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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

Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. Control embryos (Embryo Medium alone) and treated embryos (Embryo Medium with Tricaine) were incubated for 7 h starting at 17 hpf. At 24 hpf embryos were removed and control (A) and treated embryos (G) were fixed and stained immediately or put into recovery (Embryo Medium without Tricaine) and (B) control and (H) recovered embryos fixed and stained at 42 hpf. Some embryos were kept in recovery from 24 hpf up to 5 dpf before fixation and staining of both control (C) and recovered larvae (I). Relaxed immotile mutants were fixed and stained at 24 hpf (J), at 42 hpf (K), and 5 dpf (L) as well as motile control siblings at 24 hpf (D), 42 hpf (E) and 5 dpf (F). Anti-myosin antibody (F59) in (A,B,D,E,G,H,J,K), and Rhodamine phalloidin actin labeling in (C,I,F,L) reveals slow muscle fibers. For consistency, the somites imaged were taken at the level where the yolk sac and the yolk sac extension join. Scale bars corresponds to 20 μm.
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Figure 1: Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. Control embryos (Embryo Medium alone) and treated embryos (Embryo Medium with Tricaine) were incubated for 7 h starting at 17 hpf. At 24 hpf embryos were removed and control (A) and treated embryos (G) were fixed and stained immediately or put into recovery (Embryo Medium without Tricaine) and (B) control and (H) recovered embryos fixed and stained at 42 hpf. Some embryos were kept in recovery from 24 hpf up to 5 dpf before fixation and staining of both control (C) and recovered larvae (I). Relaxed immotile mutants were fixed and stained at 24 hpf (J), at 42 hpf (K), and 5 dpf (L) as well as motile control siblings at 24 hpf (D), 42 hpf (E) and 5 dpf (F). Anti-myosin antibody (F59) in (A,B,D,E,G,H,J,K), and Rhodamine phalloidin actin labeling in (C,I,F,L) reveals slow muscle fibers. For consistency, the somites imaged were taken at the level where the yolk sac and the yolk sac extension join. Scale bars corresponds to 20 μm.

Mentions: In zebrafish there are separate populations of slow and fast muscle types which develop separately. By 24 hpf, slow muscle fibers have formed as superficial monolayer in the surface of the somite and are easy to visualize using immunohistochemical staining (Devoto et al., 1996). Early paralysis (between 17 and 24 hpf) led to myofibril disruption with the appearance of the “wavy” myofibril phenotype in immotile embryos, both the chemically paralyzed (Tricaine treated) and the immotile mutant larvae (cacnb1ts25, relaxed) (Figures 1G,J), in comparison to the control embryos (Figures 1A,D). A partial structural recovery of myofibril organization was observed in embryos that had been allowed to recover movement in Embryo Medium up until 42 hpf, after an initial chemical paralysis of 7 h administered between 17 and 24 hpf (Figures 1B,H). The structural recovery was maintained at 5 dpf (Figures 1C,I) whereas the myofibrils in immotile relaxed mutant remained wavy (Figures 1E,F,K,L).


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)

Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. Control embryos (Embryo Medium alone) and treated embryos (Embryo Medium with Tricaine) were incubated for 7 h starting at 17 hpf. At 24 hpf embryos were removed and control (A) and treated embryos (G) were fixed and stained immediately or put into recovery (Embryo Medium without Tricaine) and (B) control and (H) recovered embryos fixed and stained at 42 hpf. Some embryos were kept in recovery from 24 hpf up to 5 dpf before fixation and staining of both control (C) and recovered larvae (I). Relaxed immotile mutants were fixed and stained at 24 hpf (J), at 42 hpf (K), and 5 dpf (L) as well as motile control siblings at 24 hpf (D), 42 hpf (E) and 5 dpf (F). Anti-myosin antibody (F59) in (A,B,D,E,G,H,J,K), and Rhodamine phalloidin actin labeling in (C,I,F,L) reveals slow muscle fibers. For consistency, the somites imaged were taken at the level where the yolk sac and the yolk sac extension join. Scale bars corresponds to 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814503&req=5

Figure 1: Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. Control embryos (Embryo Medium alone) and treated embryos (Embryo Medium with Tricaine) were incubated for 7 h starting at 17 hpf. At 24 hpf embryos were removed and control (A) and treated embryos (G) were fixed and stained immediately or put into recovery (Embryo Medium without Tricaine) and (B) control and (H) recovered embryos fixed and stained at 42 hpf. Some embryos were kept in recovery from 24 hpf up to 5 dpf before fixation and staining of both control (C) and recovered larvae (I). Relaxed immotile mutants were fixed and stained at 24 hpf (J), at 42 hpf (K), and 5 dpf (L) as well as motile control siblings at 24 hpf (D), 42 hpf (E) and 5 dpf (F). Anti-myosin antibody (F59) in (A,B,D,E,G,H,J,K), and Rhodamine phalloidin actin labeling in (C,I,F,L) reveals slow muscle fibers. For consistency, the somites imaged were taken at the level where the yolk sac and the yolk sac extension join. Scale bars corresponds to 20 μm.
Mentions: In zebrafish there are separate populations of slow and fast muscle types which develop separately. By 24 hpf, slow muscle fibers have formed as superficial monolayer in the surface of the somite and are easy to visualize using immunohistochemical staining (Devoto et al., 1996). Early paralysis (between 17 and 24 hpf) led to myofibril disruption with the appearance of the “wavy” myofibril phenotype in immotile embryos, both the chemically paralyzed (Tricaine treated) and the immotile mutant larvae (cacnb1ts25, relaxed) (Figures 1G,J), in comparison to the control embryos (Figures 1A,D). A partial structural recovery of myofibril organization was observed in embryos that had been allowed to recover movement in Embryo Medium up until 42 hpf, after an initial chemical paralysis of 7 h administered between 17 and 24 hpf (Figures 1B,H). The structural recovery was maintained at 5 dpf (Figures 1C,I) whereas the myofibrils in immotile relaxed mutant remained wavy (Figures 1E,F,K,L).

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