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Background: Adult muscle fibres are classified as type 1, 2A, 2X, and 2B muscle fibres based on the expression of the dominant myosin heavy chain isoform. Muscle fibre-specific gene expression and muscle fibre types change during development and in response to changes in contractile activity such as exercise, chronic electrical stimulation versus inactivity or denervation. Methods: To identify genes whose gain or loss-of-function changes type 1, 2A, 2X or 2B muscle fibre proportions in mice, we conducted a systematic review following the 2020 PRISMA guidelines and the PICO framework.Results: 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, Vgll2) whose gain or loss-of-function 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. Muscle fibre genes such as Ppaargc1a and Vgll2 increase or Mstn decrease their expression after exercise, respectively, suggesting that this contributes to muscle adaptation to exercise. Finally, there are many known DNA sequence variants of muscle fibre genes suggesting that this contributes to the large variation of muscle fibre percentages in humans. Conclusion: Muscle fibre genes are a set of genes that often regulate gene expression and where DNA variants are likely to change human muscle fibre distribution.

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.

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.

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

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.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies., Skeletal muscle conveys the beneficial effects of physical exercise but due to its heterogeneity, studying the effects of exercise on muscle fibres is challenging. Here, the authors carry out proteomic analysis of myofibres from freeze-dried muscle biopsies, show fibre-type specific changes in response to exercise, and show that the oxidative and glycolytic muscle fibers adapt differentially to exercise training.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22015-4

A thorough understanding of skeletal muscle physiology requires knowledge of the molecular composition of muscle tissue. So far, this has been mostly obtained through biochemical investigations focused on selected components of the muscle contractile and metabolic machinery. More recently, a systems biology approach, based on the emergence of omics technologies, has been introduced to get global views of all muscle components. These approaches have been used to explore the DNA (genomics and epigenomics), RNA (transcriptomics and non-coding RNA analyses), proteins (proteomics and global analyses of post-translational protein modifications, e.g. phosphoproteomics) and small molecules (metabolomics).

Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. The single fiber investigation of this important diagnostic feature has been beyond the capability of large-scale technologies so far. We used laser capture microdissection (LCM) to excise thin sections of individual muscle fibers from frozen biopsies of patients suffering from chronic progressive external ophthalmoplegia. We then applied a highly sensitive mass spectrometry (MS)-based proteomics workflow to analyze healthy and defective muscle fibers within the same biopsy. We quantified more than 4000 proteins in each patient, covering 75% of all respiratory chain subunits, and compared their expression in metabolically healthy and defective muscle fibers. Our findings show that mitochondrial disease causes extensive proteomic rearrangements, affecting the OPA1-dependent cristae remodeling pathway and mitochondrial translation. We provide fiber type-specific information showing that increased expression of fatty acid oxidation enzymes occurs in defective slow but not fast muscle fibers. Our findings shed light on compensatory mechanisms in muscle fibers that struggle with energy shortage and metabolic stress.

We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

The long awaited molecular identification of the mitochondrial calcium uniporter (MCU) in 2011 has opened an exciting phase in the study of mitochondrial calcium homeostasis. On the one hand, MCU proved to be the core of a complex signaling system, composed of a channel moiety (MCU itself and the related MCUb protein) and a family of essential regulators (the MICUs, MCUR, EMRE). On the other hand, the availability of molecular information and tools opened the possibility of directly altering mitochondrial calcium homeostasis in cell cultures or intact organisms, thus obtaining new insight into its role in physiological and pathological events. We will review here these exciting advancements, summarizing the current knowledge of the molecular composition of the MCU complex and of its role in shaping mitochondrial and cytosolic [Ca(2+)] signals.

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.

Background Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. This heterogeneity is a powerful diagnostic signpost of mitochondrial disorders. However, the single fiber investigation of its molecular basis has been beyond the capability of large-scale technologies so far. Methods We used a novel approach to single muscle fibers proteomics based on laser capture microdissection (LCM). We excised thin sections of individual muscle fibers from frozen biopsies of patients with mitochondrial disorders. Combined with a highly sensitive mass spectrometry (MS)-based proteomics workflow, this technique allowed us to analyze healthy and defective muscle fibers within the same biopsy. Findings Using less than 50 ng of muscle, we quantified more than 4000 proteins in each patient including over 75% of all respiratory chain subunits, and compared their expression in metabolically healthy and defective muscle fibers. We found a plethora of changes, most notably increases in the expression of the STOML2-PHB axis regulating inner membrane biogenesis and of the OPA1-dependent cristae remodeling pathway in defective fibers. We observed fiber type-specific information showing that increased expression of fatty acid oxidation enzymes occurs in defective slow but not fast muscle fibers. Interpretation Our findings show that mitochondrial disease causes extensive proteomic rearrangements, involving not only the expression of the respiratory chain but also critical players of mitochondrial dynamics. These findings shed light on compensatory mechanisms in muscle fibers that struggle with energy shortage and metabolic stress. Funding: Max‐Planck Society for the Advancement of Science; Louis-Jeantet Foundation; Bundesministerium fur Bildung und Forschung (BMBF); European Union Horizon 2020 Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The research project was approved by the ethics committee of the LMU Munich (N. 198-15) and the study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner.

Increased glucose transporter GLUT4 expression in skeletal muscle is an important benefit of regular exercise, resulting in improved insulin sensitivity and glucose tolerance. The Ca2+–calmodulin-dependent kinase II (CaMKII), calcineurin and AMPK pathways have been implicated in GLUT4 gene regulation based on pharmacological evidence. Here, we have used a more specific genetic approach to establish the relative role of the three pathways in fast and slow muscles. Plasmids coding for protein inhibitors of CaMKII or calcineurin were co-transfected in vivo with a GLUT4 enhancer-reporter construct either in normal mice or in mice expressing a kinase dead (KD) AMPK mutant. GLUT4 reporter activity was not inhibited in the slow soleus muscle by blocking either CaMKII or calcineurin alone, but was inhibited by blocking both pathways. GLUT4 reporter activity was likewise unchanged in the soleus of KD-AMPK mice, but was significantly reduced by incapacitation of either CaMKII or calcineurin in these mice. On the other hand, in the fast tibialis anterior (TA) muscle, calcineurin appears to exert a prominent role in the control of GLUT4 reporter activity, independent of CaMKII and AMPK. The results point to a muscle type-specific and redundant regulation of GLUT4 enhancer based on the interplay of multiple signalling pathways, all of which are known to affect myocyte enhancing factor 2 (MEF2) transcriptional activity, a point of convergence of different pathways on muscle gene regulation.

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.

et al., The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia., This work is supported by grants from the European Commission (Integrated Project MYOAGE no. 223576 to SS) and Association Francaise pour les Myopathies (AFMTelethon grant to SS).

Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTP is cleaved by three proteases: furin, ADAM 10, and -secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTP. This is followed by -secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates -catenin, an RPTP substrate. The expression of RPTP suppresses -catenin’s transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of -catenin signaling. We also found that RPTP, another MAM family member, undergoes -secretasedependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTP signaling and implicate PIC in the activation of -catenin-mediated transcription. The phosphorylation of cellular proteins on tyrosine residues is reversible and regulated by the coordinated and competing actions of two enzyme families: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). The PTP family is structurally diverse and includes both receptor-like and cytoplasmic enzymes. The majority of the receptor PTPs (RPTPs) contain two catalytic domains: a membrane-proximal domain (D1), which is responsible for mainly catalysis, and a membrane-distal domain (D2), which contains little or no phosphatase activity (45). The extracellular (E) portion exhibits broad structural variation. The MAM (meprin/A5 glycoprotein/PTPmu) family of RPTPs, including RPTP ,- ,- ,- , and PCP-2, are characterized by the presence of the MAM domain at their N termini (3, 6, 18, 29, 46). Additional structural features of the extracellular portions involve one immu

to the editor: In a recent paper, Stuart et al. ([7][1]) conclude that muscle fibers isolated by laser capture microdissection (LCM) from the human vastus lateralis muscle 1 ) are all hybrid fibers containing mixtures of fast and slow myosin heavy chain (MYH) isoforms, and 2 ) contain significant

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.

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.

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

Background: It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic ( Escherichia coli ) and eukaryotic ( Caenorhabditis elegans and Drosophila melanogaster ) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. Results In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the α 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca 2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca 2+ indicator. Conclusion GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo , it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.

The effects of anti-CD3 monoclonal antibodies on cytosolic free Ca2+ concentration, [Ca2+]i, were investigated in freshly isolated lymphocytes, T cell lines, T clones and the leukemic T cell line Jurkat with three different methodologies, i.e. classical cuvette experiments, cytofluorimetry and videoimaging. With any technique, concentrations of anti-CD3 antibodies optimal for stimulation of DNA synthesis were completely ineffective at inducing early increases of [Ca2+]i in freshly isolated lymphocytes. At supraoptimal mitogenic concentrations: (i) anti-CD3 mAb induced negligible increases of [Ca2+]i when tested in suspensions of freshly isolated lymphocytes, but the response increased progressively during in vitro culturing with IL2; (ii) most, but not all, T clones, when tested in suspension, were responsive to these concentrations of anti-CD3 antibodies in terms of [Ca2+]i; (iii) using the videoimaging technique at the single cell level, it was demonstrated that the anti-CD3 antibodies induced large increases of [Ca2+]i in lymphocytes only under conditions which allowed adherence of the antibodies (and of the cells) to the glass surface. In all T cell types investigated, the [Ca2+]i increases were most often composed by multiple, asynchronous oscillations. The buffering of [Ca2+]i increases, obtained by loading the cells with membrane permeant esters of Quin-2 and Fura-2, inhibited anti-CD3 mAb induced DNA synthesis, but this appeared entirely attributable to a toxic side effect of the ester hydrolysis. The relevance of these data is discussed in terms of their methodological and functional implications for the understanding of the role of Ca2+ in mitogenic stimulation of T cells.

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol-1,4,5-trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus-cytosol Ca2+ gradients is discussed.

ATP is released in the body from several cells under various physiological and pathological conditions. A number of authors have postulated a role for extracellular ATP (ATPo) as a neurotransmitter, a secretagogue or an inflammatory mediator. Here, we propose an additional role for ATPo, as a cytotoxic factor, and discuss in vitro experiments showing that this nucleotide causes cell death by two mechanisms: colloido-osmotic lysis and apoptosis.

Increased glucose transporter GLUT4 expression in skeletal muscle is an important benefit of regular exercise, resulting in improved insulin sensitivity and glucose tolerance. The Ca(2+)-calmodulin-dependent kinase II (CaMKII), calcineurin and AMPK pathways have been implicated in GLUT4 gene regulation based on pharmacological evidence. Here, we have used a more specific genetic approach to establish the relative role of the three pathways in fast and slow muscles. Plasmids coding for protein inhibitors of CaMKII or calcineurin were co-transfected in vivo with a GLUT4 enhancer-reporter construct either in normal mice or in mice expressing a kinase dead (KD) AMPK mutant. GLUT4 reporter activity was not inhibited in the slow soleus muscle by blocking either CaMKII or calcineurin alone, but was inhibited by blocking both pathways. GLUT4 reporter activity was likewise unchanged in the soleus of KD-AMPK mice, but was significantly reduced by incapacitation of either CaMKII or calcineurin in these mice. On the other hand, in the fast tibialis anterior (TA) muscle, calcineurin appears to exert a prominent role in the control of GLUT4 reporter activity, independent of CaMKII and AMPK. The results point to a muscle type-specific and redundant regulation of GLUT4 enhancer based on the interplay of multiple signalling pathways, all of which are known to affect myocyte enhancing factor 2 (MEF2) transcriptional activity, a point of convergence of different pathways on muscle gene regulation.

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.

Transient increases in intracellular calcium concentration activate and coordinate a wide variety of cellular processes in virtually every cell type. This review describes the main homeostatic mechanisms that control Ca(2+) transients, focusing on the mitochondrial checkpoint. We subsequently extend this paradigm to the cardiomyocyte and to the interplay between cytosol, endoplasmic reticulum and mitochondria that occurs beat-to-beat in excitation-contraction coupling. The mechanisms whereby mitochondria decode fast cytosolic calcium spikes are discussed in the light of the results obtained with recombinant photoproteins targeted to the mitochondrial matrix of contracting cardiomyocytes. Mitochondrial calcium homeostasis is then highlighted as a crucial point of convergence of the environmental signals that mediate cardiac cell death, both by necrosis and by apoptosis. Altogether we point to a role of the mitochondrion as an integrator of calcium signalling and a fundamental decision maker in cardiomyocyte metabolism and survival.

A variety of fiber types with different contractile and metabolic properties is present in mammalian skeletal muscle. The fiber-type profile is controlled by nerve activity via specific signaling pathways, whose identification may provide potential therapeutic targets for the prevention and treatment of metabolic and neuromuscular diseases.

Two members of the EGF receptor family, HER2 and HER3, act as key oncogenes in breast cancer cells. A MAb against HER2, trastuzumab, interferes with HER2 signaling and istherapeutically effective in humans. Here, we explored the biologic effects of an antibody against HER3 (α-HER3ECD) in the invasive breast cancer cell lines MCF-7ADR and MDA-MB-468. Pretreating the breast cancer cells with α-HER3ECD prior to Heregulin stimulation caused significant reduction of the migratory and proliferative properties. This reduction is due to a substantial decrease in the tyrosine phosphorylation content of HER2 and to a modification of the HER2/HER3 association, which ultimately inhibits the activity of the downstream effectors phosphatidyinositol-3-OH-kinase and c-jun-terminal kinase. Furthermore, HER3 is internalized and not activated by HRG after pretreatment with α-HER3ECD. Our data reinforce the notion that HER3 could be a key target in cancer drug design and show the great potential of anti-HER3 antibodies for the therapy of breast cancer and other malignancies characterized by overexpression of HER3. © 2005 Wiley-Liss, Inc.

Motor neurons are known to affect muscle growth and fiber type profile (fast/slow, oxidative/glycolytic) by regulating muscle gene expression. However, the mechanism by which the information contained in specific action potential patterns is decoded by the transcriptional machinery of muscle fiber nuclei remains to be established. This is a basic issue in nerve/muscle biology, which has major implications in neurology, sport medicine and aging. We describe here a general strategy aimed at identifying the signal transduction pathways mediating the effects of nerve activity. This approach is based on the overexpression of constitutively active or dominant negative transduction factors in regenerating skeletal muscle.

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation-transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras-MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.