23 research outputs found
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
Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle
Summary: Skeletal muscle is composed of different myofiber types that preferentially use glucose or lipids for ATP production. How fuel preference is regulated in these post-mitotic cells is largely unknown, making this issue a key question in the fields of muscle and whole-body metabolism. Here, we show that microRNAs (miRNAs) play a role in defining myofiber metabolic profiles. mRNA and miRNA signatures of all myofiber types obtained at the single-cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated mouse myofiber type-specific catalog of gene and miRNA expression and establishes miR-27a-3p and miR-142-3p as regulators of lipid use in skeletal muscle. : Chemello et al. characterize coding mRNAs and non-coding microRNAs expressed by myofibers of hindlimb mouse muscles, identifying complex interactions between these molecules that modulate mitochondrial functions and muscle metabolism. They demonstrate that specific short non-coding RNAs influence the contractile fiber composition of skeletal muscles by modulating muscle metabolism. Keywords: single myofiber, skeletal muscle metabolism, mitochondria, miRNAs, lipid
Microgenomic Analysis in Skeletal Muscle: Expression Signatures of Individual Fast and Slow Myofibers
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 condition
DETERMINATION OF CONTRACTILE PARAMETERS OF FIBRES FROM CARNIVORES JAW MUSCLES.
In mammalian genome, the genes coding for various MyHC isoforms
are grouped in clusters located in different chromosomes
to form 3 subfamilies: a) fast isoforms (2A, 2X, 2B, embryonic,
neonatal, extraocular); b) cardiac isoforms (beta/slow and alpha);
c) masticatory isoform (M). M-MyHC is a myosin subunit isoform
with expression restricted to muscles derived from the first branchial
arch, such as jaw closer muscles. Only sparse information is available
on the contractile properties of fibres expressing M-MyHC (M fibres).
In this study we characterized M fibres in the jaw closer muscles
(temporalis and masseter) of two species of domestic carnivores (cat
and dog) in comparison with fibres expressing slow or fast (2A, 2X
and 2B) isoforms. In each fibre isometric tension (Po) and unloaded
shortening velocity (Vo) were determined during maximal calcium
activation at 12C. To evaluate temperature effects on isometric
tension, a subgroup of fibres were activated at 25C. The results
obtained showed that in both species Vo increased regularly from
slow to 2A to 2X fibres and that M fibres had Vo value similar to 2A
fibres. M fibres showed Po values definitely greater than in any other
fibre type both in cat and dog. The diversity in Po value among fibre
types was similar at both temperatures and in all fibre types Po was
50% greater at 25C than at 12C.
The M myosin offers an optimal contractile performance and its
presence is the result of specific embryologically determined
expression programs and intense selective pressure related to feeding
habit
Masticatory myosin unveiled: first determination of contractile parameters of muscle fibers from carnivore jaw muscles
Determination of contractile parameters of muscle fibres from two species ofcarnivores: the cat and the dog.
Myosin heavy chain isoform (MyHC) expression patternin human extraocular muscles
Human extraocular muscles (EO) express a complex pattern of MyHC isoforms as the specific EO, skeletal ones (type 1, 2A, 2X), developmental isoforms (Embryonic and Perinatal, all life long), the alpha cardiac, also expressed in masseter and laryngeal, and the recently found 2B. Moreover, in EO muscles another isoform was identified, the slow tonic, but is not well characterized at genomic and protein level. Recently, Schiaffino and collaborators (Rossi et al., J Physiol 588.2, 2010) studied two new genes concluding that MYH14/7b correspond to slow tonic and MYH15 gene to ventricular isoform of chicken. Another author found, on the contrary, that the slow tonic is codified by MYH15 gene (Rahnert et al., Cell Tissues Organs 191, 2010). Our results, assessed by Realtime PCR and protein electrophoresis, comparing skeletal, laryngeal, EO, masseter and atrium human muscles, confirmed, at RNA and protein level, that MYH14/7b correspond to the slow tonic isoform. For the first time, we clarify the electrophoretic position of slow tonic MyHC between EO and cardiac bands
SINGLE MUSCLE FIBER PROTEOMICS REVEALS UNEXPECTED MITOCHONDRIAL SPECIALIZATION
To perform their diverse tasks, skeletal muscles employ four different fiber types, one type 1/slow and three different type 2/fast fibers. These multinucleated cellular units are characterized by different contractile and metabolic properties and can be combined in muscles in a dynamic way resulting in muscle plasticity. To dissect the composite and plastic metabolic properties of skeletal muscle we have developed a high sensitivity workflow based on liquid chromatography/mass spectrometry-based shotgun proteomics optimized for low abundant and high dynamic range samples. Using this procedure in mouse soleus and EDL muscles, we have obtained not only the quantification of single fiber proteomes but even of different segments of the same fiber. Our data indicate that the four fiber types significantly differ in the expression of structural and regulatory proteins which are likely to contribute to their specific physiology. In particular, specialization of mitochondria within the oxidative subset of muscle fibers emerged as one of the prominent insights revealed by this analysis, highlighting fiber type-specific patterns in the utilization of metabolic intermediates. As the single cell proteomics analysis performed here is rapid and robust, it can be applied to a wide variety of physiological and pathological conditions