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AMER. ZOOL., 39:570-579 (1999) Regulation of Muscle Growth and Sarcomeric Protein Gene Expression over the Intermolt Cycle1 ALICIA J. E L HAJ 2 School of Postgraduate Medicine, Keele University, Hartshill, Stoke on Trent ST4 7QB, UK Tel: (0)1792 554605, Fax: (0)1782 747319 SYNOPSIS. Crustacean muscle growth is associated with a hormonally mediated cyclical molt stage. The mechanisms by which fibre lengthening and hypertrophy occur in Crustaceans over the molt has been the subject of our and other researchers' investigations using histological, biochemical and molecular approaches. In this paper, we review our studies and present evidence for the different molecular mechanisms by which sarcomeric proteins are upregulated to achieve muscle sarcomere addition in lobsters during the molt. Synthesis of the sarcomeric proteins has been shown to increase during the premolt and postmolt phases in the leg and abdominal muscles coinciding with the addition of sarcomeres over ecdysis. This is in contrast to research on claw muscle demonstrating premolt atrophy. Our work and others' have investigated the factors which modulate this growth and turnover of muscle tissue in crustaceans. These changes in muscle turnover correspond with an elevated titre of circulating ecdysteroids and the role of these molting hormones in regulating sarcomeric mRNA and protein levels during cyclical muscle growth is discussed. Our results suggest that sarcomeric proteins may be controlled via both transcriptional and translational mechanisms during the molt interval and these findings are discussed in relation to previous research investigating muscle growth in Crustacea. INTRODUCTION Pioneering work by Dorothy Skinner and colleagues first began to consider changes in muscle mass through the molt cycle in the claws of the land crab, Gecarcinus lateralis, in the mid 60s (Skinner, 1965, 1966). Our work has contributed to this field with an emphasis on the growth of leg and abdominal muscles in a variety of different crustacean species. It has involved histological and biochemical analysis of muscle fibre lengthening and hypertrophy in predominantly decapod crustaceans. These studies have been extended to include investigations at the molecular level into muscle sarcomeric protein gene regulation. This review will cover these investigations and draw together some general 1 From the Symposium The Compleat Crustacean Biologist: A Symposium Recognizing the Achievements of Dorothy M. Skinner presented at the Annual Meeting of the Society for Integrative and Comparative Biology. 3-7 January 1998, at Boston. Massachusetts. 2 E-mail: [email protected] conclusions regarding muscle growth and the factors which regulate this growth. Histological studies identify the stages of muscle growth in the leg and the abdomen Fibre length and sarcomere number in the extensor muscle of the walking leg has been quantified in a number of decapod crustaceans over the intermolt cycle (El Haj et al., 1984; Houlihan and El Haj, 1985). These studies have demonstrated that muscle growth occurs by addition of new sarcomeres at ecdysis in Carcinus maenas and Homarus americanus. Autoradiographic localisation of labelled sarcomeric proteins has shown that sarcomeres may be added at the end of the fibres at the site of the attachment with the exoskeleton. This raises interesting questions regarding the ability of the muscle to function during ecdysis. When the new exoskeleton is being laid down, muscle fibres are increasing in length and reattaching. Our attempts to identify assembling sarcomeres during this period using electron microscopy have been successful in identifying shorter sarcomeres at the 570 REGULATION OF MUSCLE GROWTH ends of fibres which in some cases lack the filament organisation (Fig. la, b). However, it is not possible to confirm that these fibres are indeed not artefacts of fixation as a result of a softer cuticle. The situation is further complicated by early observations in the 60/70s of the presence of supercontracted sarcomeres in invertebrates and vertebrates by Hoyle et al. (1965), and Rice (1973). Supercontraction may be occurring in these growth regions where the muscle is forming the new attachment to the cuticle. What our evidence does confirm is that fibres are lengthening during the pre and postecdysial period by the addition of sarcomeres, but the process of assembly has yet to be characterized. Muscle hypertrophy may be occurring as a result of regulatory processes different from to those occurring during fibre lengthening. Morphometric analysis of myofibrillar surface area in leg muscles of C. maenas has demonstrated increases that result from the addition of thick and thin filaments. Analysis of size classes of myofibrils has suggested that growth of the myofibrils may be occurring by longitudinal myofibrillar splitting as in vertebrate muscle (El Haj et al., 1984). Our results have suggested that in contrast to growth occurring in length during a restricted phase of the molt cycle, muscle may be growing in bulk throughout the molt cycle by the addition of thick and thin filaments. This would account for the anecdotal observations that the muscle does not appear to fill the space in the lobster or crab immediately after the molt when the animal has expanded in size. This observation leads us to consider if the process of hypertrophy and lengthening are regulated differentially and how these processes are occurring. 571 peaks during the premolt and early postmolt (El Haj and Houlihan, 1987; El Haj et al., 1996; Mykles and Skinner, 1985). Using this technique on animals in the premolt phase, protein synthesis rates in vivo have been shown to increase in all three muscle groups, leg, abdominal and claw muscle of the American lobster, Homarus americanus (El Haj et al. 1995) (Fig. 2a). These findings have also been observed in the green shore crab in vitro, Carcinus maenas, and the crayfish, Austropotamobius pallipes (Whiteley et al, 1993; Whiteley et al., 1996). Early in vitro work by Skinner (1965) showed elevated amino acid incorporation in muscles and other tissues in the land crab in the premolt land crab, G. lateralis. El Haj et al., 1995, have suggested that the premolt elevation of protein synthesis occurs prior to the assembly of the sarcomeres during the early postmolt period and accounts for the synthesis of predominantly sarcomeric and other associated muscle proteins. In addition, Mykles (1997) proposes that the increase in synthesis at premolt reflects increased turnover of myofibullar proteins, which facilitates the rearrangements of myofilaments observed. Using cDNA probes to 18S ribosomal RNA for assessing ribosomal quantity, we have also shown that elevations in the rate of protein synthesis is not accompanied by an elevation in total ribosomal RNA in claw muscle in the American lobster (El Haj et al., 1995). In some other species, increasing ribosomal number is a mechanism for overall increased protein processing at key stages of development. However, in the lobster, increases in protein synthesis is not due to enhanced ribosomal numbers but appears to be due to enhanced ribosomal activity and increased transcription and mRNA producMuscle protein turnover during the molt tion in the muscle. cycle The premolt elevation in protein syntheThese histological observations coincide sis is not necessarily linked to an overall with biochemical experiments using la- elevation in muscle mass in all the muscle belled amino acid precursors in vivo and in groups in the animal. Evidence for this vitro. By adapting flooding dose techniques comes from studies by Skinner, Mykles and originated for mammalian turnover studies colleagues on the claw muscle. At the stage (Garlick et al., 1980), we have been able to when protein synthesis is elevated, extensor measure muscle protein turnover in vivo. and flexor muscles in the claw are underThese studies demonstrate that total muscle going up to 60% muscle atrophy (Skinner, protein synthesis rates are elevated with 1965; Mykles and Skinner, 1982). Corre- 572 ALICIA J. E L HAJ 573 REGULATION OF MUSCLE GROWTH 1.2 0.8 • Leg 1.0 0 Abdomen GDClaw ~ 0.6 '>. 0.8 -3 c I 0.6 £ 0.4 a. ~ 04 0.2 0.2 0 i C C I 1 £ c. =0 Postmoult Intermoult Injected Premoult a) Control b) FIG. 2. a) A] Rate of protein synthesis per day (K,) in three muscle groups (leg, abdomen and claw) in the lobster Homarus americanus at three stages of the moult cycle (n = 4) B] Amount of protein synthesised per day as a proportion of the total pool of RNA (KRNA) or a measure of ribosomal activity) in three muscle groups (leg, abdomen and claw) at three stages of the moult cycle. Values represent means + SEM. An asterisk indicates a significant difference from the value for the intermolt stages (P < 0.05). b) A] Rate of protein synthesis per day (K,) 3 days following an injection of a premolt concentration of 20-hydroxyecdysone (20-HE) (10~6 mol 1"') into three muscle groups in the lobster H. americanus. Controls were injected with saline (n = 6). B] Ribosomal activity in three muscle groups in the lobster 3 days after injection of 20-HE. Values represent means + SEM. An asterisk indicates a significant difference from the value for the control group (P < 0.01). sponding rates of protein degradation are also elevated in the claw tissue during the premolt. The resultant loss in muscle mass enables the claw to be drawn through the narrow basi-ischium joint (Mykles and Skinner, 1990). Whether the claw muscle is being differentially regulated compared to other muscle groups in the crab has not been determined. Mykles and Skinner (1982, 1990) proposed that there is an increase in the synthesis of Ca2+ dependant proteinases for the specific regulation of FIG. I. Longitudinal section of sarcomeres from the immediate postmolt muscle fibre in Homarus americanus. 35,000 X magnification, a) Electron micrograph showing the structure of the sarcomeres in the exoskeletal region in Homarus americanus. Note the greater proportion of thin filaments to thick filaments and the lack of organisation of the filaments into clear bands. Sarcoplasmic reticulum and mitochondria are present, although T tubule and dyad formation is not evident, b) Electron micrograph of the middle region of the fibre showing the sarcomeres with "normal" banding and typical arrangement of thick and thin filaments observed in striated muscle. Sarcomere length = 5 u.m. 574 ALICIA J. EL HAJ myofibrillar proteins during atrophy. One peak during premolt continuing through to theory is that these proteinases may be re- a lesser extent during postmolt in a number sponding to an increase in calcium titre in of different crustacean species (Chang, the haemolymph during the premolt. 1993). The elevations in protein synthesis Further evidence for differential muscle in Homarus americanus (El Haj et al., protein synthesis has been found when con- 1995) correspond to the stages during sidering the growth of the muscle in the iso- which previous measurements have shown pod, a crustacean which develops a biphasic an elevation in ecdysteroid titre, i.e., during molt. In these animals, the molt is biphasic the Dl-3 stage (Snyder and Chang, 1991). with subsequent differential growth of the Research using radiolabelled hormone two portions of the animals, the posterior binding assays has detected the presence of portion molting first followed by the ante- ecdysteroid receptors in a number of tissues rior portion (Steel, 1982). Our work on the in Carcinus maenas and Artemia sp. (El isopod has studied growth of the muscle over the biphasic molt in an attempt to un- Haj et al., 1997). In these studies the titre ravel the regulation and timing of ecdysial of ecdysteroid within the muscles has been muscle growth. Increases in size occur in shown to be low compared to other tissues two phases separated by hours or days ac- within crustaceans. This has hampered cording to the species which suggested that progress in determining the role of this horthe muscles may grow biphasically. Using mone in controlling muscle growth. Howradiolabelled phenylalanine in vivo flooding ever, ecdysteroids have been shown to have techniques, we have measured rates of pro- a direct effect on muscle turnover. Whiteley tein synthesis in the muscle and showed et al. (1991), demonstrated how doses of that they were elevated in the anterior and ecdysteroid administered in vitro to isolated the posterior portion during the premolt leg muscles in culture could have an effect compared to the intermoult rates (Whiteley on RNA synthesis rates in a dose responand El Haj, 1997). In contrast, protein syn- sive manner. Furthermore, studies have thesis rates in the ventral longitudinal mus- shown that 20 hydroxyecdysone injected in cle, which lies along the length of the body, vivo result in elevated protein synthesis in were elevated by 3 fold in the anterior half the claw, leg and abdominal muscle of the of the muscle as compared to the posterior lobster after three days (El Haj et al., 1995) half during half molt when the posterior (Fig. 2b). Ecdysteroids have been shown to exoskeleton has shed (Whiteley and El Haj, have direct effects on protein synthesis rates 1997). This difference was also measured in other crustacean tissues, such as exountil 36 hours postmolt when levels are skeleton, hypodermis and hepatopancreas equilibrated to intermolt. Interestingly, actin (Stringfellow and Skinner 1988; Traub et and myosin mRNA remained constant between biphasic molts which agrees with our al., 1987; Paulson and Skinner, 1991). findings in the lobster. Systemic levels of These results suggest that ecdysteroids are ecdysteroid were equilibrated in the anterior playing a key role in elevating protein synand posterior portions of the isopod. This thesis in preparation for the molt. More recently, ecdysteroid receptors implies that differential growth could be have been localized in the premolt muscle regulated as a function of changing receptor expression. This regulation may interact at of lobsters using a heterologous Drosophila the level of translation of proteins and sar- antibody for immunocytochemical and comeric assembly. These studies on growth Western blot techniques (El Haj et al., patterns of differing muscle groups through 1994; Koelle et al., 1991). Western blots the molt raise interesting questions regard- using the EcR antibody, hybridised with a ing how muscles may be differentially reg- 95—110 kDa protein from the lobster tissues ulated in crustaceans. examined. Sections of the eyestalk neural tissue showed cytoplasmic localisation of Ecdysteroids: Their role in regulation of the EcR in the neural cells associated with muscle growth the X-organ/sinus gland complex. MolecuEcdysteroids, the major molt regulating lar approaches have included cloning the hormones in Crustacea, have been shown to crustacean receptor for ecdysteroid from REGULATION OF MUSCLE GROWTH Uca pugilator (Durica and Hopkins, 1996) and an ecdysteroid responsive DNA binding protein from the lobster Homarus americanus (El Haj et al., 1997). The Homarid ecdysteroid responsive gene, HHR3, has a ligand binding region which contains conserved sites with the steroid receptor superfamily. In response to in vivo injection into intermolt animals of 20 hydroxyecdysone, HHR3 mRNA levels are elevated in the epidermis and leg muscle after 6 hr (Fig. 3). In contrast, the sinus gland/x organ shows an opposing pattern of expression. HHR3 expression varies in tissues from these animals at the premolt and postmolt stages which co-ordinate with other key modulations (Fig. 4). Evidence is mounting to support the theory that these ecdysteroids are acting at the level of transcription possibly through a cascade of DNA binding factors to increase sarcomeric protein synthesis during the premolt period. Further recent evidence for a cascade of ecdysteroid responsive transcription factors includes the identification of a shrimp E75 homologue, an early ecdysteroid responsive DNA binding protein, which may be involved in downstream regulation of key target proteins during development or the moult cycle in crustaceans (Chan, 1998). Regulation of sarcomeric protein genes in Crustacea Relatively few crustacean sarcomeric protein genes have been cloned and made available for use in molecular studies of crustacean growth. The majority have been cloned in the brine shrimp, Artemia sp. (El Haj, 1996). Therefore in order further to investigate the molecular basis for the control of muscle growth and development in lobsters, we along with other groups have first had to clone specific cDNAs which could be used to investigate regulatory mechanisms. Using heterologous primers and PCR, we have cloned a 736 bp fragment including the majority of the coding region of the H. gammarus sarcomeric protein, actin (Harrison and El Haj, 1994) and recognises a 1.6 kb mRNA in fast and slow fibres. In addition, cDNA probes for Homarus sp. fast and slow heavy chain myosin and tropomyosins have been cloned by the 575 group of Mykles and colleagues (Cotton and Mykles, 1994; Mykles et al, 1998). The fast myosin heavy chain clone contains the C terminus and 3' untranslated region which enables specificity for the fast isoform of MHC. We have begun to clone myosin heavy chain isoforms from various species including the Antarctic isopod, Glyptonotus antarcticus. These studies currently underway are focusing on identifying the presence of temperature related myosin isoforms in crustaceans from differing habitats and subjected to varying rearing temperatures. Little information is available on the promoter or regulatory regions present in crustacean genes. Few genomic clones have been isolated with little corresponding genomic analysis. In Artemia, analysis of the 5' end of the SERCA gene has revealed a number of common regulatory and promoter sequences including those involved in the MyoD family and MEF-2 binding sites (Escalante and Sastre, 1994). These regulatory sequences, such as MyoD and MEF 2, are involved in controlling differentiation of muscle cells. Further work is needed to identify how crustacean sarcomeric protein genes are being regulated over the molt cycle and to identify some of the key promoters responsible for their regulation. Our research has concerned the effect of regulating factors on sarcomeric protein mRNA expression such as mechanical forces, temperature and hormone levels. Work by Harrison and El Haj (1994) has demonstrated that lobster muscle is stretch responsive and that the actin mRNA up-regulation is triggered by stretch and sarcomere elongation. In this case the immobilised limbs reacted slowly and mRNA levels took up to 2 weeks to rise. This would potentially be too slow to play a functional role for a stimulus as a result of cuticle stretching over the molt (although the amount of stretch imposed may not be directly comparable). However, as has been shown for mammalian muscle (Goldspink et al., 1994), crustacean muscle fibres are stretch responsive resulting in mRNA upregulation and elevated synthesis. Varied results in different species have 576 ALICIA J. EL HAJ IN VIVO 20-HE INJECTION HOMARUS AMERICANUS 0 6 21 48 HRS POST INJECTION A) Epidermis 18S B) Leg muscle 18S 0 Sinus gland / x organ 18 S FIG. 3. Northern blot analysis of (A) epidermal, (B) leg muscle and (C) sinus gland/x-organ complex, from the eyestalk of H. americanus after injection of 20E. RNA samples were extracted from these tissues at 0, 6, 21 and 48 hr post-injection. Relative loading of the lanes was determined using a probe for 18S RNA. Leg muscle tissue was composed of extensor and flexor muscles that were dissected free of surrounding epidermal tissue. 577 REGULATION OF MUSCLE GROWTH Leg muscle 7 18 26 32 41 50 70 78 90 104 134 135 70 90 X organ /sinus gland 7 18 26 32 11 50 78 104 134 135 Days postmoult Epidermis T 0-15 0-1 _ T 005 - Post Inter Pre FIG. 4. Levels of HHR3 raRNA over the molt cycle in H. americanus in leg muscle, x-organ/sinus gland, and epidermis. Graphs represent densitometric analyses of HHR3 levels as ratios to 18S RNA. Days through the molt cycle are indicated on the x-axis and correspond to the stages (post, inter and pre) listed on the lower x-axis. been obtained regarding elevated mRNA levels throughout the molt for actin and myosin (El Haj and Whiteley, 1997; Whiteley and El Haj, 1997; Varadaraj et al., 1996). This may be partially due to the large interindividual variation in wild and laboratory reared species of Crustaceans. In muscles of Gecarcinus and Carcinus sp., actin mRNA levels are elevated during the pre and postmoult periods in comparison to intermolt levels (Whiteley et al., 1991; Varadaraj et al., 1996) In juvenile lobster, although there is a great deal of inter-individual variation, the overall trend in the levels of actin remain constant throughout the moult cycle in claw and other muscle tissues (Whiteley and El Haj, 1997). Mykles (1997) demonstrated that mRNA level of myosin HC decreases over the premolt period in the land crab even though synthesis of the protein increases about ninefold. It appears that both transcriptional and translational processes may be involved in modulation of growth of crustacean muscle; a similar finding to research on mammalian muscle (Endo and Nadal Ginard, 1987). Mammalian studies have shown that there are significant differences observed in muscle in translational efficiency, thus transcript accumulation is unlikely to be the only determinant of sarcomeric assembly (Endo and Nadal Ginard, 1987). Results from our investigations, as well as Mykles 1997, show that actin and myosin mRNA may not show co-ordinate regulation through the molt in abdominal muscle. Mykles (1997) has shown that myosin mRNA levels in the claw decline during premolt in G. lateralis when other sarcomeric proteins are elevating in expression. This is in contrast to studies of myogenesis in mammals and humans where total mRNA is maintained with similar stoichiometry in the different stages of muscle development (Wade et al., 1990). Finally, there is little information to address if there are developmental isoforms of sarcomeric proteins which may be expressed during molt induced muscle growth. Drosophila have 6 actin genes that are developmentally expressed in varying isoforms (Fyrberg et al., 1983). Varadaraj et al. (1996) have indicated 7-11 actin genes using Southern blot analysis of the G. lateralis genome. Only two or three partial actin sequences have been cloned and a full analysis of expression patterns with development and the molt has yet to be carried out. It is possible that actin isoforms may be varying in expression through the molt, but these differences are not being identified using the cDNAs available. 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