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Influence of ageing and essential amino acids on quantitative patterns of troponin T alternative splicing in human skeletal muscle.

ABSTRACT: Ageing is associated with a loss of skeletal muscle performance, a condition referred to as sarcopenia. In part, the age-related reduction in performance is due to a selective loss of muscle fiber mass, but mass-independent effects have also been demonstrated. An important mass-independent determinant of muscle performance is the pattern of expression of isoforms of proteins that participate in muscle contraction (e.g., the troponins). In the present study, we tested the hypothesis that ageing impairs alternative splicing of the pre-mRNA encoding fast skeletal muscle troponin T (TNNT3) in human vastus lateralis muscle. Furthermore, we hypothesized that resistance exercise alone or in combination with consumption of essential amino acids would attenuate age-associated effects on TNNT3 alternative splicing. Our results indicate that ageing negatively affects the pattern of TNNT3 alternative splicing in a manner that correlates quantitatively with age-associated reductions in muscle performance. Interestingly, whereas vastus lateralis TNNT3 alternative splicing was unaffected by a bout of resistance exercise 24 h prior to muscle biopsy, ingestion of a mixture of essential amino acids after resistance exercise resulted in a significant shift in the pattern of TNNT3 splice form expression in both age groups to one predicted to promote greater muscle performance. We conclude that essential amino acid supplementation after resistance exercise may provide a means to reduce impairments in skeletal muscle quality during ageing in humans.

SUBMITTER: Coble J

PROVIDER: S-EPMC4527890 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Influence of ageing and essential amino acids on quantitative patterns of troponin T alternative splicing in human skeletal muscle.

Coble Joel J Schilder Rudolf J RJ Berg Arthur A Drummond Micah J MJ Rasmussen Blake B BB Kimball Scot R SR

Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 20150323 8

Ageing is associated with a loss of skeletal muscle performance, a condition referred to as sarcopenia. In part, the age-related reduction in performance is due to a selective loss of muscle fiber mass, but mass-independent effects have also been demonstrated. An important mass-independent determinant of muscle performance is the pattern of expression of isoforms of proteins that participate in muscle contraction (e.g., the troponins). In the present study, we tested the hypothesis that ageing i ...[more]

PMID: 26201856

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Project description:BackgroundAgeing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown.MethodsStudies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ.ResultsLevels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ.ConclusionsCardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.

Project description:BackgroundMuscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.Methodology/principal findingsWe used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.Conclusions/significanceTaken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.

Dataset Information

Influence of ageing and essential amino acids on quantitative patterns of troponin T alternative splicing in human skeletal muscle.

Publications

Influence of ageing and essential amino acids on quantitative patterns of troponin T alternative splicing in human skeletal muscle.

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