Limits...
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

Co-immunofluorescence analysis of 6-day differentiated myotubes.(A) Triple staining was performed for α-actinin (green), F-actin (red) and M-band epitope of titin (magenta) (A); (B) myomesin (green), F-actin (red) and Z-disc epitope of titin (magenta); (C) myomesin (green), F-actin (red) and A/I junction epitope of titin (magenta). The results were visualised with a Zeiss Axio Observer microscope (Carl Zeiss AG, Germany) at 63× magnification. All nuclei were counterstained with DAPI (blue). The Z-disc can be seen clearly with α-actinin and Z-disc epitope of titin, and the M-band with myomesin, M-band epitope of titin (insets). Note the strikingly thinner myotubes in the patient compared to the cells in the control. The bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4636365&req=5

pone.0142094.g005: Co-immunofluorescence analysis of 6-day differentiated myotubes.(A) Triple staining was performed for α-actinin (green), F-actin (red) and M-band epitope of titin (magenta) (A); (B) myomesin (green), F-actin (red) and Z-disc epitope of titin (magenta); (C) myomesin (green), F-actin (red) and A/I junction epitope of titin (magenta). The results were visualised with a Zeiss Axio Observer microscope (Carl Zeiss AG, Germany) at 63× magnification. All nuclei were counterstained with DAPI (blue). The Z-disc can be seen clearly with α-actinin and Z-disc epitope of titin, and the M-band with myomesin, M-band epitope of titin (insets). Note the strikingly thinner myotubes in the patient compared to the cells in the control. The bars represent 10 μm.

Mentions: We investigated the proper localisation of myosin at the A-band and the alternating band patterns of the sarcomere. Results from triple immunostaining for α-actinin, actin, and myosin or M-protein, α-actinin, and myosin indicated that there was no disruptive effect on sarcomere organisation and there was proper formation of A-band, M-band, and Z-disc in both control myotubes and patient myotubes (Fig 4A–4C). Results from further double and triple immunostaining indicated proper formation of M-band, where the myosin-containing thick filaments are anchored, in both control myotubes and patient myotubes (S7 Fig and Fig 5A–5C). Moreover, immunostaining for myomesin and M-band epitope of titin indicated their proper co-localisation at the M-band region of the sarcomere (Fig 4C). Triple-immunostaining for either α-actinin, actin, and M-band epitope of titin, or Z-disc epitope of titin, actin and myomesin additionally revealed the alternating staining patterns of Z-discs and M-bands in the patient and control 3-day myotubes (Fig 5A and 5B). The appearance of correctly alternating band patterns of the sarcomere was further demonstrated in 3- and 6-day myotubes of both patient and control by triple-immunostaining for myomesin, actin and A/I junction epitope of titin (Fig 5C).


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)

Co-immunofluorescence analysis of 6-day differentiated myotubes.(A) Triple staining was performed for α-actinin (green), F-actin (red) and M-band epitope of titin (magenta) (A); (B) myomesin (green), F-actin (red) and Z-disc epitope of titin (magenta); (C) myomesin (green), F-actin (red) and A/I junction epitope of titin (magenta). The results were visualised with a Zeiss Axio Observer microscope (Carl Zeiss AG, Germany) at 63× magnification. All nuclei were counterstained with DAPI (blue). The Z-disc can be seen clearly with α-actinin and Z-disc epitope of titin, and the M-band with myomesin, M-band epitope of titin (insets). Note the strikingly thinner myotubes in the patient compared to the cells in the control. The bars represent 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142094.g005: Co-immunofluorescence analysis of 6-day differentiated myotubes.(A) Triple staining was performed for α-actinin (green), F-actin (red) and M-band epitope of titin (magenta) (A); (B) myomesin (green), F-actin (red) and Z-disc epitope of titin (magenta); (C) myomesin (green), F-actin (red) and A/I junction epitope of titin (magenta). The results were visualised with a Zeiss Axio Observer microscope (Carl Zeiss AG, Germany) at 63× magnification. All nuclei were counterstained with DAPI (blue). The Z-disc can be seen clearly with α-actinin and Z-disc epitope of titin, and the M-band with myomesin, M-band epitope of titin (insets). Note the strikingly thinner myotubes in the patient compared to the cells in the control. The bars represent 10 μm.
Mentions: We investigated the proper localisation of myosin at the A-band and the alternating band patterns of the sarcomere. Results from triple immunostaining for α-actinin, actin, and myosin or M-protein, α-actinin, and myosin indicated that there was no disruptive effect on sarcomere organisation and there was proper formation of A-band, M-band, and Z-disc in both control myotubes and patient myotubes (Fig 4A–4C). Results from further double and triple immunostaining indicated proper formation of M-band, where the myosin-containing thick filaments are anchored, in both control myotubes and patient myotubes (S7 Fig and Fig 5A–5C). Moreover, immunostaining for myomesin and M-band epitope of titin indicated their proper co-localisation at the M-band region of the sarcomere (Fig 4C). Triple-immunostaining for either α-actinin, actin, and M-band epitope of titin, or Z-disc epitope of titin, actin and myomesin additionally revealed the alternating staining patterns of Z-discs and M-bands in the patient and control 3-day myotubes (Fig 5A and 5B). The appearance of correctly alternating band patterns of the sarcomere was further demonstrated in 3- and 6-day myotubes of both patient and control by triple-immunostaining for myomesin, actin and A/I junction epitope of titin (Fig 5C).

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