25 research outputs found

    FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

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    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotube

    FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation

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    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes. Keywords: FSHD, vimentin, myogenic differentiation, proteomics.ΠŸΠ»Π΅Ρ‡ΠΎ-Π»ΠΎΠΏΠ°Ρ‚ΠΊΠΎΠ²ΠΎ-Π»ΠΈΡ†Π΅Π²Π° м’язова дистрофія (міодистрофія Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π°) Ρ” аутосомним Π΄ΠΎΠΌΡ–Π½Π°Π½Ρ‚Π½ΠΎ-успадковуваним нСйром’язовим Π·Π°Ρ…Π²ΠΎΡ€ΡŽΠ²Π°Π½Π½ΡΠΌ. Π”ΠΎ ΠΊΠ»Ρ–Π½Ρ–Ρ‡Π½ΠΈΡ… ΠΎΠ·Π½Π°ΠΊ Π΄Π°Π½ΠΎΠ³ΠΎ Ρ‚ΠΈΠΏΡƒ м’язової дистрофії Π½Π°Π»Π΅ΠΆΠ°Ρ‚ΡŒ ΡΠ»Π°Π±ΠΊΡ–Ρ‚ΡŒ Ρ– атрофія Π»ΠΈΡ†Π΅Π²ΠΈΡ… м’язів ΠΏΠ»Π΅Ρ‡ΠΎΠ²ΠΎΠ³ΠΎ пояса, Π΄ΠΎ яких Π½Π° ΠΏΡ–Π·Π½Ρ–ΡˆΠΈΡ… стадіях Π·Π°Ρ…Π²ΠΎΡ€ΡŽΠ²Π°Π½Π½Ρ Π΄ΠΎΠ΄Π°ΡŽΡ‚ΡŒΡΡ м’язи пояса Π½ΠΈΠΆΠ½Ρ–Ρ… ΠΊΡ–Π½Ρ†Ρ–Π²ΠΎΠΊ. ΠΠ΅Π·Π²Π°ΠΆΠ°ΡŽΡ‡ΠΈ Π½Π° Ρ‚Π΅, Ρ‰ΠΎ міобласти, Π²ΠΈΠ΄Ρ–Π»Π΅Π½Ρ– Ρ–Π· Ρ…Π²ΠΎΡ€ΠΈΡ… Π½Π° ΠΌΡ–ΠΎΠ΄ΠΈΡΡ‚Ρ€ΠΎΡ„Ρ–ΡŽ Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π°, Π·Π΄Π°Ρ‚Π½Ρ– Π΄ΠΎ Π΄ΠΈΡ„Π΅Ρ€Π΅Π½Ρ†Ρ–ΡŽΠ²Π°Π½Π½Ρ in vitro, ΠΌΡ–ΠΎΡ‚Ρ€ΡƒΠ±ΠΊΠΈ, які Π²ΠΈΠ½ΠΈΠΊΠ»ΠΈ Π· Π½ΠΈΡ…, ΠΌΠ°ΡŽΡ‚ΡŒ Π½ΠΈΠ·ΠΊΡƒ ΠΌΠΎΡ€Ρ„ΠΎΠ»ΠΎΠ³Ρ–Ρ‡Π½ΠΈΡ… Π°Π½ΠΎΠΌΠ°Π»Ρ–ΠΉ. ΠœΠ΅Ρ‚Π°. ΠœΠ΅Ρ‚Π° Π΄Π°Π½ΠΎΡ— Ρ€ΠΎΠ±ΠΎΡ‚ΠΈ полягає Π² ΠΏΠΎΡˆΡƒΠΊΡƒ ΠΏΡ€ΠΈΡ‡ΠΈΠ½ΠΈ ΠΌΠΎΡ€Ρ„ΠΎΠ»ΠΎΠ³Ρ–Ρ‡Π½ΠΈΡ… Π°Π½ΠΎΠΌΠ°Π»Ρ–ΠΉ ΠΌΡ–ΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ ΠΏΠ°Ρ†Ρ–Ρ”Π½Ρ‚Ρ–Π² Π· ΠΌΡ–ΠΎΠ΄ΠΈΡΡ‚Ρ€ΠΎΡ„Ρ–Ρ”ΡŽ Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π°. ΠœΠ΅Ρ‚ΠΎΠ΄ΠΈ. Π†Π· використанням ростового сСрСдовища Π· низьким вмістом сироватки ΠΌΠΈ Ρ–Π½Π΄ΡƒΠΊΡƒΠ²Π°Π»ΠΈ м’язовС Π΄ΠΈΡ„Π΅Ρ€Π΅Π½Ρ†Ρ–ΡŽΠ²Π°Π½Π½Ρ Π½ΠΎΡ€ΠΌΠ°Π»ΡŒΠ½ΠΈΡ… міобластів Ρ– міобластів ΠΏΠ°Ρ†Ρ–Ρ”Π½Ρ‚Ρ–Π² Π· ΠΌΡ–ΠΎΠ΄ΠΈΡΡ‚Ρ€ΠΎΡ„Ρ–Ρ”ΡŽ Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π° Ρ‚Π° ΠΏΡ€ΠΎΠ°Π½Π°Π»Ρ–Π·ΡƒΠ²Π°Π»ΠΈ Π±Ρ–Π»ΠΊΠΎΠ²ΠΈΠΉ склад ΠΌΡ–ΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ, які Π²ΠΈΠ½ΠΈΠΊΠ»ΠΈ Π· Π½ΠΈΡ…, ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ Π‘Π”Π‘-ΠŸΠΠΠ“ Π· Π½Π°ΡΡ‚ΡƒΠΏΠ½ΠΎΡŽ Ρ–Π΄Π΅Π½Ρ‚ΠΈΡ„Ρ–ΠΊΠ°Ρ†Ρ–Ρ”ΡŽ Π±Ρ–Π»ΠΊΡ–Π² ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ масс-спСктромСтрії. Π Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚ΠΈ. Π’ прСдставлСній Ρ€ΠΎΠ±ΠΎΡ‚Ρ– Π²ΠΏΠ΅Ρ€ΡˆΠ΅ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ, Ρ‰ΠΎ Π² ΠΌΡ–ΠΎΡ‚Ρ€ΡƒΠ±ΠΊΠ°Ρ… ΠΏΠ°Ρ†Ρ–Ρ”Π½Ρ‚Ρ–Π² Π· ΠΌΡ–ΠΎΠ΄ΠΈΡΡ‚Ρ€ΠΎΡ„Ρ–Ρ”ΡŽ Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π° ΠΏΡ–Π΄Π²ΠΈΡ‰Π΅Π½Π° СкспрСсія Π³Π΅Π½Π° Π²Ρ–ΠΌΠ΅Π½Ρ‚ΠΈΠ½Ρƒ. Висновки. Π’Ρ–ΠΌΠ΅Π½Ρ‚ΠΈΠ½ ΠΌΠΎΠΆΠ½Π° застосовувати як Π³Π΅Π½ – ΠΌΠ°Ρ€ΠΊΠ΅Ρ€ ΠΌΡ–ΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ Ρ…Π²ΠΎΡ€ΠΈΡ… Π½Π° ΠΌΡ–ΠΎΠ΄ΠΈΡΡ‚Ρ€ΠΎΡ„Ρ–ΡŽ Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π°. ΠšΠ»ΡŽΡ‡ΠΎΠ²Ρ– слова: міодистрофія Π›Π°Π½Π΄ΡƒΠ·Ρ–-Π”Π΅ΠΆΠ΅Ρ€Ρ–Π½Π°, Π²Ρ–ΠΌΠ΅Π½Ρ‚ΠΈΠ½, м’язовС Π΄ΠΈΡ„Π΅Ρ€Π΅Π½Ρ†Ρ–ΡŽΠ²Π°Π½Π½Ρ, ΠΏΡ€ΠΎΡ‚Π΅ΠΎΠΌΡ–ΠΊΠ°.Π›ΠΈΡ†Π΅-Π»ΠΎΠΏΠ°Ρ‚ΠΎΡ‡Π½ΠΎ-бСдрСнная ΠΌΡ‹ΡˆΠ΅Ρ‡Π½Π°Ρ дистрофия (миодистрофия Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π°) являСтся аутосомным Π΄ΠΎΠΌΠΈΠ½Π°Π½Ρ‚Π½ΠΎ-наслСдуСмым Π½Π΅ΠΉΡ€ΠΎΠΌΡ‹ΡˆΠ΅Ρ‡Π½Ρ‹ΠΌ Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΠ΅ΠΌ. ΠšΠ»ΠΈΠ½ΠΈΡ‡Π΅ΡΠΊΠ°Ρ ΠΊΠ°Ρ€Ρ‚ΠΈΠ½Π° Π΄Π°Π½Π½ΠΎΠ³ΠΎ Ρ‚ΠΈΠΏΠ° ΠΌΡ‹ΡˆΠ΅Ρ‡Π½ΠΎΠΉ дистрофии Π²ΠΊΠ»ΡŽΡ‡Π°Π΅Ρ‚ ΡΠ»Π°Π±ΠΎΡΡ‚ΡŒ ΠΈ Π°Ρ‚Ρ€ΠΎΡ„ΠΈΡŽ Π»ΠΈΡ†Π΅Π²Ρ‹Ρ… ΠΌΡ‹ΡˆΡ† ΠΈ ΠΌΡ‹ΡˆΡ† ΠΏΠ»Π΅Ρ‡Π΅Π²ΠΎΠ³ΠΎ пояса, ΠΊ ΠΊΠΎΡ‚ΠΎΡ€Ρ‹ΠΌ Π½Π° Π±ΠΎΠ»Π΅Π΅ ΠΏΠΎΠ·Π΄Π½ΠΈΡ… стадиях заболСвания Π΄ΠΎΠ±Π°Π²Π»ΡΡŽΡ‚ΡΡ ΠΌΡ‹ΡˆΡ†Ρ‹ пояса Π½ΠΈΠΆΠ½ΠΈΡ… конСчностСй. НСсмотря Π½Π° Ρ‚ΠΎ, Ρ‡Ρ‚ΠΎ миобласты, Π²Ρ‹Π΄Π΅Π»Π΅Π½Π½Ρ‹Π΅ ΠΈΠ· Π±ΠΎΠ»ΡŒΠ½Ρ‹Ρ… миодистрофиСй Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π°, способны ΠΊ Π΄ΠΈΡ„Ρ„Π΅Ρ€Π΅Π½Ρ†ΠΈΡ€ΠΎΠ²ΠΊΠ΅ in vitro, Π²ΠΎΠ·Π½ΠΈΠΊΠ°ΡŽΡ‰ΠΈΠ΅ ΠΈΠ· Π½ΠΈΡ… ΠΌΠΈΠΎΡ‚Ρ€ΡƒΠ±ΠΊΠΈ ΠΈΠΌΠ΅ΡŽΡ‚ ряд морфологичСских Π°Π½ΠΎΠΌΠ°Π»ΠΈΠΉ. ЦСль. ЦСлью Π΄Π°Π½Π½ΠΎΠΉ Ρ€Π°Π±ΠΎΡ‚Ρ‹ являСтся поиск ΠΏΡ€ΠΈΡ‡ΠΈΠ½Ρ‹ морфологичСских Π°Π½ΠΎΠΌΠ°Π»ΠΈΠΉ ΠΌΠΈΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ ΠΏΠ°Ρ†ΠΈΠ΅Π½Ρ‚ΠΎΠ² с миодистрофиСй Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π°. ΠœΠ΅Ρ‚ΠΎΠ΄Ρ‹. Π˜ΡΠΏΠΎΠ»ΡŒΠ·ΡƒΡ Ρ€ΠΎΡΡ‚ΠΎΠ²ΡƒΡŽ срСду с Π½ΠΈΠ·ΠΊΠΈΠΌ содСрТаниСм сыворотки, ΠΌΡ‹ ΠΈΠ½Π΄ΡƒΡ†ΠΈΡ€ΠΎΠ²Π°Π»ΠΈ ΠΌΡ‹ΡˆΠ΅Ρ‡Π½ΡƒΡŽ Π΄ΠΈΡ„Ρ„Π΅Ρ€Π΅Π½Ρ†ΠΈΡ€ΠΎΠ²ΠΊΡƒ Π½ΠΎΡ€ΠΌΠ°Π»ΡŒΠ½Ρ‹Ρ… миобластов ΠΈ миобластов ΠΏΠ°Ρ†ΠΈΠ΅Π½Ρ‚ΠΎΠ² с миодистрофиСй Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π° ΠΈ ΠΏΡ€ΠΎΠ°Π½Π°Π»ΠΈΠ·ΠΈΡ€ΠΎΠ²Π°Π»ΠΈ Π±Π΅Π»ΠΊΠΎΠ²Ρ‹ΠΉ состав Π²ΠΎΠ·Π½ΠΈΠΊΡˆΠΈΡ… ΠΈΠ· Π½ΠΈΡ… ΠΌΠΈΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ Π‘Π”Π‘-ΠŸΠΠΠ“ с ΠΏΠΎΡΠ»Π΅Π΄ΡƒΡŽΡ‰Π΅ΠΉ ΠΈΠ΄Π΅Π½Ρ‚ΠΈΡ„ΠΈΠΊΠ°Ρ†ΠΈΠ΅ΠΉ Π±Π΅Π»ΠΊΠΎΠ² ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ масс-спСктромСтрии. Π Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Ρ‹. Π’ Π΄Π°Π½Π½ΠΎΠΉ Ρ€Π°Π±ΠΎΡ‚Π΅ Π²ΠΏΠ΅Ρ€Π²Ρ‹Π΅ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ, Ρ‡Ρ‚ΠΎ Π² ΠΌΠΈΠΎΡ‚Ρ€ΡƒΠ±ΠΊΠ°Ρ… ΠΏΠ°Ρ†ΠΈΠ΅Π½Ρ‚ΠΎΠ² с миодистрофиСй Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π° ΡƒΠ²Π΅Π»ΠΈΡ‡Π΅Π½Π° экспрСссия Π³Π΅Π½Π° Π²ΠΈΠΌΠ΅Π½Ρ‚ΠΈΠ½Π°. Π’Ρ‹Π²ΠΎΠ΄Ρ‹. Π’ΠΈΠΌΠ΅Π½Ρ‚ΠΈΠ½ ΠΌΠΎΠΆΠ΅Ρ‚ Π±Ρ‹Ρ‚ΡŒ использован Π² качСствС Π³Π΅Π½Π° – ΠΌΠ°Ρ€ΠΊΠ΅Ρ€Π° ΠΌΠΈΠΎΡ‚Ρ€ΡƒΠ±ΠΎΠΊ Π±ΠΎΠ»ΡŒΠ½Ρ‹Ρ… миодистрофиСй Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π°. ΠšΠ»ΡŽΡ‡Π΅Π²Ρ‹Π΅ слова: миодистрофия Π›Π°Π½Π΄ΡƒΠ·ΠΈ-Π”Π΅ΠΆΠ΅Ρ€ΠΈΠ½Π°, Π²ΠΈΠΌΠ΅Π½Ρ‚ΠΈΠ½, ΠΌΡ‹ΡˆΠ΅Ρ‡Π½Π°Ρ Π΄ΠΈΡ„Ρ„Π΅Ρ€Π΅Π½Ρ†ΠΈΡ€ΠΎΠ²ΠΊΠ°, ΠΏΡ€ΠΎΡ‚Π΅ΠΎΠΌΠΈΠΊΠ°

    Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes

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    Background: miRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis. Results: Here, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation. Conclusions: This simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions

    A Functional Role for 4qA/B in the Structural Rearrangement of the 4q35 Region and in the Regulation of FRG1 and ANT1 in Facioscapulohumeral Dystrophy

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    The number of D4Z4 repeats in the subtelomeric region of chromosome 4q is strongly reduced in patients with Facio-Scapulo-Humeral Dystrophy (FSHD). We performed chromosome conformation capture (3C) analysis to document the interactions taking place among different 4q35 markers. We found that the reduced number of D4Z4 repeats in FSHD myoblasts was associated with a global alteration of the three-dimensional structure of the 4q35 region. Indeed, differently from normal myoblasts, the 4qA/B marker interacted directly with the promoters of the FRG1 and ANT1 genes in FSHD cells. Along with the presence of a newly identified transcriptional enhancer within the 4qA allele, our demonstration of an interaction occurring between chromosomal segments located megabases away on the same chromosome 4q allows to revisit the possible mechanisms leading to FSHD

    A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

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    There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (BΓ©camel et al., EMBO J., 21(10): 2332, 2002)

    DUX4c Is Up-Regulated in FSHD. It Induces the MYF5 Protein and Human Myoblast Proliferation

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    Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology

    Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation

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    <p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (<it>ANT1</it>), FSHD-related gene 1 (<it>FRG1</it>), <it>FRG2 </it>and <it>DUX4c</it>, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (<it>DUX4</it>) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing <it>FRG1 </it>has been generated, displaying skeletal muscle defects.</p> <p>Results</p> <p>In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and <it>FRG1 </it>gene promoter, and <it>FRG1 </it>expression, in control and FSHD cells. The <it>FRG1 </it>gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of <it>FRG1 </it>expression. Using chromosome conformation capture (3C) technology, we revealed that the <it>FRG1 </it>promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the <it>FRG1</it>/4q-D4Z4 array loop in myotubes. The <it>FRG1 </it>promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.</p> <p>Conclusion</p> <p>We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of <it>in cis </it>chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.</p

    Gene expression during normal and FSHD myogenesis

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    <p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, <it>DUX4</it>, that can encode a protein containing two homeodomains. A <it>DUX4 </it>transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.</p> <p>Methods</p> <p>Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.</p> <p>Results</p> <p>Many of the ~17,000 examined genes were differentially expressed (> 2-fold, <it>p </it>< 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked <it>DUX4 </it>RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.</p> <p>Conclusions</p> <p><it>DUX4</it>'s pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated <it>DUX4 </it>expression at the myoblast or myotube stages. Our model could explain why <it>DUX4</it>'s inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.</p

    Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells

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    The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient
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