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Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle.

Mazelet L, Parker MO, Li M, Arner A, Ashworth R - Front Physiol (2016)

Bottom Line: Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf.In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes.The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position, capping the actin pointed ends and ultimately regulating actin length.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Chemical Sciences, Queen Mary, University of London London, UK.

ABSTRACT
Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1 (ts25) ) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position, capping the actin pointed ends and ultimately regulating actin length. This study demonstrates that both contraction and contractile-independent mechanisms are important for the regulation of myofibril organization, which in turn is necessary for establishing proper skeletal muscle structure and function during development in vivo in zebrafish.

No MeSH data available.


Related in: MedlinePlus

Tropomodulin 1 localization within the fast skeletal muscle from 17 hpf up to 42 hpf in immotile and control embryos. In control embryos (A–C) Tmod1 is initially located in the nucleus of the fast muscle cell at 17 hpf (orange arrow, inserts) (A) and then migrate into the skeletal muscle cell cytosol at 24 hpf (empty nuclei, white arrow, insert) (B) where it stays and get organized linearly by 42 hpf (orange rectangle, insert) (C). Tricaine treated immotile embryos (D,E) display a nuclear localization of Tmod1 at both 24 hpf (D) and 42 hpf (E) (orange arrows, inserts). Recovered motile embryos (F) display at 42 hpf a similar cytosolic alignment of Tmod1 as control embryos (orange rectangle, insert). Controls siblings motile embryos (G,H) display a cytosolic pattern of Tmod1 from 20 hpf (G) (empty nuclei, white arrow, inserts) which is linearized by 42 hpf (H) (orange box, insert) as control and recovered embryos. Immotile relaxed mutants embryos (I,J) display a similar nuclear localization of Tmod1 at both 20 hpf (I) and 42 hpf (J) as Tricaine treated embryos (orange arrows, inserts). Right hand corner inserts shown at a magnification of X5 compared to main image.
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Figure 7: Tropomodulin 1 localization within the fast skeletal muscle from 17 hpf up to 42 hpf in immotile and control embryos. In control embryos (A–C) Tmod1 is initially located in the nucleus of the fast muscle cell at 17 hpf (orange arrow, inserts) (A) and then migrate into the skeletal muscle cell cytosol at 24 hpf (empty nuclei, white arrow, insert) (B) where it stays and get organized linearly by 42 hpf (orange rectangle, insert) (C). Tricaine treated immotile embryos (D,E) display a nuclear localization of Tmod1 at both 24 hpf (D) and 42 hpf (E) (orange arrows, inserts). Recovered motile embryos (F) display at 42 hpf a similar cytosolic alignment of Tmod1 as control embryos (orange rectangle, insert). Controls siblings motile embryos (G,H) display a cytosolic pattern of Tmod1 from 20 hpf (G) (empty nuclei, white arrow, inserts) which is linearized by 42 hpf (H) (orange box, insert) as control and recovered embryos. Immotile relaxed mutants embryos (I,J) display a similar nuclear localization of Tmod1 at both 20 hpf (I) and 42 hpf (J) as Tricaine treated embryos (orange arrows, inserts). Right hand corner inserts shown at a magnification of X5 compared to main image.

Mentions: In control embryos, Tmod1 is located in the nucleus of skeletal muscle fibers prior to movement at 17 hpf (Figure 7A) but by 24 hpf the distribution of the protein has changed, it was less prominent in the nucleus and was mainly found in the cytosol (Figure 7B). By 42 hpf the staining appears punctate and arranged in lines within the cytoplasm of the fibers themselves (Figure 7C). In contrast to control embryos, Tricaine treated immotile embryos display nuclear localization of Tmod1 at both 24 hpf (Figure 7D) and 42 hpf (Figure 7E). Whereas, in recovered motile embryos (Figure 7F) at 42 hpf, Tmod1 displays a similar cytosolic staining pattern to control embryos. In summary, prior to movement (at 17 hpf) and in immotile embryos (at 24 and 42 hpf) Tmod1 staining is observed at the nucleus, whilst in motile embryos it is located in the cytoplasm. These observations were investigated further using the immotile relaxed mutant line. Control siblings motile embryos display cytosolic Tmod1 staining at 20 hpf (Figure 7G) and had a similar patterns to control and recovered embryos by 42 hpf (Figure 7H). Mutant immotile relaxed embryos display a nuclear localization of Tmod1 at both 20 hpf (Figure 7I) and 42 hpf (Figure 7J), similar to that observed in Tricaine treated immotile embryos. In summary these results suggest that the relocation of Tmod1 from the nucleus to the cytoplasm is driven by embryonic active movement.


Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle.

Mazelet L, Parker MO, Li M, Arner A, Ashworth R - Front Physiol (2016)

Tropomodulin 1 localization within the fast skeletal muscle from 17 hpf up to 42 hpf in immotile and control embryos. In control embryos (A–C) Tmod1 is initially located in the nucleus of the fast muscle cell at 17 hpf (orange arrow, inserts) (A) and then migrate into the skeletal muscle cell cytosol at 24 hpf (empty nuclei, white arrow, insert) (B) where it stays and get organized linearly by 42 hpf (orange rectangle, insert) (C). Tricaine treated immotile embryos (D,E) display a nuclear localization of Tmod1 at both 24 hpf (D) and 42 hpf (E) (orange arrows, inserts). Recovered motile embryos (F) display at 42 hpf a similar cytosolic alignment of Tmod1 as control embryos (orange rectangle, insert). Controls siblings motile embryos (G,H) display a cytosolic pattern of Tmod1 from 20 hpf (G) (empty nuclei, white arrow, inserts) which is linearized by 42 hpf (H) (orange box, insert) as control and recovered embryos. Immotile relaxed mutants embryos (I,J) display a similar nuclear localization of Tmod1 at both 20 hpf (I) and 42 hpf (J) as Tricaine treated embryos (orange arrows, inserts). Right hand corner inserts shown at a magnification of X5 compared to main image.
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Related In: Results  -  Collection

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Figure 7: Tropomodulin 1 localization within the fast skeletal muscle from 17 hpf up to 42 hpf in immotile and control embryos. In control embryos (A–C) Tmod1 is initially located in the nucleus of the fast muscle cell at 17 hpf (orange arrow, inserts) (A) and then migrate into the skeletal muscle cell cytosol at 24 hpf (empty nuclei, white arrow, insert) (B) where it stays and get organized linearly by 42 hpf (orange rectangle, insert) (C). Tricaine treated immotile embryos (D,E) display a nuclear localization of Tmod1 at both 24 hpf (D) and 42 hpf (E) (orange arrows, inserts). Recovered motile embryos (F) display at 42 hpf a similar cytosolic alignment of Tmod1 as control embryos (orange rectangle, insert). Controls siblings motile embryos (G,H) display a cytosolic pattern of Tmod1 from 20 hpf (G) (empty nuclei, white arrow, inserts) which is linearized by 42 hpf (H) (orange box, insert) as control and recovered embryos. Immotile relaxed mutants embryos (I,J) display a similar nuclear localization of Tmod1 at both 20 hpf (I) and 42 hpf (J) as Tricaine treated embryos (orange arrows, inserts). Right hand corner inserts shown at a magnification of X5 compared to main image.
Mentions: In control embryos, Tmod1 is located in the nucleus of skeletal muscle fibers prior to movement at 17 hpf (Figure 7A) but by 24 hpf the distribution of the protein has changed, it was less prominent in the nucleus and was mainly found in the cytosol (Figure 7B). By 42 hpf the staining appears punctate and arranged in lines within the cytoplasm of the fibers themselves (Figure 7C). In contrast to control embryos, Tricaine treated immotile embryos display nuclear localization of Tmod1 at both 24 hpf (Figure 7D) and 42 hpf (Figure 7E). Whereas, in recovered motile embryos (Figure 7F) at 42 hpf, Tmod1 displays a similar cytosolic staining pattern to control embryos. In summary, prior to movement (at 17 hpf) and in immotile embryos (at 24 and 42 hpf) Tmod1 staining is observed at the nucleus, whilst in motile embryos it is located in the cytoplasm. These observations were investigated further using the immotile relaxed mutant line. Control siblings motile embryos display cytosolic Tmod1 staining at 20 hpf (Figure 7G) and had a similar patterns to control and recovered embryos by 42 hpf (Figure 7H). Mutant immotile relaxed embryos display a nuclear localization of Tmod1 at both 20 hpf (Figure 7I) and 42 hpf (Figure 7J), similar to that observed in Tricaine treated immotile embryos. In summary these results suggest that the relocation of Tmod1 from the nucleus to the cytoplasm is driven by embryonic active movement.

Bottom Line: Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf.In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes.The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position, capping the actin pointed ends and ultimately regulating actin length.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Chemical Sciences, Queen Mary, University of London London, UK.

ABSTRACT
Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1 (ts25) ) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position, capping the actin pointed ends and ultimately regulating actin length. This study demonstrates that both contraction and contractile-independent mechanisms are important for the regulation of myofibril organization, which in turn is necessary for establishing proper skeletal muscle structure and function during development in vivo in zebrafish.

No MeSH data available.


Related in: MedlinePlus