Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging

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

GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells

<p>Abstract</p> <p>Background</p> <p>We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells.</p> <p>Results</p> <p>Mutation or deletion of GATA elements induces a strong transcriptional activation of the cTnI promoter in regenerating skeletal muscle and in cultured skeletal muscle cells. Electrophoretic mobility shift assays show that proteins present in nuclear extracts of C2C12 muscle cells bind the GATA motifs present in the cTnI promoter. However, GATA protein complex formation is neither reduced nor supershifted by antibodies specific for GATA-2, -3 and -4, the only GATA transcripts present in muscle cells.</p> <p>Conclusion</p> <p>These findings indicate that the cTnI gene promoter is repressed in skeletal muscle cells by GATA-like factors and open the way to further studies aimed at identifying these factors.</p

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

Role of Strain Elastography and Shear-Wave Elastography in a Multiparametric Clinical Approach to Indeterminate Cytology Thyroid Nodules

BACKGROUND In thyroid nodules with indeterminate cytology, further clinical assessment aimed at ruling out malignancy is often mandatory. Ancillary imaging techniques and genetic mutation analysis can improve the risk stratification of such lesions, thereby facilitating the clinician's decision to undertaken surgery or simple follow-up. The aim of this study was to evaluate the diagnostic performance of shear-wave elastography (SW), strain elastography (ELX 2/1), conventional ultrasound (US), contrast-enhanced ultrasound (CEUS), and BRAF V600E mutation analysis in the aforementioned lesions. MATERIAL AND METHODS We enrolled 81 patients, each with 1 indeterminate-cytology thyroid nodule. Thyroid function, thyroperoxidase antibodies and calcitonin were known in each case. SW, ELX 2/1, US, CEUS, and BRAF mutation analysis were subsequently performed, followed by a second FNAB. If the lesion was not downgraded to benign, surgery was recommended and histological reports collected. RESULTS There were 28 nodules (34%) that proved benign on the second FNAB; 38 nodules (47%) underwent surgery (17 benign, 21 malignant), and 15 nodules (19%) refused surgery. The only techniques related to histological outcome were US (AUC=0,766), ELX 2/1 (AUC=0.701), and BRAF analysis (AUC=0.762). ELX 2/1 and SW reports were not correlated with each other (P=0.45). A scoring system taking into account all the variables considered performed better than the single variables alone (AUC=0.831). CONCLUSIONS In indeterminate-cytology thyroid lesions, repeating FNAB can avoid unnecessary surgery. ELX 2/1 seems to perform better than SW in distinguishing malignancy; these techniques could, however, be complementary in describing such lesions. A multiparametric approach appears the most accurate in predicting nodule histology

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

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

Structure and regulation of the mouse cardiac troponin I gene

The gene coding for mouse cardiac troponin I (TnI) has been cloned and sequenced. The cardiac TnI gene contains 8 exons and has an exon-intron organization similar to the quail fast skeletal TnI gene except for the region of exons 1-3, which is highly divergent. Comparative analysis suggests that cardiac TnI exon 1 corresponds to fast TnI exons 1 and 2 and that cardiac exon 3, which codes for most of the cardiac-specific amino-terminal extension and has no counterpart in the fast gene, evolved by exon insertion/deletion. The amino acid sequence of cardiac TnI exon 4 shows limited homology (36% identity) with fast TnI exon 4 but is remarkably similar (79% identity) to the corresponding sequence of slow TnI, possibly reflecting an isoform-specific TnC-binding site. The cardiac TnI gene is one of the very few contractile protein genes expressed exclusively in cardiac muscle. To identify the regulatory sequences responsible for the cardiac-specific expression of this gene we transfected cultured cardiac and skeletal muscle cells with fragments up to 4.0 kilobases of the 5'-flanking region linked to a reporter gene. Deletion analysis reveals four major regions in the 5'-flanking sequence, a minimal promoter region, which directs expression at low level in cardiac and skeletal muscle cells, and two upstream cardiac-specific positive regions separated by a negative region

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS:We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression

Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles

The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals

Aging: a portrait from gene expression profile in blood cells

The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43 +/- 3 and 68 +/- 4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process