Developmental and diaphragm
changes in hindlimb of sheep
DAVID I. FINKELSTEIN, PETER ANDRIANAKIS, ANTHONY R. LUFF, AND DAVID W. WALKER Department of Physiology, Monash University, Clayton, Finkelstein, David I., Peter Andrianakis, Anthony R. Luff, and David W. Walker. Developmental changes in hindlimb muscles and diaphragm of sheep. Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): R900-R908, 1992. -In this study, plasmathyroxine, contractile and histochemical (adenosinetriphosphataseand NADH) characteristics of soleus(SOL), medial gastrocnemius(MG), and extensor digitorum longus (EDL) were examined in 140-day-gestationfetal sheepand in 2-, 5, and 30-day-old lambsand adult ewes.Electrophoretic separationof myosin heavy chainswasalsodone on all musclesand the diaphragm. There were no differencesin the twitch contraction and relaxation times of MG and EDL at the different ages;in contrast SOL contraction times were significantly shorter in the fetus and newborn than in the adult. Fast glycolytic fibers first appeared in EDL, MG, and diaphragm at 5, 30, and 5 days after birth, respectively. The proportion of slow oxidative fibers decreasedafter birth and with postnatal development in EDL, whereasthey increasedin MG and diaphragm. Plasma thyroxine concentrations were higher in the fetus and day-old lambs than in 2-, 5, and 30-day-old lambsor adult sheep.It is suggestedthat contractile specialization of the fast-twitch diaphragm, MG, and EDL is largely achievedin utero and is probably mediatedby thyroid hormone. In contrast, SOL changedpostnatally, probably influenced by the altered neural drive. muscle contraction; muscle differentiation; myosin; development; fetus MAMMALS SUCH AS HUMANS, cats,andratsareunableto walk for some time after birth and are termed altricial. It is well established that the limb muscles from these species are functionally and structurally not fully differentiated at birth and, over time, differentiate into either the typical fast- or slow-twitch muscle types. For exampie, the twitch cNontraction time of the fast-twitch muscles from altricial animals decreases to adult values by 3 wk in the rat and by 5 wk in the cat. In contrast, there is little change in the twitch contraction time of the slow-twitch soleus muscle after birth (3, 13, 29). The emergence of muscle fibers exhibiting different characteristics and expressing different myosin isozymes during early development is myogenically determined (17). In altricial species such as the cat and rat the different neuronal firing patterns associated with adult fast- and slow-twitch muscle start to appear after the first postnatal week (29). After this point these patterns are strong determining influences on muscle characteristics (3, 25). In contrast to altricial species, sheep are relatively mature at birth and are able to run and stand. This sugges ts that considerable development of skeletal muscles w ith different functional characteristics has occurred before birth. However, little is known of the in utero development of fast and slow muscles in a precocial mammalian species such as the sheep. An earlier study found that at 1east two fibe r types could be identified halfway through gestatio n in the selmiten diR900
0363-6119/92
$2.00
Copyright
muscles
Victoria
3168, Australia
nosus muscle, a mixed fast-twitch muscle of the hindlimb ( 1). Because these limb muscles pro bably do not bear load in utero, it was concluded that the quantity of muscle contractions was not the major determining factor for the differentiation of muscles 0 f this species (I) . Some other nonmammalian vertebrates are also able to move soon after birth. For example, the development of fiber types in the chicken embryo has been found to be influenced by both neural and myogenic factors. Unlike in mammals, the neurogenic influence is exerted primarily in the first week of embryonic life (8, 16). In all species th .e diaph ragm has to be fully functional from birth and as such is of intere st when investigating neural and hormonal control of muscle development. In the rat diaphragm the transition from embryonic to fetal myosins precedes their appearance in limb muscles (5, 20). Despite the neural activity in utero, substantial development of the diaphragm still occurs postpartum (5, 20). This indicates that neural activity in utero does not account for all the developmental changes that occur in the diaphragm. In sheep the diaphragm is more or less in constant use before birth (2), but little is known of the development of fiber types in the diaphragm of this species. Thyroid hormones provide an important influence on myosin formation and myosin transition with different responses occurring in the various muscle fiber types (5, 19). Scow (26, 27) first showed the importance of thyroid and growth hormones for normal-muscle development, these acting synergistically to increase muscle mass and the myosin content of muscle. Thyroid hormone has subsequently been shown to alter the isometric contractile properties of adult muscle (10, 11). In the dev ,eloping animal thyroid hormone can act directly on the fast-tw *itch mu scle cell, triggering the expression of the adult fast-type myosin even in the denervated muscle (9). Therefore, the expression of the fast myosin is not influenced by innervation, whereas the expression of adult slow myosin only occurs while innervated. In the rat the normal increase in thyroid hormone levels during postnatal development (7) is coincident with shortening of the twitch contraction time of fast-twitch muscle (4). The aim of this study was to investigate the development of skeletal muscle in a precocial mammal. Fetal sheep develop an autonomously regulated hypothalamopituitary-thyroid axis by -75 days of gestation (term is - 147 days; Ref. 7). However, the developmental changes that occur in fast and slow muscle fibers have not been described fully in this species, nor is it known if the hypothalamus-pituitary-thyroid axis has a role in the differentiation of muscle fibers. The sheep offers an ideal experimental model in which to investigate the influences of thyroid hormone on muscle fiber
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
SKELETAL
MUSCLE
DEVELOPMENT
development in utero and the postnatal increase in neural drive that accompanies postnatal exercise. In this study, contractile and histochemical characteristics of the soleus (SOL), medial gastrocnemius (MG) and extensor digitorum longus (EDL) muscles were examined in 140-day-gestation fetal sheep and in 2-, 5-, and 30day-old lambs and adult ewes. Plasma thyroxine was measured in each age group. Fiber typing and electophoretic separation of heavy myosin chains were performed on each of these muscles as well as the diaphragm. It was found that contractile specialization of the mixed fast-twitch muscles such as the diaphragm, MC, and EDL is largely complete at birth, even though the proportions of the different fiber types and the myosin isozymes continue to change postnatally. This in utero maturation of the fast-twitch muscles is probably caused by the high level of thyroid hormone found in this species. In contrast, soleus muscle exhibited significant changes of twitch contraction time and myosin isozymes postnatally, probably influenced by the increase in neural drive. METHODS
Animals, surgery, and experimental protocol. The ewesand lambswere anesthetized with pentobarbitone sodium (35 mg/ kg- iv) and maintained by intermittent infusion of 3-5 mg/kg sothat only a weak cornea1reflex could be elicited. Five different agegroupswere investigated: 140-day fetus (n = 7; term is 147-150 days), lambs aged2-3 days (n = 3), &day lambs (n = 5), 30-day lambs (n = 3), and adult ewes(n = 5). When the fetus was studied, each ewewas anesthetized, intubated, and placed in a lateral recumbent position, and the abdomenwasopenedin the midline. The fetus was then removed from the uterus through a 6- to 8cm incision, taking care not to stretch the umbilical cord, and wrapped in a heating blanket controlled by a rectal thermistor to maintain the body temperature at 38 39°C. A catheter wasinserted into one carotid artery of the fetus from which blood sampleswere drawn intermittently for measurementof blood PO,? Pco~, and pH usingan ABL3 Blood Gas Analyzer (Radiometer, Copenhagen). The method of preparing the musclesof the hindlimb for study was essentiallythe samefor the fetuses,lambs,and ewes. One of the hindlimbs wasextended and the SOL, EDL, and MG muscleswere dissectedfree from surrounding tissues,taking care to maintain the nerve and blood supply. All other muscle nerves in the hindlimb were sectioned. The hindlimb was secured to a rigid frame by inserting pins into either end of the tibia. The muscleswere covered with warm mineral oil and agar-soakedswabscontained by skin flaps and maintained at 37 t 0.5”C by radiant heating lamps. The distal tendon of insertion of each musclewas cut and tied to a purpose-madeforce transducer that had a sensitivity of ~0.78 g/mV. The nerve to each musclewas stimulated with pulsesof 0.05 ms duration at supramaximal voltage. Each muscle was set at the optimum length for the isometric twitch. The maximum twitch force, time to peak force of the twitch contraction (t,), and time to half relaxation (tl& were determined from the records obtained at this length. Then an isometric tetanic force-length curve was constructed using a 400-msstimulus train at a stimulation frequency of 100 Hz. The musclewas set to optimum length for tetanic force (L,) and the force-frequency relationship determined for frequencies between 10 and 500 Hz to obtain the maximum tetanic force. In the fetusesand lambs the fatigue index of the muscle was calculated as the ratio of the force generatedon the 1st and 120th tetanus, with eachtetanus producedby stimulating the muscleat 40 Hz for 330ms once every
IN
SHEEP
R901
second. The fatigue index of the adult soleuswas calculated from the ratio of forces produced by the 1st to the 121ststimulus train, with the 121st train being delivered 2-3 s after the last stimulus train to allow the muscletime to relax to baseline. This procedure was necessarybecauseof the long relaxation halftime of this muscle (RESULTS). Single muscletwitches and tetanuses were stored in a 8088-basedmicrocomputer using purpose-written software (Capricorn Scientific Software). Twitches were digitized at 2 kHz and tetanusesat 0.5 kHz. No recordswere obtained from the adult MG musclebecause the developedforce was in excessof the capacity of the strain gauge.For the adult EDL, maximum isometric force was estimated by constructing a twitch force-length curve, setting the muscleat optimum length, and then paring the nerve so that only a fraction of the nerve was stimulated. The maximum tetanic force causedby this fractional stimulation of the nerve was obtained and the tetanus-twitch ratio of the fraction calculated. Whole muscletetanic force wasthen estimatedby multiplying the whole muscle twitch force by the tetanus-twitch ratio obtained from the fractional stimulation of the nerve. It haspreviously been found that if a fraction exceeds15% of the whole muscleforce, then that fraction accurately reflects whole musclecharacteristics (14). Histochemistry. When the contractile recording was completed, each musclewas removed from each leg, set at approximately the resting in vivo length usingcalipers,and after being coated with talcum powder wasplunged in isopentanecooledin liquid Nz. The muscleswere then wrappedin aluminum foil and stored at -70°C u til processing.A costal strip of the diaphragm was also obt “a ined. The musclesfrom each age group were treated as follows. Serial sections (IO-12 pm) were cut on a freezing microtome and processedusing the myosin adenosinetriphosphatase(ATPase) reaction at pH 4.3 (23) and the NADH-tetrazolium reductasemethod (22). With the useof the ATPase and NADH reactions, fibers were identified as slow oxidative (SO), fast oxidative glycolytic (FOG), or fast glycolytic (FG) using the classification schemeand nomenclature of Peter et al. (24). This method was chosenbecauseof the difficulties of typing fibers in developing muscle (12). Type SO fibers were identified by their intense reaction to ATPase (at pH 4.3) and to NADH in the serial sections.The FOG fibers produced a dark reaction product to NADH and light for the ATPase (pH 4.3). In contrast the FG fibers produced light reaction products to both histochemical reactions. Figure 4 illustrates how fibers were classified. Area of musclefibers was measuredby projecting representative fields onto a Zeiss-MOP graphicstablet via a front-silvered mirror using a total magnification of x330. A digitizing pen and computer were then used to determine the areawithin the perimeterstraced around each fiber type. These data were stored on computer disk for later analysis. Electrophoresis. A portion of each musclewas usedto characterize the heavy chain myosinspresent in the sample.A piece of each muscle weighing 0.5-l g was cooled in liquid N2 and then powdered with a chilled mortar and pestle. The methods usedwere basedon the work of Hoh et al. (18). To extract the myosins,the powderedmusclewas homogenizedin 10 volumes of a solution of 100mM Na4P207,5 mM ethylene glycol-bis(Paminoethyl ether) -N,N,N’,N’-tetraacetic acid (EGTA), 2 mM 2mercaptoethanol (pH 8.8) and centrifuged at 48,000g at 4°C for 4 h in a Beckman L5-65 ultracentrifuge. The supernatant wasmixed with an equalvolume of glycerol and stored at -20°C until analysis.A volume containing 8-9 pg of protein wasloaded onto 0.5-cm-diameter rods (total monomer concentration 4%) containing 3% N,N’-methylene-bis-acrylamide). A voltage gradient of 14 V/cm was applied to the gelsfor 23 h at 1-4°C in a solution of 20 mM Na4P207, 10% glycerol, and 0.01% (vol/vol) 2mercaptoethanol at pH 8.8. The gelswerethen stainedfor 3 h
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
R902
SKELETAL
MUSCLE
DEVELOPMENT
at 37°C in CoomassieBlue (0.05%wt/vol in 45% methanol, 10% glacial acetic acid, 45% distilled water) and destained in 10% glacial acetic acid. The gels were scanned with a laser densitometer(LKB 2202)to determine the position and relative densitiesof each band in the gel. The slowly migrating myosin has beenfound to correlate with the type I or SO musclefibers (seeRef. 18). Radioimmunoassay for thyroxine. Blood sampleswere taken from the fetuses, lambs, and ewesto determine plasma total thyroxine (TJ concentrations by radioimmunoassayusing an Amerlex-M T4 RIA kit (Amersham, IM3014). A blood sample wasalsoobtained from sevenof the lambson the first postnatal day. The cross-reactivity of the antiserum usedwas 12% with 3,5,3’-triiodothyronine and
changes in hindlimb of sheep
DAVID I. FINKELSTEIN, PETER ANDRIANAKIS, ANTHONY R. LUFF, AND DAVID W. WALKER Department of Physiology, Monash University, Clayton, Finkelstein, David I., Peter Andrianakis, Anthony R. Luff, and David W. Walker. Developmental changes in hindlimb muscles and diaphragm of sheep. Am. J. Physiol. 263 (Regulatory Integrative Cornp. Physiol. 32): R900-R908, 1992. -In this study, plasmathyroxine, contractile and histochemical (adenosinetriphosphataseand NADH) characteristics of soleus(SOL), medial gastrocnemius(MG), and extensor digitorum longus (EDL) were examined in 140-day-gestationfetal sheepand in 2-, 5, and 30-day-old lambsand adult ewes.Electrophoretic separationof myosin heavy chainswasalsodone on all musclesand the diaphragm. There were no differencesin the twitch contraction and relaxation times of MG and EDL at the different ages;in contrast SOL contraction times were significantly shorter in the fetus and newborn than in the adult. Fast glycolytic fibers first appeared in EDL, MG, and diaphragm at 5, 30, and 5 days after birth, respectively. The proportion of slow oxidative fibers decreasedafter birth and with postnatal development in EDL, whereasthey increasedin MG and diaphragm. Plasma thyroxine concentrations were higher in the fetus and day-old lambs than in 2-, 5, and 30-day-old lambsor adult sheep.It is suggestedthat contractile specialization of the fast-twitch diaphragm, MG, and EDL is largely achievedin utero and is probably mediatedby thyroid hormone. In contrast, SOL changedpostnatally, probably influenced by the altered neural drive. muscle contraction; muscle differentiation; myosin; development; fetus MAMMALS SUCH AS HUMANS, cats,andratsareunableto walk for some time after birth and are termed altricial. It is well established that the limb muscles from these species are functionally and structurally not fully differentiated at birth and, over time, differentiate into either the typical fast- or slow-twitch muscle types. For exampie, the twitch cNontraction time of the fast-twitch muscles from altricial animals decreases to adult values by 3 wk in the rat and by 5 wk in the cat. In contrast, there is little change in the twitch contraction time of the slow-twitch soleus muscle after birth (3, 13, 29). The emergence of muscle fibers exhibiting different characteristics and expressing different myosin isozymes during early development is myogenically determined (17). In altricial species such as the cat and rat the different neuronal firing patterns associated with adult fast- and slow-twitch muscle start to appear after the first postnatal week (29). After this point these patterns are strong determining influences on muscle characteristics (3, 25). In contrast to altricial species, sheep are relatively mature at birth and are able to run and stand. This sugges ts that considerable development of skeletal muscles w ith different functional characteristics has occurred before birth. However, little is known of the in utero development of fast and slow muscles in a precocial mammalian species such as the sheep. An earlier study found that at 1east two fibe r types could be identified halfway through gestatio n in the selmiten diR900
0363-6119/92
$2.00
Copyright
muscles
Victoria
3168, Australia
nosus muscle, a mixed fast-twitch muscle of the hindlimb ( 1). Because these limb muscles pro bably do not bear load in utero, it was concluded that the quantity of muscle contractions was not the major determining factor for the differentiation of muscles 0 f this species (I) . Some other nonmammalian vertebrates are also able to move soon after birth. For example, the development of fiber types in the chicken embryo has been found to be influenced by both neural and myogenic factors. Unlike in mammals, the neurogenic influence is exerted primarily in the first week of embryonic life (8, 16). In all species th .e diaph ragm has to be fully functional from birth and as such is of intere st when investigating neural and hormonal control of muscle development. In the rat diaphragm the transition from embryonic to fetal myosins precedes their appearance in limb muscles (5, 20). Despite the neural activity in utero, substantial development of the diaphragm still occurs postpartum (5, 20). This indicates that neural activity in utero does not account for all the developmental changes that occur in the diaphragm. In sheep the diaphragm is more or less in constant use before birth (2), but little is known of the development of fiber types in the diaphragm of this species. Thyroid hormones provide an important influence on myosin formation and myosin transition with different responses occurring in the various muscle fiber types (5, 19). Scow (26, 27) first showed the importance of thyroid and growth hormones for normal-muscle development, these acting synergistically to increase muscle mass and the myosin content of muscle. Thyroid hormone has subsequently been shown to alter the isometric contractile properties of adult muscle (10, 11). In the dev ,eloping animal thyroid hormone can act directly on the fast-tw *itch mu scle cell, triggering the expression of the adult fast-type myosin even in the denervated muscle (9). Therefore, the expression of the fast myosin is not influenced by innervation, whereas the expression of adult slow myosin only occurs while innervated. In the rat the normal increase in thyroid hormone levels during postnatal development (7) is coincident with shortening of the twitch contraction time of fast-twitch muscle (4). The aim of this study was to investigate the development of skeletal muscle in a precocial mammal. Fetal sheep develop an autonomously regulated hypothalamopituitary-thyroid axis by -75 days of gestation (term is - 147 days; Ref. 7). However, the developmental changes that occur in fast and slow muscle fibers have not been described fully in this species, nor is it known if the hypothalamus-pituitary-thyroid axis has a role in the differentiation of muscle fibers. The sheep offers an ideal experimental model in which to investigate the influences of thyroid hormone on muscle fiber
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
SKELETAL
MUSCLE
DEVELOPMENT
development in utero and the postnatal increase in neural drive that accompanies postnatal exercise. In this study, contractile and histochemical characteristics of the soleus (SOL), medial gastrocnemius (MG) and extensor digitorum longus (EDL) muscles were examined in 140-day-gestation fetal sheep and in 2-, 5-, and 30day-old lambs and adult ewes. Plasma thyroxine was measured in each age group. Fiber typing and electophoretic separation of heavy myosin chains were performed on each of these muscles as well as the diaphragm. It was found that contractile specialization of the mixed fast-twitch muscles such as the diaphragm, MC, and EDL is largely complete at birth, even though the proportions of the different fiber types and the myosin isozymes continue to change postnatally. This in utero maturation of the fast-twitch muscles is probably caused by the high level of thyroid hormone found in this species. In contrast, soleus muscle exhibited significant changes of twitch contraction time and myosin isozymes postnatally, probably influenced by the increase in neural drive. METHODS
Animals, surgery, and experimental protocol. The ewesand lambswere anesthetized with pentobarbitone sodium (35 mg/ kg- iv) and maintained by intermittent infusion of 3-5 mg/kg sothat only a weak cornea1reflex could be elicited. Five different agegroupswere investigated: 140-day fetus (n = 7; term is 147-150 days), lambs aged2-3 days (n = 3), &day lambs (n = 5), 30-day lambs (n = 3), and adult ewes(n = 5). When the fetus was studied, each ewewas anesthetized, intubated, and placed in a lateral recumbent position, and the abdomenwasopenedin the midline. The fetus was then removed from the uterus through a 6- to 8cm incision, taking care not to stretch the umbilical cord, and wrapped in a heating blanket controlled by a rectal thermistor to maintain the body temperature at 38 39°C. A catheter wasinserted into one carotid artery of the fetus from which blood sampleswere drawn intermittently for measurementof blood PO,? Pco~, and pH usingan ABL3 Blood Gas Analyzer (Radiometer, Copenhagen). The method of preparing the musclesof the hindlimb for study was essentiallythe samefor the fetuses,lambs,and ewes. One of the hindlimbs wasextended and the SOL, EDL, and MG muscleswere dissectedfree from surrounding tissues,taking care to maintain the nerve and blood supply. All other muscle nerves in the hindlimb were sectioned. The hindlimb was secured to a rigid frame by inserting pins into either end of the tibia. The muscleswere covered with warm mineral oil and agar-soakedswabscontained by skin flaps and maintained at 37 t 0.5”C by radiant heating lamps. The distal tendon of insertion of each musclewas cut and tied to a purpose-madeforce transducer that had a sensitivity of ~0.78 g/mV. The nerve to each musclewas stimulated with pulsesof 0.05 ms duration at supramaximal voltage. Each muscle was set at the optimum length for the isometric twitch. The maximum twitch force, time to peak force of the twitch contraction (t,), and time to half relaxation (tl& were determined from the records obtained at this length. Then an isometric tetanic force-length curve was constructed using a 400-msstimulus train at a stimulation frequency of 100 Hz. The musclewas set to optimum length for tetanic force (L,) and the force-frequency relationship determined for frequencies between 10 and 500 Hz to obtain the maximum tetanic force. In the fetusesand lambs the fatigue index of the muscle was calculated as the ratio of the force generatedon the 1st and 120th tetanus, with eachtetanus producedby stimulating the muscleat 40 Hz for 330ms once every
IN
SHEEP
R901
second. The fatigue index of the adult soleuswas calculated from the ratio of forces produced by the 1st to the 121ststimulus train, with the 121st train being delivered 2-3 s after the last stimulus train to allow the muscletime to relax to baseline. This procedure was necessarybecauseof the long relaxation halftime of this muscle (RESULTS). Single muscletwitches and tetanuses were stored in a 8088-basedmicrocomputer using purpose-written software (Capricorn Scientific Software). Twitches were digitized at 2 kHz and tetanusesat 0.5 kHz. No recordswere obtained from the adult MG musclebecause the developedforce was in excessof the capacity of the strain gauge.For the adult EDL, maximum isometric force was estimated by constructing a twitch force-length curve, setting the muscleat optimum length, and then paring the nerve so that only a fraction of the nerve was stimulated. The maximum tetanic force causedby this fractional stimulation of the nerve was obtained and the tetanus-twitch ratio of the fraction calculated. Whole muscletetanic force wasthen estimatedby multiplying the whole muscle twitch force by the tetanus-twitch ratio obtained from the fractional stimulation of the nerve. It haspreviously been found that if a fraction exceeds15% of the whole muscleforce, then that fraction accurately reflects whole musclecharacteristics (14). Histochemistry. When the contractile recording was completed, each musclewas removed from each leg, set at approximately the resting in vivo length usingcalipers,and after being coated with talcum powder wasplunged in isopentanecooledin liquid Nz. The muscleswere then wrappedin aluminum foil and stored at -70°C u til processing.A costal strip of the diaphragm was also obt “a ined. The musclesfrom each age group were treated as follows. Serial sections (IO-12 pm) were cut on a freezing microtome and processedusing the myosin adenosinetriphosphatase(ATPase) reaction at pH 4.3 (23) and the NADH-tetrazolium reductasemethod (22). With the useof the ATPase and NADH reactions, fibers were identified as slow oxidative (SO), fast oxidative glycolytic (FOG), or fast glycolytic (FG) using the classification schemeand nomenclature of Peter et al. (24). This method was chosenbecauseof the difficulties of typing fibers in developing muscle (12). Type SO fibers were identified by their intense reaction to ATPase (at pH 4.3) and to NADH in the serial sections.The FOG fibers produced a dark reaction product to NADH and light for the ATPase (pH 4.3). In contrast the FG fibers produced light reaction products to both histochemical reactions. Figure 4 illustrates how fibers were classified. Area of musclefibers was measuredby projecting representative fields onto a Zeiss-MOP graphicstablet via a front-silvered mirror using a total magnification of x330. A digitizing pen and computer were then used to determine the areawithin the perimeterstraced around each fiber type. These data were stored on computer disk for later analysis. Electrophoresis. A portion of each musclewas usedto characterize the heavy chain myosinspresent in the sample.A piece of each muscle weighing 0.5-l g was cooled in liquid N2 and then powdered with a chilled mortar and pestle. The methods usedwere basedon the work of Hoh et al. (18). To extract the myosins,the powderedmusclewas homogenizedin 10 volumes of a solution of 100mM Na4P207,5 mM ethylene glycol-bis(Paminoethyl ether) -N,N,N’,N’-tetraacetic acid (EGTA), 2 mM 2mercaptoethanol (pH 8.8) and centrifuged at 48,000g at 4°C for 4 h in a Beckman L5-65 ultracentrifuge. The supernatant wasmixed with an equalvolume of glycerol and stored at -20°C until analysis.A volume containing 8-9 pg of protein wasloaded onto 0.5-cm-diameter rods (total monomer concentration 4%) containing 3% N,N’-methylene-bis-acrylamide). A voltage gradient of 14 V/cm was applied to the gelsfor 23 h at 1-4°C in a solution of 20 mM Na4P207, 10% glycerol, and 0.01% (vol/vol) 2mercaptoethanol at pH 8.8. The gelswerethen stainedfor 3 h
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
R902
SKELETAL
MUSCLE
DEVELOPMENT
at 37°C in CoomassieBlue (0.05%wt/vol in 45% methanol, 10% glacial acetic acid, 45% distilled water) and destained in 10% glacial acetic acid. The gels were scanned with a laser densitometer(LKB 2202)to determine the position and relative densitiesof each band in the gel. The slowly migrating myosin has beenfound to correlate with the type I or SO musclefibers (seeRef. 18). Radioimmunoassay for thyroxine. Blood sampleswere taken from the fetuses, lambs, and ewesto determine plasma total thyroxine (TJ concentrations by radioimmunoassayusing an Amerlex-M T4 RIA kit (Amersham, IM3014). A blood sample wasalsoobtained from sevenof the lambson the first postnatal day. The cross-reactivity of the antiserum usedwas 12% with 3,5,3’-triiodothyronine and
