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Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Carlson EJ, Ramirez F, Keene DR, Sakai LY - PLoS Genet. (2015)

Bottom Line: These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy.In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development.New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America; Shriners Hospital for Children, Portland, Oregon, United States of America.

ABSTRACT
Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

No MeSH data available.


Related in: MedlinePlus

Myosin heavy chain 8, a perinatal myosin, in Fbn2  forelimb muscle and in C2C12 cultures.(A) Pepsin-resistant fragments extracted from P0 –P8 forelimb muscles, visualized after Ponceau staining (top) and immunoblotting with anti-fibrillin-1 (bottom). Asterisk indicates pepsin-resistant fibrillin-1 fragment. Open stars mark pepsin-resistant bands that are likely to be collagen I. Myosin heavy chain 8 was identified by N-terminal Edman sequencing. (B) Expression of Myh8 during C2C12 myoblast to myotube differentiation while Fbn2 was knocked down by RNAi. (C) Expression of Myh8 in C2C12 cells treated with 100 ng/ml BMP-7 or 50 ng/ml BMP-4. Results in (B) and (C) were obtained from three independent experiments, and each experiment was performed in triplicates. Error bars indicate mean ± SD.
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pgen.1005340.g004: Myosin heavy chain 8, a perinatal myosin, in Fbn2 forelimb muscle and in C2C12 cultures.(A) Pepsin-resistant fragments extracted from P0 –P8 forelimb muscles, visualized after Ponceau staining (top) and immunoblotting with anti-fibrillin-1 (bottom). Asterisk indicates pepsin-resistant fibrillin-1 fragment. Open stars mark pepsin-resistant bands that are likely to be collagen I. Myosin heavy chain 8 was identified by N-terminal Edman sequencing. (B) Expression of Myh8 during C2C12 myoblast to myotube differentiation while Fbn2 was knocked down by RNAi. (C) Expression of Myh8 in C2C12 cells treated with 100 ng/ml BMP-7 or 50 ng/ml BMP-4. Results in (B) and (C) were obtained from three independent experiments, and each experiment was performed in triplicates. Error bars indicate mean ± SD.

Mentions: To assess possible effects on fibrillin-1, forearm muscles from Fbn2 and wildtype mice were dissected and digested with pepsin, following an established protocol [24], and pepsin-resistant fibrillin-1 fragments were analyzed over the first 8 days of postnatal life. We identified a pepsin-resistant fragment of fibrillin-1 with a Mr of 70 kDa (Fig 4A) similar to previously characterized fibrillin-1 fragments from human amnion [24]. At P0, when contractures were severe and muscle mass was reduced, no pepsin-resistant fibrillin-1 fragments were detected. However, from P1 to P8, fibrillin-1 bands (asterisk) of equal intensities were detected in wildtype, Fbn2+/-, Fbn2-/- muscle, indicating that the amount of fibrillin-1 in forearm muscle tissue was not significantly altered by Fbn2 genotype (consistent with qPCR data shown in S2 Fig). Since no pepsin-resistant fibrillin-1 fragments were identified in P0 tissue digests, fibrillin-1 microfibrils may undergo maturation processes after birth that are necessary to stabilize certain fibrillin-1 peptides to pepsin digestion. As a positive control for the pepsin digestions, Ponceau staining showed two bands (open stars), most likely the α1 and α2 chains of type I collagen, that were present in all extracts from P0 to P8.


Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Carlson EJ, Ramirez F, Keene DR, Sakai LY - PLoS Genet. (2015)

Myosin heavy chain 8, a perinatal myosin, in Fbn2  forelimb muscle and in C2C12 cultures.(A) Pepsin-resistant fragments extracted from P0 –P8 forelimb muscles, visualized after Ponceau staining (top) and immunoblotting with anti-fibrillin-1 (bottom). Asterisk indicates pepsin-resistant fibrillin-1 fragment. Open stars mark pepsin-resistant bands that are likely to be collagen I. Myosin heavy chain 8 was identified by N-terminal Edman sequencing. (B) Expression of Myh8 during C2C12 myoblast to myotube differentiation while Fbn2 was knocked down by RNAi. (C) Expression of Myh8 in C2C12 cells treated with 100 ng/ml BMP-7 or 50 ng/ml BMP-4. Results in (B) and (C) were obtained from three independent experiments, and each experiment was performed in triplicates. Error bars indicate mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482570&req=5

pgen.1005340.g004: Myosin heavy chain 8, a perinatal myosin, in Fbn2 forelimb muscle and in C2C12 cultures.(A) Pepsin-resistant fragments extracted from P0 –P8 forelimb muscles, visualized after Ponceau staining (top) and immunoblotting with anti-fibrillin-1 (bottom). Asterisk indicates pepsin-resistant fibrillin-1 fragment. Open stars mark pepsin-resistant bands that are likely to be collagen I. Myosin heavy chain 8 was identified by N-terminal Edman sequencing. (B) Expression of Myh8 during C2C12 myoblast to myotube differentiation while Fbn2 was knocked down by RNAi. (C) Expression of Myh8 in C2C12 cells treated with 100 ng/ml BMP-7 or 50 ng/ml BMP-4. Results in (B) and (C) were obtained from three independent experiments, and each experiment was performed in triplicates. Error bars indicate mean ± SD.
Mentions: To assess possible effects on fibrillin-1, forearm muscles from Fbn2 and wildtype mice were dissected and digested with pepsin, following an established protocol [24], and pepsin-resistant fibrillin-1 fragments were analyzed over the first 8 days of postnatal life. We identified a pepsin-resistant fragment of fibrillin-1 with a Mr of 70 kDa (Fig 4A) similar to previously characterized fibrillin-1 fragments from human amnion [24]. At P0, when contractures were severe and muscle mass was reduced, no pepsin-resistant fibrillin-1 fragments were detected. However, from P1 to P8, fibrillin-1 bands (asterisk) of equal intensities were detected in wildtype, Fbn2+/-, Fbn2-/- muscle, indicating that the amount of fibrillin-1 in forearm muscle tissue was not significantly altered by Fbn2 genotype (consistent with qPCR data shown in S2 Fig). Since no pepsin-resistant fibrillin-1 fragments were identified in P0 tissue digests, fibrillin-1 microfibrils may undergo maturation processes after birth that are necessary to stabilize certain fibrillin-1 peptides to pepsin digestion. As a positive control for the pepsin digestions, Ponceau staining showed two bands (open stars), most likely the α1 and α2 chains of type I collagen, that were present in all extracts from P0 to P8.

Bottom Line: These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy.In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development.New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America; Shriners Hospital for Children, Portland, Oregon, United States of America.

ABSTRACT
Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

No MeSH data available.


Related in: MedlinePlus