Cell Tissue Res. 203, 503-506 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Short Communication Ultrastructural Evidence for Disuse Atrophy in Insect Skeletal Muscle M. Anderson Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham, England
Summary. The changes which occur in the ultrastructure of the mesothoracic retractor unguis muscle of the cockroach Periplaneta americana as a result of disuse are described. Breakdown of myofibrils, sarcoplasmic reticulum, and mitochondria are all marked, this degeneration only being apparent 9 weeks after the operation. Key words: Muscle - Disuse atrophy - Insects - Ultrastructure - Electron microscopy.
Disuse atrophy is a c o m m o n phenomenon in vertebrate skeletal muscle (see Gutmann, 1962, 1976). Previous studies on the effects of disuse on insect skeletal muscle have been confined to the flight muscles of Drosophila (Sohal, 1975; Deak, 1976) and, in spite of slight structural changes in the muscles of the flightless individuals (Sohal, 1975), there is no evidence for extensive degeneration or loss of function of the muscles due to disuse (Deak, 1976). The structural changes in skeletal muscle of Schistocerca following denervation have been studied by Rees and Usherwood (1972) who were able to draw close parallels between their results and the work on vertebrate muscle. The present paper reports on preliminary studies on disuse atrophy in a leg muscle of the cockroach Periplaneta induced by preventing normal working of the muscle.
Materials and Methods Adult cockroaches, Periplaneta americana, from departmental stock were operated upon so as to prevent the retractor unguis muscle in one of the mesothoracic legs from functioning. This operation involved the removal of the pretarsus of the leg, since the apodeme of the retractor unguis muscle Send offprint requests to: Dr. M. Anderson, Department of Zoology and Comparative Physiology, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, England
0302-766X/79/0203/0503/$01.00
504
M. Anderson
attaches to the unguitractorplate, responsiblefor the movementof the pretarsal claws,and to a plate at the junction of the 4th tarsal segment and the pretarsus, which causes the movementof the pretarsus. This operation preventsthe retractor unguis musclefrom performingany effectivework. The retractor unguis muscle from the other mesothoracic leg provided the control muscle. At various intervals followingthe operation,the retractor unguis muscleswerefixed,embeddedin epoxyresin,and ultrathin sections were examinedin an electron microscope.
Results and Discussion
Fig. I a shows part of a transverse section through a retractor unguis muscle from the mesothoracic leg of a cockroach that had been operated upon 9 weeks previously. At this stage the differences between this inactivated muscle and the control (Fig. lb) are clearly seen. These changes take place gradually and the precise timing of the disruption of the various organelles is at present being studied. In the experimental muscle fibres the sarcoplasmic reticulum (S.R) shows signs of disintegration with the result that the division of the muscle fibres into myofibrils is indistinct, w i t h the S.R. spread unevenly throughout the fibres. The control muscle fibres reveal two or three layers of S.R. clearly dividing the fibres into myofibrils. There appears to be little change in the transverse tubular system (T.T.S.) in the experimental muscles, and there is certainly no sign of the granulated sacs in the T.T.S. that are one of the signs of degeneration apparent in denervated muscle in insects (Rees and Usherwood, 1972). The mitochondria in the atrophied muscle have split into many smaller fragments that are widely dispersed amongest the myofibrils (Fig. 1 a), and not concentrated around the Z-bands which is the case in the control muscles. This fragmentation of the mitochondria was also apparent in denervated muscle (Rees and Usherwood, 1972). In the atrophied muscle the myofibrillar structure has begun to break up with the result that there are spaces in the muscle fibres which contain only microtubules and relatively large areas surrounding the nuclei devoid of any contractile material. The microtubules resemble the primary filaments which are associated with myofilament synthesis in developing insect muscle (Auber, 1969). Associated with this breakdown in the contractile material there is a reduction in the diameter of the muscle fibres; this together with the breaking up of the myofibrils is also a feature of denervation atrophy (Rees and Usherwood, 1972). The studies of Sohal (1975) and Deak (1976) on the flight muscles of Drosophila revealed no significant structural changes in the flight muscles after periods of flightlessness. It is possible that the periods of flightlessness to which they subjected their experimental animals were not sufficiently long for atrophy to become apparent, or that their experimental procedures did not completely eliminate isometric contractions which may be sufficient to maintain the ultrastructural integrity of the muscle fibres. It appears from the present study that normal innervation, normal tracheation, and normal haemolymph supply will not in themselves preserve the structure of the retractor unguis of Periplaneta. It seems probable that, like in the case of vertebrate skeletal muscle, active stretching of the muscle is necessary for maintenance of muscle structure.
Disuse Atrophy in Insect Muscle
505
Fig. 1. Transverse sections through muscle fibres from retractor unguis muscle of a an experimental, and b a control mesothoracic leg. Compare sarcoplasmic reticulum (S.R.), mitochondria (M), myofibrils (My), and note large spaces (S) in a containing microtubules (m) devoid of any myofilaments. Scale bar 1 txm. x 10,000
506
M. Anderson
Further experiments are in progress to induce disuse a t r o p h y in the retractor unguis musele by methods other than severance o f the apodeme. These experiments are designed to prevent m o v e m e n t o f the various joints operated by the retractor unguis by simulated "plaster casts" made f r o m various materials including dental wax and cyanocrylate adhesives. These experiments are proving reasonably successful and the results support those o f the pretarsal removal experiments. In addition to these structural studies, the effects o f disuse on the electrophysiological properties o f the muscle are being studied and some interesting results are being found which seem to follow closely the effects o f denervation on muscle physiology.
References Auber, J.: La myofibrillogen6sede muscle stri~ - 1. Insectes. J. Microsc. 8, 197-232 (1969) Deak, I.I.: Use of Drosophila mutants to investigate the effect oi~disuseon the maintenance of muscle. J. Insect Physiol. 22, 1159-1165 (1976) Gutmann, E.: Denervation and disuse atrophy in cross-striated muscle. Rev. Can. Biol. 21, 353-365 (1962) Gutmann, E.: Neurotrophic relations. Annu. Rev. Physiol. 38, 177-216 (1976) Rees, D., Usherwood, P.N.R.: Effects of denervation on the ultrastructure of insect muscle. J. Cell Sci. 10, 667~582 (1972) Sohal, R.S.: Mitochondrial changes in flight muscles of normal and flightless Drosophila melanogaster with age. J. Morphol. 145, 337-354 (1975) Accepted September 12, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
Short Communication Ultrastructural Evidence for Disuse Atrophy in Insect Skeletal Muscle M. Anderson Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham, England
Summary. The changes which occur in the ultrastructure of the mesothoracic retractor unguis muscle of the cockroach Periplaneta americana as a result of disuse are described. Breakdown of myofibrils, sarcoplasmic reticulum, and mitochondria are all marked, this degeneration only being apparent 9 weeks after the operation. Key words: Muscle - Disuse atrophy - Insects - Ultrastructure - Electron microscopy.
Disuse atrophy is a c o m m o n phenomenon in vertebrate skeletal muscle (see Gutmann, 1962, 1976). Previous studies on the effects of disuse on insect skeletal muscle have been confined to the flight muscles of Drosophila (Sohal, 1975; Deak, 1976) and, in spite of slight structural changes in the muscles of the flightless individuals (Sohal, 1975), there is no evidence for extensive degeneration or loss of function of the muscles due to disuse (Deak, 1976). The structural changes in skeletal muscle of Schistocerca following denervation have been studied by Rees and Usherwood (1972) who were able to draw close parallels between their results and the work on vertebrate muscle. The present paper reports on preliminary studies on disuse atrophy in a leg muscle of the cockroach Periplaneta induced by preventing normal working of the muscle.
Materials and Methods Adult cockroaches, Periplaneta americana, from departmental stock were operated upon so as to prevent the retractor unguis muscle in one of the mesothoracic legs from functioning. This operation involved the removal of the pretarsus of the leg, since the apodeme of the retractor unguis muscle Send offprint requests to: Dr. M. Anderson, Department of Zoology and Comparative Physiology, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, England
0302-766X/79/0203/0503/$01.00
504
M. Anderson
attaches to the unguitractorplate, responsiblefor the movementof the pretarsal claws,and to a plate at the junction of the 4th tarsal segment and the pretarsus, which causes the movementof the pretarsus. This operation preventsthe retractor unguis musclefrom performingany effectivework. The retractor unguis muscle from the other mesothoracic leg provided the control muscle. At various intervals followingthe operation,the retractor unguis muscleswerefixed,embeddedin epoxyresin,and ultrathin sections were examinedin an electron microscope.
Results and Discussion
Fig. I a shows part of a transverse section through a retractor unguis muscle from the mesothoracic leg of a cockroach that had been operated upon 9 weeks previously. At this stage the differences between this inactivated muscle and the control (Fig. lb) are clearly seen. These changes take place gradually and the precise timing of the disruption of the various organelles is at present being studied. In the experimental muscle fibres the sarcoplasmic reticulum (S.R) shows signs of disintegration with the result that the division of the muscle fibres into myofibrils is indistinct, w i t h the S.R. spread unevenly throughout the fibres. The control muscle fibres reveal two or three layers of S.R. clearly dividing the fibres into myofibrils. There appears to be little change in the transverse tubular system (T.T.S.) in the experimental muscles, and there is certainly no sign of the granulated sacs in the T.T.S. that are one of the signs of degeneration apparent in denervated muscle in insects (Rees and Usherwood, 1972). The mitochondria in the atrophied muscle have split into many smaller fragments that are widely dispersed amongest the myofibrils (Fig. 1 a), and not concentrated around the Z-bands which is the case in the control muscles. This fragmentation of the mitochondria was also apparent in denervated muscle (Rees and Usherwood, 1972). In the atrophied muscle the myofibrillar structure has begun to break up with the result that there are spaces in the muscle fibres which contain only microtubules and relatively large areas surrounding the nuclei devoid of any contractile material. The microtubules resemble the primary filaments which are associated with myofilament synthesis in developing insect muscle (Auber, 1969). Associated with this breakdown in the contractile material there is a reduction in the diameter of the muscle fibres; this together with the breaking up of the myofibrils is also a feature of denervation atrophy (Rees and Usherwood, 1972). The studies of Sohal (1975) and Deak (1976) on the flight muscles of Drosophila revealed no significant structural changes in the flight muscles after periods of flightlessness. It is possible that the periods of flightlessness to which they subjected their experimental animals were not sufficiently long for atrophy to become apparent, or that their experimental procedures did not completely eliminate isometric contractions which may be sufficient to maintain the ultrastructural integrity of the muscle fibres. It appears from the present study that normal innervation, normal tracheation, and normal haemolymph supply will not in themselves preserve the structure of the retractor unguis of Periplaneta. It seems probable that, like in the case of vertebrate skeletal muscle, active stretching of the muscle is necessary for maintenance of muscle structure.
Disuse Atrophy in Insect Muscle
505
Fig. 1. Transverse sections through muscle fibres from retractor unguis muscle of a an experimental, and b a control mesothoracic leg. Compare sarcoplasmic reticulum (S.R.), mitochondria (M), myofibrils (My), and note large spaces (S) in a containing microtubules (m) devoid of any myofilaments. Scale bar 1 txm. x 10,000
506
M. Anderson
Further experiments are in progress to induce disuse a t r o p h y in the retractor unguis musele by methods other than severance o f the apodeme. These experiments are designed to prevent m o v e m e n t o f the various joints operated by the retractor unguis by simulated "plaster casts" made f r o m various materials including dental wax and cyanocrylate adhesives. These experiments are proving reasonably successful and the results support those o f the pretarsal removal experiments. In addition to these structural studies, the effects o f disuse on the electrophysiological properties o f the muscle are being studied and some interesting results are being found which seem to follow closely the effects o f denervation on muscle physiology.
References Auber, J.: La myofibrillogen6sede muscle stri~ - 1. Insectes. J. Microsc. 8, 197-232 (1969) Deak, I.I.: Use of Drosophila mutants to investigate the effect oi~disuseon the maintenance of muscle. J. Insect Physiol. 22, 1159-1165 (1976) Gutmann, E.: Denervation and disuse atrophy in cross-striated muscle. Rev. Can. Biol. 21, 353-365 (1962) Gutmann, E.: Neurotrophic relations. Annu. Rev. Physiol. 38, 177-216 (1976) Rees, D., Usherwood, P.N.R.: Effects of denervation on the ultrastructure of insect muscle. J. Cell Sci. 10, 667~582 (1972) Sohal, R.S.: Mitochondrial changes in flight muscles of normal and flightless Drosophila melanogaster with age. J. Morphol. 145, 337-354 (1975) Accepted September 12, 1979
