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Developmental MYH3 Myopathy Associated with Expression of Mutant Protein and Reduced Expression Levels of Embryonic MyHC.

Pokrzywa M, Norum M, Lengqvist J, Ghobadpour M, Abdul-Hussein S, Moslemi AR, Tajsharghi H - PLoS ONE (2015)

Bottom Line: We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins.Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased.We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Medical Genetics, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT

Objective: An essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy.

Methods and results: We determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres.

Conclusion: In conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.

No MeSH data available.


Related in: MedlinePlus

Detection and relative quantification of wild-type and mutated embryonic MyHC.(A and B) Extracted ion chromatograms (XICs) for wild-type and (C and D) mutant embryonic MyHC tryptic peptides eluting at 28.6 and 39.9 minutes are shown as indicated. Note that panels A and C have been adjusted to equal intensity, as have panels B and D.
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pone.0142094.g003: Detection and relative quantification of wild-type and mutated embryonic MyHC.(A and B) Extracted ion chromatograms (XICs) for wild-type and (C and D) mutant embryonic MyHC tryptic peptides eluting at 28.6 and 39.9 minutes are shown as indicated. Note that panels A and C have been adjusted to equal intensity, as have panels B and D.

Mentions: At retention times of 28.6 and 39.9 minutes, common (y2-y7 and b2-b6) and sequence- specific (y8-y13) fragment ions were observed for wild-type (Fig 3A and 3C) and mutant peptides (Fig 3B and 3D), respectively. Peptide identification was apparent by high precursor (< 2 ppm, S2 and S3 Figs) and fragment mass accuracy (RMS < 1.8 ppm for 12 (wild-type) and 13 (mutant) y-ion fragments, S4 and S5 Figs). Importantly, the p.Thr178Ile substitution was apparent from six unique fragment ions in each case. Measured relative intensities of common (non-unique) fragment ions indicated a 1:1 ratio of wild type to mutant (c.f. Fig 3A and 3C).


Developmental MYH3 Myopathy Associated with Expression of Mutant Protein and Reduced Expression Levels of Embryonic MyHC.

Pokrzywa M, Norum M, Lengqvist J, Ghobadpour M, Abdul-Hussein S, Moslemi AR, Tajsharghi H - PLoS ONE (2015)

Detection and relative quantification of wild-type and mutated embryonic MyHC.(A and B) Extracted ion chromatograms (XICs) for wild-type and (C and D) mutant embryonic MyHC tryptic peptides eluting at 28.6 and 39.9 minutes are shown as indicated. Note that panels A and C have been adjusted to equal intensity, as have panels B and D.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636365&req=5

pone.0142094.g003: Detection and relative quantification of wild-type and mutated embryonic MyHC.(A and B) Extracted ion chromatograms (XICs) for wild-type and (C and D) mutant embryonic MyHC tryptic peptides eluting at 28.6 and 39.9 minutes are shown as indicated. Note that panels A and C have been adjusted to equal intensity, as have panels B and D.
Mentions: At retention times of 28.6 and 39.9 minutes, common (y2-y7 and b2-b6) and sequence- specific (y8-y13) fragment ions were observed for wild-type (Fig 3A and 3C) and mutant peptides (Fig 3B and 3D), respectively. Peptide identification was apparent by high precursor (< 2 ppm, S2 and S3 Figs) and fragment mass accuracy (RMS < 1.8 ppm for 12 (wild-type) and 13 (mutant) y-ion fragments, S4 and S5 Figs). Importantly, the p.Thr178Ile substitution was apparent from six unique fragment ions in each case. Measured relative intensities of common (non-unique) fragment ions indicated a 1:1 ratio of wild type to mutant (c.f. Fig 3A and 3C).

Bottom Line: We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins.Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased.We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Medical Genetics, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT

Objective: An essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy.

Methods and results: We determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres.

Conclusion: In conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.

No MeSH data available.


Related in: MedlinePlus