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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. (A) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of treatment, F(1, 32) = 77.45, p < 0.001, but no main effect of age, F(1, 32) < 1. There was, however, an age × treatment interaction, F(1, 32) = 13.27, p < 0.01. As is clear from (A), this interaction is characterized by a steeper increase in the ratio for treated individuals at 24 hpf than at 42 hpf. (length: controls n = 6, treated n = 6 and width: controls n = 17, treated n = 36). (B) A factorial ANOVA with age (2-levels: 24 hpf and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of genotype, F(1, 116) = 49.81, p < 0.001, but no main effect of age, F(1, 116) = 3.46, p = 0.07. There was, however, an age × genotype interaction, F(1, 116) = 9.42, p < 0.01. As is clear from (B), this interaction is characterized by a steeper increase in the ratio for homozygous individuals at 24 hpf than at 42 hpf. (length: controls n = 18, treated n = 6, width: controls n = 57, treated n = 23). (C) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the width of myofibrils. This revealed significant main effect of treatment, F(1, 129) = 30.06, p < 0.001, and of age, F(1, 129) = 19.08, p < 0.001. There was no age × treatment interaction, F(1, 129) < 1. As is clear from (C), Tricaine-treated embryos showed robust reductions in myofibril width. In addition, older embryos (42 hpf vs. 24 hpf) had wider myofibrils, regardless of Tricaine treatment. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 10, rr n = 10). (D) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the width of myofibrils. This revealed significant main effects of genotype, F(1, 478) = 200.06, p < 0.001, and of age, F(1, 116) = 18.76, p < 0.001. There was also an age × genotype interaction, F(1, 478) = 46.79, p < 0.001. As is clear from (D), this interaction is characterized by a steeper increase in the width of myofibrils in homozygous individuals at 24 hpf than at 42 hpf. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 13, rr n = 13).
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Figure 2: Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. (A) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of treatment, F(1, 32) = 77.45, p < 0.001, but no main effect of age, F(1, 32) < 1. There was, however, an age × treatment interaction, F(1, 32) = 13.27, p < 0.01. As is clear from (A), this interaction is characterized by a steeper increase in the ratio for treated individuals at 24 hpf than at 42 hpf. (length: controls n = 6, treated n = 6 and width: controls n = 17, treated n = 36). (B) A factorial ANOVA with age (2-levels: 24 hpf and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of genotype, F(1, 116) = 49.81, p < 0.001, but no main effect of age, F(1, 116) = 3.46, p = 0.07. There was, however, an age × genotype interaction, F(1, 116) = 9.42, p < 0.01. As is clear from (B), this interaction is characterized by a steeper increase in the ratio for homozygous individuals at 24 hpf than at 42 hpf. (length: controls n = 18, treated n = 6, width: controls n = 57, treated n = 23). (C) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the width of myofibrils. This revealed significant main effect of treatment, F(1, 129) = 30.06, p < 0.001, and of age, F(1, 129) = 19.08, p < 0.001. There was no age × treatment interaction, F(1, 129) < 1. As is clear from (C), Tricaine-treated embryos showed robust reductions in myofibril width. In addition, older embryos (42 hpf vs. 24 hpf) had wider myofibrils, regardless of Tricaine treatment. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 10, rr n = 10). (D) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the width of myofibrils. This revealed significant main effects of genotype, F(1, 478) = 200.06, p < 0.001, and of age, F(1, 116) = 18.76, p < 0.001. There was also an age × genotype interaction, F(1, 478) = 46.79, p < 0.001. As is clear from (D), this interaction is characterized by a steeper increase in the width of myofibrils in homozygous individuals at 24 hpf than at 42 hpf. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 13, rr n = 13).

Mentions: The effect of paralysis on myofibril length and end-end length (as shown in Table 1, data used to calculate index for straightness) and width was assessed in Tricaine treated (17–24 hpf) embryos at 24 hpf and after recovery of movement at 42 hpf. Results showed that early paralysis (between 17 and 24 hpf) does affect both the myofibril length/end-end ratio and the width, whereas these measurements showed a recovery close to control values by 42 hpf [Figures 2A,C, factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated)]. Similar results were observed in relaxed immotile mutant [Figures 2B,D, factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control siblings (RR/Rr) and relaxed immotile mutants (rr)]. In conclusion, the initial contractile events prior to 24 hpf are important in driving the early structural organization of the myofibrils. However, as recovery can occur independently of contraction by 42 hpf movement does not seem as critical for the organization of myofibril structure (myofibril length/end-end ratio and the width) during this later stage.


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. (A) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of treatment, F(1, 32) = 77.45, p < 0.001, but no main effect of age, F(1, 32) < 1. There was, however, an age × treatment interaction, F(1, 32) = 13.27, p < 0.01. As is clear from (A), this interaction is characterized by a steeper increase in the ratio for treated individuals at 24 hpf than at 42 hpf. (length: controls n = 6, treated n = 6 and width: controls n = 17, treated n = 36). (B) A factorial ANOVA with age (2-levels: 24 hpf and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of genotype, F(1, 116) = 49.81, p < 0.001, but no main effect of age, F(1, 116) = 3.46, p = 0.07. There was, however, an age × genotype interaction, F(1, 116) = 9.42, p < 0.01. As is clear from (B), this interaction is characterized by a steeper increase in the ratio for homozygous individuals at 24 hpf than at 42 hpf. (length: controls n = 18, treated n = 6, width: controls n = 57, treated n = 23). (C) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the width of myofibrils. This revealed significant main effect of treatment, F(1, 129) = 30.06, p < 0.001, and of age, F(1, 129) = 19.08, p < 0.001. There was no age × treatment interaction, F(1, 129) < 1. As is clear from (C), Tricaine-treated embryos showed robust reductions in myofibril width. In addition, older embryos (42 hpf vs. 24 hpf) had wider myofibrils, regardless of Tricaine treatment. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 10, rr n = 10). (D) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the width of myofibrils. This revealed significant main effects of genotype, F(1, 478) = 200.06, p < 0.001, and of age, F(1, 116) = 18.76, p < 0.001. There was also an age × genotype interaction, F(1, 478) = 46.79, p < 0.001. As is clear from (D), this interaction is characterized by a steeper increase in the width of myofibrils in homozygous individuals at 24 hpf than at 42 hpf. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 13, rr n = 13).
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Figure 2: Disruption of myofibril organization during paralysis is reversed by restoring movement in developing skeletal muscle. (A) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of treatment, F(1, 32) = 77.45, p < 0.001, but no main effect of age, F(1, 32) < 1. There was, however, an age × treatment interaction, F(1, 32) = 13.27, p < 0.01. As is clear from (A), this interaction is characterized by a steeper increase in the ratio for treated individuals at 24 hpf than at 42 hpf. (length: controls n = 6, treated n = 6 and width: controls n = 17, treated n = 36). (B) A factorial ANOVA with age (2-levels: 24 hpf and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the ratios of myofibril length to end-to-end measurement. This revealed significant main effect of genotype, F(1, 116) = 49.81, p < 0.001, but no main effect of age, F(1, 116) = 3.46, p = 0.07. There was, however, an age × genotype interaction, F(1, 116) = 9.42, p < 0.01. As is clear from (B), this interaction is characterized by a steeper increase in the ratio for homozygous individuals at 24 hpf than at 42 hpf. (length: controls n = 18, treated n = 6, width: controls n = 57, treated n = 23). (C) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated) was carried out on the width of myofibrils. This revealed significant main effect of treatment, F(1, 129) = 30.06, p < 0.001, and of age, F(1, 129) = 19.08, p < 0.001. There was no age × treatment interaction, F(1, 129) < 1. As is clear from (C), Tricaine-treated embryos showed robust reductions in myofibril width. In addition, older embryos (42 hpf vs. 24 hpf) had wider myofibrils, regardless of Tricaine treatment. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 10, rr n = 10). (D) A factorial ANOVA with age (2-levels: 24 and 42 hpf) and genotype (2-levels: Rr/RR and rr) was carried out on the width of myofibrils. This revealed significant main effects of genotype, F(1, 478) = 200.06, p < 0.001, and of age, F(1, 116) = 18.76, p < 0.001. There was also an age × genotype interaction, F(1, 478) = 46.79, p < 0.001. As is clear from (D), this interaction is characterized by a steeper increase in the width of myofibrils in homozygous individuals at 24 hpf than at 42 hpf. (length: Rr/RR n = 5, rr n = 5 and width: Rr/RR n = 13, rr n = 13).
Mentions: The effect of paralysis on myofibril length and end-end length (as shown in Table 1, data used to calculate index for straightness) and width was assessed in Tricaine treated (17–24 hpf) embryos at 24 hpf and after recovery of movement at 42 hpf. Results showed that early paralysis (between 17 and 24 hpf) does affect both the myofibril length/end-end ratio and the width, whereas these measurements showed a recovery close to control values by 42 hpf [Figures 2A,C, factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control and treated)]. Similar results were observed in relaxed immotile mutant [Figures 2B,D, factorial ANOVA with age (2-levels: 24 and 42 hpf) and treatment (2-levels: control siblings (RR/Rr) and relaxed immotile mutants (rr)]. In conclusion, the initial contractile events prior to 24 hpf are important in driving the early structural organization of the myofibrils. However, as recovery can occur independently of contraction by 42 hpf movement does not seem as critical for the organization of myofibril structure (myofibril length/end-end ratio and the width) during this later stage.

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