROUND: Human skeletal muscle is composed of three major fiber types, referred to as type 1, 2A, and 2X fibers. This heterogeneous cellular composition complicates the interpretation of studies based on whole skeletal muscle lysate. A single-fiber proteomics approach is required to obtain a fiber-type resolved quantitative information on skeletal muscle pathophysiology. METHODS: Single fibers were dissected from vastus lateralis muscle biopsies of young adult males and processed for mass spectrometry-based single-fiber proteomics. We provide and analyze a resource dataset based on relatively pure fibers, containing at least 80% of either MYH7 (marker of slow type 1 fibers), MYH2 (marker of fast 2A fibers), or MYH1 (marker of fast 2X fibers). RESULTS: In a dataset of more than 3800 proteins detected by single-fiber proteomics, we selected 404 proteins showing a statistically significant difference among fiber types. We identified numerous type 1 or 2X fiber type-specific protein markers, defined as proteins present at 3-fold or higher levels in these compared to other fiber types. In contrast, we could detect only two 2A-specific protein markers in addition to MYH2. We observed three other major patterns: proteins showing a differential distribution according to the sequence 1 > 2A > 2X or 2X > 2A > 1 and type 2-specific proteins expressed in 2A and 2X fibers at levels 3 times greater than in type 1 fibers. In addition to precisely quantifying known fiber type-specific protein patterns, our study revealed several novel features of fiber type specificity, including the selective enrichment of components of the dystrophin and integrin complexes, as well as microtubular proteins, in type 2X fibers. The fiber type-specific distribution of some selected proteins revealed by proteomics was validated by immunofluorescence analyses with specific antibodies. CONCLUSION: We here show that numerous muscle proteins, including proteins whose function is unknown, are selectively enriched in specific fiber types, pointing to potential implications in muscle pathophysiology. This reinforces the notion that single-fiber proteomics, together with recently developed approaches to single-cell proteomics, will be instrumental to explore and quantify muscle cell heterogeneity.

Fiber type diversity in skeletal muscle explored by mass spectrometry-based single fiber proteomics.

Mammalian skeletal muscles are composed of a variety of muscle fibers with specialized functional properties. Slow fibers are suited for long lasting and low intensity contractile activity, while various subtypes of fast fibers are optimized to produce high force and power even with a significant fatigue. The functional specialization of muscle fibers is based on selective gene expression regulation, which provides each fiber with a specific protein complement. The recent refinement of small-scale sample preparation, combined with the development of mass spectrometers characterized by high sensitivity, sequencing speed and mass accuracy, has allowed the characterization of the proteome of single muscle fibers with an unprecedented resolution. In the last few years, the first studies on the global proteomics of individual fibers of different types have been published. In this short review we discuss the methodological advancements which have opened the way to single fiber proteomics and the discovery power of this approach. We provide examples of how specific features of single fibers can be overlooked when whole muscle or multi-fiber samples are analyzed and can only be detected when a single fiber proteome is analyzed. Thus, novel subtype-specific metabolic features, most prominently mitochondrial specialization of fiber types have been revealed by single fiber proteomics. In the same way, specific adaptive responses of single fibers to aging or loss of neural input have been detected when single fibers were individually analyzed. We conclude that the fiber type-resolved proteomes represent a powerful tool which can be applied to a variety of physiological and pathological conditions.

Calsequestrins New Calcium Store Markers of Adult Zebrafish Cerebellum and Optic Tectum.

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.