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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

Paralysis leads to actin lengthening in both relaxed mutant and Tricaine treated embryos, subsequent movement restoration in recovered embryos leads to actin length complete rescue. (A,B) H-H measurement. Confocal images of actin filaments stained with phalloidin (1/40) were taken (upper panel 24 hpf control embryo and lower panel 24 hpf Tricaine treated embryo) and measurements between H bands, across Z discs and sarcomeres, were made. (C) 17–24 hpf paralysis caused a significant lengthening of actin filaments H-H at 24 hpf (*p < 0.05) (n = 20 in 5 controls, n = 15 in 3 treated embryos, t-test). At 42 hpf, recovered embryos (17–24 hpf paralysis) showed a full recovery (n = 50 in 10 control embryos, n = 40 in 8 recovered embryos, unpaired t-test), in contrast with treated embryos (17–42 hpf paralysis) which did not recover (n = 50 in 10 control embryos, n = 135 in 27 treated,*p < 0.05). (D) Immotile relaxed mutants, rr, showed a very significant lengthening of actin filaments H-H at 24 hpf (***p < 0.001) (n = 115 in 23 immotile relaxed mutants rr, n = 120 in 24 motile control siblings Rr/RR and at 42hpf (***p < 0.001) (n = 105 in 21 immotile relaxed mutants rr, n = 135 in 27 motile control siblings Rr/RR). Interestingly, in contrast with the Tricaine treated immotile embryos, the immotile relaxed mutant (rr) actin is still very significantly longer than the motile control siblings (Rr/RR) actin at 42 hpf. It is noticeable that in the complete absence of movement up to 42 hpf actin filaments showed a significant shortening between 24 and 42 hpf in between both treated and control embryos and immotile relaxed mutants and motile control siblings, respectively (n = 15 in 3 24 hpf treated, n = 135 in 27 42 hpf treated embryos, *p < 0.05 unpaired t-test; and n = 115 in 23 24 hpf immotile relaxed mutants rr, n = 105 in 21 42 hpf immotile relaxed mutants rr, *p < 0.05 unpaired t-test).
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Figure 5: Paralysis leads to actin lengthening in both relaxed mutant and Tricaine treated embryos, subsequent movement restoration in recovered embryos leads to actin length complete rescue. (A,B) H-H measurement. Confocal images of actin filaments stained with phalloidin (1/40) were taken (upper panel 24 hpf control embryo and lower panel 24 hpf Tricaine treated embryo) and measurements between H bands, across Z discs and sarcomeres, were made. (C) 17–24 hpf paralysis caused a significant lengthening of actin filaments H-H at 24 hpf (*p < 0.05) (n = 20 in 5 controls, n = 15 in 3 treated embryos, t-test). At 42 hpf, recovered embryos (17–24 hpf paralysis) showed a full recovery (n = 50 in 10 control embryos, n = 40 in 8 recovered embryos, unpaired t-test), in contrast with treated embryos (17–42 hpf paralysis) which did not recover (n = 50 in 10 control embryos, n = 135 in 27 treated,*p < 0.05). (D) Immotile relaxed mutants, rr, showed a very significant lengthening of actin filaments H-H at 24 hpf (***p < 0.001) (n = 115 in 23 immotile relaxed mutants rr, n = 120 in 24 motile control siblings Rr/RR and at 42hpf (***p < 0.001) (n = 105 in 21 immotile relaxed mutants rr, n = 135 in 27 motile control siblings Rr/RR). Interestingly, in contrast with the Tricaine treated immotile embryos, the immotile relaxed mutant (rr) actin is still very significantly longer than the motile control siblings (Rr/RR) actin at 42 hpf. It is noticeable that in the complete absence of movement up to 42 hpf actin filaments showed a significant shortening between 24 and 42 hpf in between both treated and control embryos and immotile relaxed mutants and motile control siblings, respectively (n = 15 in 3 24 hpf treated, n = 135 in 27 42 hpf treated embryos, *p < 0.05 unpaired t-test; and n = 115 in 23 24 hpf immotile relaxed mutants rr, n = 105 in 21 42 hpf immotile relaxed mutants rr, *p < 0.05 unpaired t-test).

Mentions: Given the observations that paralysis alters myofibril arrangement and shortens sarcomere length in developing zebrafish embryos, actin remodeling was assessed by measuring actin myofilament length during paralysis at 24 and 42 hpf and after movement recovery at 42 hpf, in both Tricaine treated and immotile relaxed mutant embryos. For this purpose, we estimated the thin filament length by determining the distance between the H zones in the sarcomeres in Alexa488 phalloidin stained larvae (Figures 5A,B).


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)

Paralysis leads to actin lengthening in both relaxed mutant and Tricaine treated embryos, subsequent movement restoration in recovered embryos leads to actin length complete rescue. (A,B) H-H measurement. Confocal images of actin filaments stained with phalloidin (1/40) were taken (upper panel 24 hpf control embryo and lower panel 24 hpf Tricaine treated embryo) and measurements between H bands, across Z discs and sarcomeres, were made. (C) 17–24 hpf paralysis caused a significant lengthening of actin filaments H-H at 24 hpf (*p < 0.05) (n = 20 in 5 controls, n = 15 in 3 treated embryos, t-test). At 42 hpf, recovered embryos (17–24 hpf paralysis) showed a full recovery (n = 50 in 10 control embryos, n = 40 in 8 recovered embryos, unpaired t-test), in contrast with treated embryos (17–42 hpf paralysis) which did not recover (n = 50 in 10 control embryos, n = 135 in 27 treated,*p < 0.05). (D) Immotile relaxed mutants, rr, showed a very significant lengthening of actin filaments H-H at 24 hpf (***p < 0.001) (n = 115 in 23 immotile relaxed mutants rr, n = 120 in 24 motile control siblings Rr/RR and at 42hpf (***p < 0.001) (n = 105 in 21 immotile relaxed mutants rr, n = 135 in 27 motile control siblings Rr/RR). Interestingly, in contrast with the Tricaine treated immotile embryos, the immotile relaxed mutant (rr) actin is still very significantly longer than the motile control siblings (Rr/RR) actin at 42 hpf. It is noticeable that in the complete absence of movement up to 42 hpf actin filaments showed a significant shortening between 24 and 42 hpf in between both treated and control embryos and immotile relaxed mutants and motile control siblings, respectively (n = 15 in 3 24 hpf treated, n = 135 in 27 42 hpf treated embryos, *p < 0.05 unpaired t-test; and n = 115 in 23 24 hpf immotile relaxed mutants rr, n = 105 in 21 42 hpf immotile relaxed mutants rr, *p < 0.05 unpaired t-test).
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Related In: Results  -  Collection

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

Figure 5: Paralysis leads to actin lengthening in both relaxed mutant and Tricaine treated embryos, subsequent movement restoration in recovered embryos leads to actin length complete rescue. (A,B) H-H measurement. Confocal images of actin filaments stained with phalloidin (1/40) were taken (upper panel 24 hpf control embryo and lower panel 24 hpf Tricaine treated embryo) and measurements between H bands, across Z discs and sarcomeres, were made. (C) 17–24 hpf paralysis caused a significant lengthening of actin filaments H-H at 24 hpf (*p < 0.05) (n = 20 in 5 controls, n = 15 in 3 treated embryos, t-test). At 42 hpf, recovered embryos (17–24 hpf paralysis) showed a full recovery (n = 50 in 10 control embryos, n = 40 in 8 recovered embryos, unpaired t-test), in contrast with treated embryos (17–42 hpf paralysis) which did not recover (n = 50 in 10 control embryos, n = 135 in 27 treated,*p < 0.05). (D) Immotile relaxed mutants, rr, showed a very significant lengthening of actin filaments H-H at 24 hpf (***p < 0.001) (n = 115 in 23 immotile relaxed mutants rr, n = 120 in 24 motile control siblings Rr/RR and at 42hpf (***p < 0.001) (n = 105 in 21 immotile relaxed mutants rr, n = 135 in 27 motile control siblings Rr/RR). Interestingly, in contrast with the Tricaine treated immotile embryos, the immotile relaxed mutant (rr) actin is still very significantly longer than the motile control siblings (Rr/RR) actin at 42 hpf. It is noticeable that in the complete absence of movement up to 42 hpf actin filaments showed a significant shortening between 24 and 42 hpf in between both treated and control embryos and immotile relaxed mutants and motile control siblings, respectively (n = 15 in 3 24 hpf treated, n = 135 in 27 42 hpf treated embryos, *p < 0.05 unpaired t-test; and n = 115 in 23 24 hpf immotile relaxed mutants rr, n = 105 in 21 42 hpf immotile relaxed mutants rr, *p < 0.05 unpaired t-test).
Mentions: Given the observations that paralysis alters myofibril arrangement and shortens sarcomere length in developing zebrafish embryos, actin remodeling was assessed by measuring actin myofilament length during paralysis at 24 and 42 hpf and after movement recovery at 42 hpf, in both Tricaine treated and immotile relaxed mutant embryos. For this purpose, we estimated the thin filament length by determining the distance between the H zones in the sarcomeres in Alexa488 phalloidin stained larvae (Figures 5A,B).

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