PHYSIOLOGY AND R E P R O D U C T I O N Myosin Isoform Expression in Skeletal Muscles of Turkeys at Various Ages 1 K. MARUYAMA and N. KANEMAKI2 USDA Agricultural Research Service, Avian Physiology Laboratory, Beltsville, Maryland 20705
ABSTRACT The appearance of myosin isoforms in skeletal muscles of turkey embryos, poults, and torus was studied, using monoclonal antibodies raised against myosin isoforms in chicken fast-twitch muscle {Pectoralis). The myosin extract was prepared by repeated salt extraction-precipitation. The reactivity of monoclonal antibodies with turkey myosin isoforms was tested by an enzyme linked immunosorbent assay using alkaline phosphatase-conjugated antibody and detection by color development with p-nitrophenyl phosphate. Detection was also effected by protein slot blotting using peroxidase-conjugated antibody and color development with 3,3'-diaminobenzidine tetrahydrochloride. The monoclonal antibody AB8 was found to be specific for the adult myosin isoform, present in Pectoralis muscle of 14-day-old and adult turkeys and adult chickens. Subsequent peptide mapping also indicated that the adult myosin isoform of turkey Pectoralis muscle was nearly identical to the adult isoform from chickens. The monoclonal antibody 2E9 reacted with the myosin extract only from poults at ages of 7 days and 14 days posthatch, indicating that 2E9 is specific for the neonatal myosin isoform. The reactivity of 2E9 was noted with the muscle of the mixed fiber type (the thigh muscle group) as well as with the fast-twitch muscle (Pectoralis). Monoclonal antibodies EB165 and AG6 were found to react with the myosin extractfromall ages tested. Based on the reactivity with monoclonal antibodies, it was concluded that myosin in turkey muscles existed as at least three discrete isoforms that were expressed sequentially in the course of muscle development. (Key words: turkey, muscle development, myofibrillar proteins, myosin heavy chain isoform, gene expression) 1991 Poultry Science 70:1748-1757 INTRODUCTION
Skeletal muscles are classified into fast- and slow-twitch muscles based on their contraction speed. In both fast- and slow-twitch muscles, myosin and actin make up the bulk of myofibrillar proteins. Myosin, the largest component, constitutes approximately 44% of myofibrillar proteins in chicken skeletal muscle (Khalili and Zarkadas, 1988). Different forms (isoforms) of myosin are known to be present in smooth, cardiac, and skeletal muscles. In skeletal muscles, myosin was reported to exist as isoforms specific for the muscle fiber type (Hon et al, 1976; d'Albis et al, 1979). These muscle fiber-specific isoforms of myosin were classified into slow myosin
1 This work was in part supported by Biomedical Research Support Grant S07 RR07026 awarded by Biomedical Research Support Grant Program, Division of Research Sources, National Institutes of Health, Washington, DC. 2 A visiting scientistfromAzabu University, Kanagawa, Japan.
isoform, fast myosin isoform, and intermediate myosin isoform (Fitzsimons and Hoh, 1983) and more recendy into HCI, HCIIa, HCIIb, and HCIId isoforms (Termin et al, 1989). Three muscle fiber types of slow, fast, and intermediate (Type I, Type HA, and Type HB, and intermediate between Type HA and Type HB, respectively) have been identified in chicken thigh muscles (Aberle et al, 1979; Aberle and Stewart, 1983; Suzuki et al, 1985). The breast muscle of chickens, Pectoralis, was shown to contain mostly fast-twitch white fiber (Type HB) and to a minor extent slow-twitch and intermediate fibers (Rosser and George, 1986). The myosin molecule consists of six peptide subunits, including two heavy chains of about 200 kDa. In addition to muscle fiberspecific isoforms, myosin from chicken Pectoralis muscle exists as developmental stagespecific isoforms. The myosin heavy chain prepared from me Pectoralis muscle of adult chickens contained an isoform discrete from the isoforms found in the Pectoralis muscle of 12-day-old embryos and newly hatched chicks
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(Received for publication December 17, 1990)
MYOSIN ISOFORM EXPRESSION
MATERIALS AND METHODS
Animals and Myosin Extraction A flock of Large White turkey hens (BUTA 8 line) were artificially inseminated with semen from Large White male turkeys (BUTA 6 line).3 Fertilized eggs were incubated in a forced-draft incubator at the dry-bulb temperature of 37.5 C and the relative humidity of 54.5%. On the 25th day of incubation, eggs were transferred to a hatcher in hatching baskets and incubated at the dry-bulb temperature of 37 C and the relative humidity of 53.5%. The hatching was completed
British United Turkeys of America, Lewisburg, WV 24901. 4 Brinkmann Instruments, Inc., Westbury, NY 11590.
by 29 days of incubation. On Day 24 of the incubation, nine developing embryos (24-day embryos) were killed by exsanguination to excise breast muscle (Pectoralis) and thigh muscles (Semitendinosus, Semimembranosus, and Quadriceps femoris). The breast muscle and the thigh muscles were also collected from eight poults on day of hatch, 7 days posthatch, and 14 days posthatch. Six male turkeys (BUTA 6 line) were trilled with an overdose of sodium pentobarbital at 44 wk of age (adults) to excise Pectoralis, Gastrocnemius, Sartorius, and Posterior latissimus dorsi muscles. Muscles from embryos, poults, and adult turkeys were quickly frozen in solid CO2 and stored at -70 C until protein preparation. Muscle samples were cut into pieces and homogenized in a homogenizing buffer containing .2 M KCl, 20 mM K 2 HP0 4 , and 10 mM ethyleneglycol-bis-(P-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) (pH 6.8), with a Polytron PT-10 homogenizer.4 The myosin fraction was extracted by a modified procedure of Baril et al. (1966), using repeated precipitation in the homogenizing (low ionic strength) buffer and extraction in an extraction (high ionic strength) buffer containing .6 M KCl, 10 mMK 2 HP0 4 ,1 mMNa 2 P20 5 ,1 mMMgCl 2 , 1 mM dithiothreitol, and 1 mM EGTA (pH 8.8). The myosin extract obtained was dialyzed overnight against the homogenizing buffer and centrifuged at 10,000 x g for 20 min. The pellet was dissolved in a buffer containing 80 mM Na 2 P 2 0 5 , 5 mM MgCl2, and 2 mM EGTA (pH 8.8). After 1 h at 4 C, the solution was centrifuged at 10,000 x g for 10 min. An equal volume of glycerol was added to the supernatant for storage at -70 C until analysis. In the course of the present study, muscle samples were collected from chickens (Indian River broiler chicks and White Leghorn hens) as needed, to prepare myosin extracts that served as the reference samples for monoclonal antibodies. Monoclonal Antibodies Monoclonal antibodies used for the ELISA and protein slot blotting were EB165, AG6,2E9, and AB8 in ascites fluid provided by Everett Bandman at the University of California-Davis. These monoclonal antibodies are specific for myosin heavy chain isoforms of fast-twitch muscles of chickens as follows: EB165 for embryonic and adult myosin heavy chain isoforms, AB8 for adult myosin heavy chain
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(Bandman et al., 1982). These developmental stage-specific isoforms of the myosin heavy chain have been successfully identified with monoclonal antibodies. The sequential appearance of the embryonic isoform, the neonatal isoform, and the adult isoform in the Pectoralis muscle was demonstrated as chickens developed from embryos to chicks and to adults (Bandman, 1985; Cerny and Bandman, 1987). It has been shown that the myosin heavy chain isoforms were encoded by genes of a multigene family containing at least seven genes and expressed in a developmental stagespecific manner (Robbins et al., 1982, 1986), suggesting the possible expression of more isoforms than has been detected by monoclonal antibodies. The Pectoralis muscle of turkeys is composed primarily of fast-twitch white (Type IIB) muscle fiber, as observed in chickens. Whereas in thigh muscles, the Sartorius muscle is composed of nearly equal amounts of fast and slow fibers and the Semitendinosus muscle is composed of fast white fibers and fast red fibers (Wiskus et al., 1976). In the present investigation, it was hypothesized that in skeletal muscles of turkeys, the myosin heavy chain exists as isoforms and the expression of isoforms is dependent on the developmental stage, as well as the muscle fiber type. In the current study, monoclonal antibodies from chickens were evaluated on their reactivity with the crude myosin preparation from skeletal muscles of turkeys at various developmental stages.
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MARUYAMA AND KANEMAK3
Immunoblotting
Peptide Mapping
Using an immunoblotting technique, the specificity of monoclonal antibodies for myosin isoforms determined by ELISA was verified and the expression of developmental stage-specific isoforms was studied in Pectoralis muscle (mostly Type IIB muscle fiber) and muscles of the mixed muscle fiber types from 24-day-old embryos, day-old poults, 7-day-old poults, 14-day-old poults, and adult turkeys. For this purpose, the thigh muscle group (Semitendinosus, Biceps flexor cruris, Extensor femoris, and Sartorius muscles), in addition to Pectoralis muscle, were collected from turkey embryos and poults, and Gastrocnemius, Sartorius, and Posterior latissimus dorsi muscles were collected from adult turkeys. The myosin extract
Fifty microliters of the myosin extract was diluted with an equal volume of a denaturing buffer containing 62.5 mAf Tris, 2% SDS, 5% 2-mercaptoethanol (MCE), and .001% bromophenol blue (pH 6.8) in a 1.5 mL microcentrifuge tube. Then the tube was placed in boiling water for 4 min to denature proteins. The denatured sample was subjected to SDSPAGE. The sample volume for SDS-PAGE was adjusted to give 100 |ig protein per lane for neonatal samples and 80 ug protein per lane for adult samples. Electrophoresis was performed in a discontinuous slab gel system described by Laemmli (1970). The SDS-PAGE procedure used was a modification of Fritz et al. (1989), in which the upper reservoir buffer contained 5 raM MCE. The stacking gel was prepared with 3% (wt/vol) acrylamide and the resolving gel with 10% (wt/ vol) acrylamide with the dimension of 14 cm x 13.5 cm. The gels were run at a constant current of 30 mA per gel at room temperature for
Enzyme-linked Immunosorbent Assay
jSigma Chemical Co., St. Louis, MO 63178-9916. "Model EL309, Bio-Tek Instruments, WinoosM, VT 05404. 7 Bio-Rad Laboratories, Richmond, CA 94804.
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To evaluate the cross-reactivity of monoclonal antibodies with turkey myosin heavy chain isoforms, an ELISA was performed using anti-mouse IgG conjugated with alkaline phosphatase5 for detection of bound antibody and pnitrophenyl phosphate as the substrate for color development. Pectoralis muscles from nine embryos, eight neonatal poults, and six adult turkeys were pooled to yield respective myosin extracts for each age group. Each ELISA well of a microtiter plate was coated with the myosin extract containing .5 jig protein (50 uL total volume) and incubated at 4 C overnight. Each well was then sequentially incubated with skim milk for blocking nonspecific binding, a monoclonal antibody, the second antibody-enzyme conjugate, and me substrate for color development. Each sample was run in quadruplicate and the absorbance was measured at 405 nm with a microtiter plate reader.6 The ELISA system was tested witii a commercially available monoclonal anti-skeletal myosin (fast) antibody (Clone MY32).5
was transferred to membranes using the protein slot blotting technique, and the isoform of interest was visualized by immunological detection. The protein slot blotting is carried out using a vacuum-assisted microfiltration apparatus (BioDot® SF7) and a nitrocellulose blotting membrane. The blotting procedure prepared by BioRad Laboratories'was used. A pair of protein blotting membranes were prepared for each monoclonal antibody. One membrane was stained for protein with amido black and the other was immunologically stained for a specific isoform. The immunological detection was done by the double antibody method using anti-mouse IgG conjugated with peroxidase5 as the second antibody. The substrate for peroxidase was 3,3 '-diaminobenzidine tetrahydrochloride (DAB). The working dilution of monoclonal antibodies was set at 2,000-fold for immunological detection. When the myosin extract was transferred to membranes by protein slot blotting, sample protein contents per well were adjusted to .25 and .5 u.g per slot. The sample protein content of .25 ug per slot was found to be sufficient for amido black staining for total protein to assure the transfer of proteins to membranes (data not shown), but was insufficient for the detection of isoforms by the monoclonal antibody-peroxidase-DAB staining.
isoform, 2E9 for neonate myosin heavy chain isoform, and AG6 for embryonic, neonate, and adult myosin heavy chain isoforms (Bandman, 1985).
1751
MYOSIN ISOFORM EXPRESSION
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(1977). The composition of the stacking gel was 3% (wt/vol) acrylamide, 1 mMEDTA, and .07% (vol/vol) N,N,N',N'-tetramethylethylenediamine (TEMED), and that of the resolving gel was 17.5% (wt/vol) acrylamide, 1 vaM EDTA, and .07% (vol/vol) TEMED with the dimension of 14 cm x 13.5 cm. The proteolytic digestion of gel slices was carried out by adding Protease Type XVTf-B from Staphylococcus aureus Strain V8 (EC 3.4.21.19)5 to a sample well at the amounts of .5, 1.0, and 2.5 ug per lane (well).
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FIGURE 1. The ELISA curves of the myosin extract from Pectoralis muscle of 24-day embryos ( • ) , 7-day-old poults (•), and adult male turkeys (A). The ELISA was performed using monoclonal antibodies, 2E9 (A), AB8 (B), AG6 (Q, and EB165 (D) and the absorbance (A405) was measured at the wavelength of 405 nm.
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approximately 5 h to separate the myosin heavy chain band. The gel was stained with Coomassie Brilliant Blue R250,5 destained and rinsed. The myosin heavy chain band was carved out and trimmed to a section 4 mm wide. The gel slice was placed in a sample well 6 mm wide in the peptide mapping gel. The sample well was filled with a buffer containing 20% glycerol, . 1 % SDS, 10 mM EDTA in .125 M Tris buffer (pH 6.8), and the peptide mapping was carried out by the procedure outlined by Cleveland et al.
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MARUYAMA AND KANEMAK3
The electrophoresis was run at 30 mA per gel for 40 min, stopped for 30 min to incubate and run at 30 mA per gel for 5 h to complete the run. The gel was stained with Coomassie brilliant blue R250 and the absorbance of individual bands was read at the wavelength of 633 nm with a laser beam densitometer.8
1C), and EB165 (Figure ID). The monoclonal antibody 2E9 was found to react selectively with the myosin extract from neonatal (7-day) poults (Figure 1A). In chickens, 2E9 was shown to have specificity for the neonatal isoform of the myosin heavy chain (Bandman, 1985) and the myosin in Pectoralis muscle of 20-day-old chickens was composed exclusively of the neonatal isoform of myosin. RESULTS AND DISCUSSION In the present investigation, the myosin extract was also prepared from Pectoralis Reactivity of Monoclonal Antibodies muscle of chickens to verify specificity of with Myosin from Pectoralis Muscle monoclonal antibodies for developmental stageof Embryos, Neonate, and Adult Turkeys specific myosin isoforms. It was confirmed that 2E9 reacted selectively with myosin from The reactivity of chicken monoclonal antiPectoralis muscle of 10-day-old chicks (Figure bodies with the myosin isoforms from turkey 2A), suggesting that in Pectoralis muscle of Pectoralis muscle was examined by ELISA (Figure 1). The reactivity of commercially turkeys, an isoform or isoforms comparable to the chicken neonatal isoform were expressed by prepared anti-skeletal myosin monoclonal anti7 days posthatch. hi order to learn the identity of body with the myosin extract indicated satisfacthe isoform, the myosin heavy chain peptide tory extraction of myosin from skeletal muscle maps were prepared from Pectoralis muscles of samples by the current procedure (data not 7-day-old poults and 10-day-old chicks simulshown). taneously. The peptide maps obtained from The myosin extract prepared from Pectoralis poults and chicks showed nearly identical muscle of embryos, neonatal poults, and adult cleavage fragments by the protease (EC turkeys was reacted with monoclonal antibodies, 3.4.21.19) (Figure 3). When the myosin heavy 2E9 (Figure 1 A), AB8 (Figure IB), AG6 (Figure chain band was digested by .5 |xg protease in the peptide mapping procedure, seven distinct cleavage fragments were noted in the peptide maps (Figure 3, Lanes 1 and 2). When the 8 UltroScans XL Laser Densitometer, Pharmacia LKB amount of the protease was increased to 2.5 ug Biotechnology, Inc., Piscataway, NJ 08855.
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FIGURE 2. The ELISA curves of the myosin extract from Pectoralis muscle of day-old chicks (O), 10-day-old chicks (D), and adult hens (A). The ELISA was performed using monoclonal antibodies, 2E9 (A) and AB8 (B) and the absorbance (A405) was measured at the wavelength of 405 nm.
1753
MYOSIN ISOFORM EXPRESSION
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FIGURE 3. Peptide maps of myosin heavy chains from turkey Pectoralis muscle and chicken Pectoralis muscle. The myosin heavy chain was electrophoretically prepared from the crude myosin extract of Pectoralis muscles and subjected to peptide mapping by proteolytic digestion as described. Lanes 1,5, and 9 were from 7-day-old turkey muscles and Lanes 2,6, and 10 were from 10-day-old chicken muscles. Lane 3, 7, and 11 were from adult turkey muscles and Lanes 4, 8 and 12 were from adult chicken muscles. The amounts of the protease (EC 3.4.21.19) per well were .5 \lg (Lanes 1 through 4), 1.0 Hg (Lanes 5 through 8), and 2.5 |ig (Lanes 9 through 12). Lane 13 was the blank (no sample). Arrows next to Lane 1 point seven major cleavage fragments of myosin from the breast muscle of 7-day-old turkeys.
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heavy chain by AB8 was not detected until the age of 20 days posthatch in chickens (Cerny and Bandman, 1987). As a reference, ELISA for AB8 was performed with the myosin extract from day-old, 10-day-old, and adult chickens (Figure 2B). It was observed that the absorbance (ELISA color development) with the myosin extract from 10-day-old chicks was about .10 of the absorbance recorded with the myosin extract from adult chickens. This observation with chicken myosin extracts was similar to the observation with turkey myosin extracts. Hence, it was concluded that the weak color development observed wim myosin extracts from neonatal poults and chicks was an indication of very weak expression of the adult myosin isoform as early as 7-day posthatch in turkeys. For the purpose of verifying the specificity of AB8 and learning the identity of the adult
the said seven cleavage fragments were still recognizable by a visual examination, as well as by densitometer scanning (Figure 3, Lanes 9 and 10). These observations suggested that turkeys and chickens had a similar, if not identical, isoform of the myosin heavy chain expressed during a relatively short period after hatch. The monoclonal antibody AB8 exhibited a strong reactivity with the myosin extract from Pectoralis muscle of adult turkeys and to a considerably lesser extent, with that of 7-day-old poults when tested in ELISA. The absorbance at the wavelength of 405 nm (ELISA color development) with the myosin extract from 7-day-old poults was approximately .10 of the absorbance observed with the adult myosin extract. It was reported that AB8 was specific for the adult myosin heavy chain isoform and the expression of the adult isoform of the myosin
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MARUYAMA AND KANEMAKI
(Figure IB). However, the specificity of EB165 for turkey myosin isoforms, particularly for the neonatal isoform, remains undefined at present. Developmental Stage-Specific Myosin Isoforms in Fast-Twitch (Pectoralis) Muscle and Mixed Fiber Muscles in Turkeys As the monoclonal antibody AB8 was determined to be specific for the adult myosin isoform from Pectoralis muscle by ELISA and peptide mapping, it was used to detect the presence of the adult isoform in turkey skeletal muscles. In protein slot blotting, AB8 exhibited strong reactivity with myosin extracts collected from Pectoralis muscle of adult turkeys and 14-day-old turkeys, indicating the expression of die adult isoform in Pectoralis muscle at 14 days of age (Figure 4A). The i ntensity of immunostaining appeared to be less with the myosin extract from pectoralis muscle of 14-day-old turkeys when compared with immunostaining with the myosin extract from adult turkeys, but die extent of immunostaining observed was sufficient to warrant the conclusion mat the adult myosin isoform was expressed by 14 days of age in turkeys, in contrast to 20 days of age reported in chickens (Cerny and Bandman, 1987). In turkeys, sizable populations of Type I muscle fibers were noted in Gastrocnemius and Sartorius muscles, but Type IIB muscle fibers were also found in these muscle (Wiskus et al., 1976). Despite me presence of Type IIB muscle fibers in Gastrocnemius and Sartorius muscles, no immunological reaction witii AB8 was observed with die myosin extracts from mese muscles. In the present investigation, me specificity of AB8 was clearly demonstrated to be limited to the adult myosin isoform of Pectoralis
FIGURE 4. Immunoblotting of the myosin extract from skeletal muscles of the fast-twitch white fiber type (Type ITB; Pectoralis muscle) and the mixed fiber types (the thigh muscle group, Gastrocnemius, Sartorius, and Posterior latissimus dorsi muscles) in turkeys at various developmental stages (24-day embryo, at hatch, 7-day posthatch, 14-day posthatch, and adult). The myosin extract was blotted onto membranes by protein slot blotting and myosin isoforms were detected with monoclonal antibodies, anti-mouse IgG conjugated wim peroxidase, and 3,3'-diaminobenzidine tetrahydrochloride as described. Immunoblots detected with monoclonal antibodies AB8 (A), 2E9 (B), AG6 (C), and EB165 (D). Each immunoblot contained slots in the following matrix. Column a: Rows 1 and 2, breast muscle from 24-day embryos; Rows 3 and 4, the thigh muscle group from 24-day embryos; Rows 5 and 6, breast muscle from day-old poults; Rows 7 and 8, the thigh muscle group from day-old poults. The amounts of protein blotted are .5 ug per slot (Rows 1, 3, 5, and 7) and .25 fig per slot (Rows 2, 4, 6, and 8). Column b: Rows 1 and 2, breast muscle from 7-day-old poults; Rows 3 and 4, the thigh muscle group from 7-day-old poults; Rows 5 and 6, breast muscle from 14-day-poults; Rows 7 and 8, the thigh muscle group from 14-day-old poults. The amounts of protein blotted are J5 ug per slot (Rows 1, 3,5, and 7) and .25 ug per slot (Rows 2,4,6, and 8). Column c: Rows 1 and 2, Gastrocnemius muscle from adult turkeys; Rows 3 and 4, Sartorius muscle from adult turkeys; Rows 5 and 6, Posterior latissimus dorsi muscle from adult turkeys: Rows 7 and 8, Pectoralis musclefromadult turkeys. The amounts of protein blotted are .5 ug per slot (Rows 1,3,5, and 7) and .25 ug per slot (Rows 2, 4, 6, and 8).
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myosin isoform, the peptide maps of Pectoralis muscle were simultaneously prepared from 44-wk-old turkeys and 18-mo-old chickens (Figure 3, Lanes 3 and 4). Nearly identical cleavage fragments in 44-wk-old turkeys and 18-mo-old chickens indicated that the adult isoforms of the myosin heavy chain in both species were closely related. The monoclonal antibody AG6 was shown in ELISA to react with myosin prepared from Pectoralis muscles of 24-day embryos, 7-day-old poults and 44-wk-old turkeys (Figure 1C). The absorbance at 405 nm measured in ELISA was the highest with die adult myosin extract, followed by the embryonic myosin extract, and the least with the neonatal myosin extract. It is concluded that AG6, as in chickens, reacted witii all diree developmental stagespecific isoforms of myosin in Pectoralis muscle of turkeys. The monoclonal antibody EB165 exhibited reactivity with the myosin extract from all three developmental stages (Figure ID). In ELISA, the absorbance observed with the myosin extract from 7-day-old poults was substantially less than those observed with adult and embryonic myosin extracts. The specificity of this monoclonal antibody was reported to be for embryonic and adult myosin heavy chain of fast-twitch muscle, with a notable absence of reactivity with the myosin extract from 8- and 20-day-old chickens (Cerny and Bandman, 1987). The reactivity of EB165 observed with the myosin extract from 7-day-old poults could be due to a small population of the embryonic isoform expressed concurrently with the neonatal isoform and not due to the cross-reactivity of EB165 with the neonatal isoform in turkeys. This conclusion was partially supported by the observations with 2E9 (Figure 1A) and AB8
MYOSIN ISOPORM BXPRESSION
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MARUYAMA AND KANEMAKI
Sequential Expression of Myosin Isoforms and Genetic Programming for Muscle Development In the present investigation, three isoforms of myosin, nearly identical to the developmental stage-specific isoforms reported in chickens, were detected. Li mammals, the expression of
myosin heavy chain isoforms proceeds in an orderly manner during skeletal muscle development (Whalen et al., 1981). In chickens, a similar developmental programming for the fast-fiber myosin heavy chain isoform was reported. In Pectoralis muscle, the embryonic isoform was replaced with neonatal isoform sometime after hatch and eventually replaced with the adult isoform. In Sartorius muscle (mixed-fiber muscle), the expression of the embryonic isoform persisted throughout development, with the exception that the neonatal isoform was expressed briefly during the neonatal period (Crow and Stockdale, 1986). The difference in timing of the transition from neonatal isoform expression to adult isoform expression observed in muscle fiber types is particularly important. In rats, it was reported that all muscles with varying muscle fiber populations displayed the sequential transitions from embryonic to neonatal and then to adult isoforms, but at varying speeds. The difference between muscles was found in the timing of the transition from the neonatal isoform expression to the adult isoform expression, rather than in the transition from the embryonic isoform expression to the neonatal isoform expression (d'Albis et al., 1989b). These observations suggest that each species, each muscle type, or both are subject to a specific developmental program for protein synthesis. It was also reported that when the fast muscle, Gastrocnemius muscle, of adult rats was injured, embryonic and neonatal isoforms were the myosin isoforms expressed first and then they were gradually replaced with the adult isoform just as observed in a normal developmental process of skeletal muscle (d'Albis et al., 1989a). The expression of these isoforms has been reported to be influenced by the hormonal environment (Gambke et al., 1983; ButlerBrowne et al., 1984) and by work-induced hypertrophy (Periasamy et al., 1989). Hence, it is plausible to explain the continuous and marked fall in the rate of muscle protein synthesis during embryonic development and the neonatal period in chickens (Maruyama et al., 1978; Muramatsu and Okumura, 1985; Muramatsu et al, 1987) as the result of the transition from the expression of one isoform to the expression of the other isoform until this transition process reaches the expression of the adult isoform or the last isoform gene in the developmental sequence.
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muscle in turkeys. These observations are comparable to the observation of AB8 being unreactive with chicken Gastrocnemius muscle (Bandman and Bennett, 1988). The neonatal myosin heavy chain isoform specific monoclonal antibody 2E9 was found to react with not only the myosin extract from Pectoralis muscle from 7-day-old turkeys, but also the myosin extract from thigh muscles of 7-day-old turkeys and 14-day-old turkeys. The 2E9 showed no reactivity with either Pectoralis muscle or the thigh muscles of 24-day embryos, day-old poults, or adults (Figure 4B). Based on the observations in the present study, it is concluded that the neonatal myosin isoform similar to chicken neonatal isoform is expressed in Pectoralis muscle and muscles of the mixed fiber type during the period of 7 to 14 days posthatch in turkeys. In chickens, 2E9 has been shown to react with not only Pectoralis muscle, but also Biceps brachii, Gastrocnemius, and Posterior latissimus dorsi muscles during the period of 17 through 31 days posthatch (Bandman and Bennett, 1988). The monoclonal antibody AG6 showed reactivity with the myosin extract prepared from all muscles studied (Figure 4C). The monoclonal antibody, EB165 was also found to react with all muscles studied (Figure 4D). The specificity of EB165 for the neonatal myosin heavy chain isoform remained uncertain in turkeys, due to the complication arising from the concurrent expression of neonatal and adult isoforms around 14 days posthatch. In the absence of a monoclonal antibody specific for a putative embryonic myosin isoform in turkeys, it appears likely that an embryonic isoform is expressed at 7 days posthatch along with the neonatal isoform. Because AG6 and EB165 were reactive with the myosin extract prepared from muscles of embryos and hatchlings, these can be used in combination with AB8 and 2E9 to indicate the population of mixed embryonic and adult isoforms in turkey skeletal muscles. However, no direct detection meuiod for embryonic myosin isoform was used in the present study.
MYOSIN ISOFORM EXPRESSION ACKNOWLEDGMENTS
REFERENCES Aberle, E. D.t P. B. Addis, and R. N. Shoffner, 1979. Fiber types in skeletal muscles of broiler- and layer-type chickens. Poultry Sci. 58:1210-1212. Aberle, E. D., and T. S. Stewart, 1983. Growth of fiber types and apparent fiber number in skeletal muscle of broiler- and layer-type chickens. Growth 47: 135-144. Bandman, E., 1985. Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle. Science 227:780-782. Bandman, E., and T. Bennett, 1988. Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscle in normal and dystrophic chickens. Dev. Biol. 130:220-231. Bandman, E., R. Matsuda, and R. C. Strohman, 1982. Developmental appearance of myosin heavy chain and light chain isoforms in vivo and in vitro in chicken skeletal muscle. Dev. Biol. 93:508-518. Baril, E. F., D. S. Love, and H. Herrmann, 1966. Investigation of myosin heterogeneity observed during chromatography on diethylaminoethyl cellulose. J. Biol. Chem. 241:822-830. Butler-Browne, G. S., D. Herlicoviez, and R. G. Whalen, 1984. Effects of hypothyroidism on myosin isozyme transitions in developing rat muscle. Fed. Eur. Biochem. Soc. Lett. 166:71-75. Cerny, L. C , and E. Bandman, 1987. Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle. Dev. Biol. 119:350-362. Cleveland, D. W., S. G. Fisher, M. W. Kirschner, and U. K. Laemmli, 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. Crow, M. T., and F. E. Stockdale, 1986. The developmental program of fast myosin heavy chain expression in avian skeletal muscles. Dev. Biol. 118:333-342. d'Albis, A., R. Couteaux, C. Janmot, and J. C. Mira, 1989a. Myosin isoform transitions in regression of fast and slow muscles during postnatal development of the rat. Dev. Biol. 135:320-325. d'Albis, A., R. Couteaux, C. Janmot, and A. Roulet, 1989b. Specific programs of myosin expression in the postnatal development of rat muscles. Eur. J. Biochem. 183:583-590. d'Albis, A., C. Pantaloni, and J.-J. Bechet, 1979. An electrophoreric study of native myosin isozymes and
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The authors wish to express appreciation to Beverly Russell and Duane Bartley for technical assistance; to Everett Bandman, Department of Food Science and Technology, University of California, Davis, CA 95616, for providing monoclonal antibodies; and to Robert Rosebrough, USDA, ARS, Nonruminant Nutrition Laboratory, Beltsville, MD 20705, and Mary Ann Ottinger, Department of Poultry Science, University of Maryland, College Park, MD 20742, for providing chickens for this study.
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