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Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.

ABSTRACT: Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro function. The Met8Arg mutation reduces tropomyosin affinity for regulated actin 30- to 100-fold. Ca(2+)-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca(2+) is reduced. The poor activation may reflect weakened actin affinity or reduced effectiveness in switching the thin filament to the open, force-producing state. The presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impair end-to-end association between TMs on the thin filament. In muscle, the mutation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end of the filament. The mutation may also reduce force generation during activation.

SUBMITTER: Moraczewska J

PROVIDER: S-EPMC1301196 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.

Moraczewska J J Greenfield N J NJ Liu Y Y Hitchcock-DeGregori S E SE

Biophysical journal 20001201 6

Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg ...[more]

PMID: 11106625

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Project description:BackgroundCongenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown.MethodsHistopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated.ResultsWhile TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment.ConclusionsThese results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Project description:Purpose: Congenital myopathies (CM) are a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A novel and de novo mutation in tropomyosin 3 [TPM3(E151G)] was identified from a 7-year-old boy with clinically diagnosed CM. TPM3(E151A) mutation was previously reported as causing CM. In this study, we created transgenic zebrafish to investigate the pathogenicity and find potential treatment. The goals of this study are to describe the RNA contents in muscle specimens derived from TPM3(WT), TPM3(E151A), TPM3(E152G) at embryos and adult stages and to compare the differences TPM3(E151A), TPM3(E151G) mutants and TPM3(WT). The biological function of L-carnitine treatment TPM3(E151G) versus without treatment are also predicted and evaluated. Methods: Muscle mRNA profiles of 6 months adult zebrafish from TPM3(WT), TPM3(E151A) and TPM3(E151G) were extracted and performed deep sequencing, in duplicate, using Illumina Novaseq6000. We also obtained the muscle mRNA profiles of 2 day's zebrafish embryos from TPM3(WT), TPM3(E151A), TPM3(E151G) and TPM3(E151G) treated with L-carnitine. RNA profiles of adult and embryos were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we mapped about 40 million sequence reads per sample to the zebrafish genome (GRCz11). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with zebrafish genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first detailed analysis of zebrafish muscle transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles between TPM3(WT), TPM3(E151A) and TPM3(E151G) and L-carnitine treatment. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Dataset Information

Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.

Publications

Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.

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