Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training LUC E. GOSSELIN, MICHAEL AND D. PAUL THOMAS
BETLACH,
ARTHUR
C. VAILAS,
MARION
L. GREASER,
Biodynamics and Muscle Biology Laboratories, University of Wisconsin, Madison, Wisconsin 53706; and Human Energy Research Laboratory, University of Wyoming, Laramie, Wyoming 82071 GOSSELIN,LucE., MICHAELBETLACH,ARTHURC.VAILAS, MARION L. GREASER,AND D. PAUL THOMAS. Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training. J. Appl. Physiol. 73(4): 1282-1286, 1992.-Increases in aerobic capacity in both young and senescent rats consequent to endurance exercise training are now known to occur not only in locomotor skeletal muscle but also in diaphragm. In the current study the effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Exercise training [treadmill running at 75% maximal oxygen consumption (1 h/day, 5 day/wk, X 10 wk)] resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23 mo) trained animals (P < 0.05). Computerized densitometric image analysis of fast and slow MHC bands revealed the ratio of fast to slow MHC to be significantly higher (P < 0.005) in the crural compared with costal diaphragm region in both age groups. In addition, a significant age-related increase (P < 0.05) in percentage of slow MHC was observed in both diaphragm regions. However, exercise training failed to change the relative proportion of slow MHC in either the costal or crural region. aging; Fischer 344
of plasticity in response to changing physiological demand. These changes can occur not only in young animals but in senescent ones as well (12). Previous studies have reported alterations in the biochemical properties of the diaphragm in response to increased resistive loads (1, 15) and increased metabolic demand resulting from chronic locomotor exercise (12). Endurance exercise training has been associated with changes not only in aerobic enzyme capacity of limb skeletal muscle but also in myosin isoform expression (3, 25). Recent evidence indicates similar alterations can occur in the diaphragm muscle from young rats after endurance swim training (31). It is not known if such changes can occur in the diaphragm from senescent animals. Therefore, the purposes of this study were threefold: 1) to determine if differences in MHC composition exist between costal and crural regions of the rodent diaphragm, 2) to see if aging alters MHC composition in these same two regions of the diaphragm, and 3) to determine if chronic treadmill exercise causes alterations in the relative proportion of fast and slow MHC in both diaphragm regions from young and senescent rats. METHODS
THE MYOSIN HEAVY CHAIN (MHC)
composition of a muscle fiber has been shown to correlate both with its velocity of shortening (7, 24) and peak isometric force (7). In addition, the relative proportions of the various myosin heavy chains in a muscle appear to correlate with the relative area occupied by specific fiber types (17). Hence, regions within a muscle with distinct differences in fiber type composition are expected to exhibit different physiological characteristics as well as MHC composition. The diaphragm has two distinct regions, costal and crural. These two regions have different mechanical actions on the rib cage (6) as well as different biochemical properties (23) and fiber type profile (22). Type II fiber atrophy in senescent skeletal muscle has been observed (l7,18) and results in a reduced contribution by these fibers to the total cross-sectional area. In addition, the relative proportion of fast MHC isoform decreases with age and correlates with the concomitant decrease in relative area occupied by the respective fiber type (17). The extent to which aging influences MHC composition in the diaphragm has not been previously examined. The diaphragm, like other skeletal muscles, is capable 1282
0161-7567192
$2.00
Copyright
Animals. Young adult (2.5 mo) and old (20 mo) specific pathogen-free female Fischer 344 rats were obtained from the National Institute on Aging Colony (Indianapolis, IN). The rats were individually caged and maintained in the small animal laboratory at the Psychology Building, University of Wisconsin, according to University of Wisconsin Research Animal Resource Center guidelines. The animals were maintained on an alternating 12:12-h light-dark photoperiod. Purina Rat Chow and water were provided ad libitum. Training. All animals were familiarized with walking on a motor-driven treadmill (lo-15 m/min, 10 min/day) over a l-wk period. At the end of this period, the animals from each age group were weight matched and randomly assigned to either a sedentary control or exercise training group. Hence, the four groups consisted of 1) young control (YC), 2) young trained (YT), 3) old control (OC), and 4) old trained (OT) rats. The YT and OT rats began progressive treadmill running at increasing grade and speed for 1 h/day, 5 days/wk, for 10 wk. By the end of the training period, the young animals were running at 30 m/min, 15% grade, whereas the old animals were running at 15 m/min, 15% grade. These work loads are rela-
0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/jappl at Georgetown Univ Med Ctr (141.161.091.014) on August 24, 2019.
RAT
DIAPHRAGM
MUSCLE
MYOSIN
tively similar in intensity (-75% of maximal oxygen consumption) for the two age groups (21). Killing of the animals and tissue removal. At the end of the lo-wk training regimen, the animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The soleus (SOL), plantaris and extensor digitorum longus (EDL) muscles were removed and quickly frozen in isopentane precooled with liquid nitrogen. The diaphragm was then exposed, and portions of both the midcostal and crural area were removed and frozen. All tissue was stored in a -70°C freezer. Biochemical analysis. Citrate synthase activity was measured on plantaris muscle to document a peripheral training effect. The plantaris muscle was kept on ice while cleaned of connective tissue and minced with scissors. The minced tissue samples were mixed with homogenizing medium (1:19) and homogenized on ice using a glass pestle driven by a Wheaton stirring motor. The homogenizing medium consisted of (in mM) 50 potassium phosphate (pH 7.4), 1 EDTA, 2 MgCl,, 2 ADP, and 0.5 dithiothreitol. Citrate synthase activity was measured spectrophotometrically at a wavelength of 412 nm,
31°C (30). Gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate MHC isoforms from homogenized whole muscle. Gel and sample preparation were based on a modification of the procedure by Fritz et al. (11) and Carraro and Catani (5). Briefly, -10-20 mg of frozen whole muscle were pulverized in liquid nitrogen and dissolved (1:600) in sample buffer A [8 M urea, 2 M thiourea, 0.05 M tris(hydroxymethyl)aminomethane (pH 6.8), 75 mM dithiothreitol, 3.0% SDS, and 0.05% bromophenol blue]. The mixture was homogenized using a glass pestle driven by a Wheaton stirring motor. The sample was heated to 100°C for 4 min and then allowed to cool to room temperature. After further homogenization, the samples were stored at -7OOC and thawed just before the electrophoresis procedure. A 3.5% acrylamide concentration (pH 6.8) was used in the stacking gel, whereas the resolving gel (8 X 10 cm in size, 0.75 mm thick, Hoefer SE250) consisted of 8% acrylamide concentration (pH 8.8) with 25% (vol/vol) glycerol. The samples were run at constant current (20 mA/gel) until the tracking dye reached the bottom of the gel (- 1.75 h). To optimize resolution of the MHC bands, the sample loads were kept small at -250 ng myosin/ lane. After completion of the gel run, the gels were removed from the plates and silver-stained according to a modification of the procedure of Tunon and Johansson (32). Following staining, the gels were placed on a light box (Fotodyne) and imaged on a computerized image processing system. Briefly, this system consisted of Kohu camera and television zoom lens (18-108 mm, F-stop 2.5) connected to a Sony monitor. This monitor was interfaced with a Magnavox computer for both the processing and storage of images (JAVA software). Obtained images were digitized into an array of pixel elements that had a total of 256 possible gray levels (GL). Identification of the fast and slow isoforms was accomplished by comigration of SOL and EDL muscle samples. These two muscle samples consist of primarily slow and fast MHC isoforms, respectively. Relative contribu-
HEAVY
CHAIN
1283
COMPOSITION
YC
YT
oc
OT
1. Effect of training on plantaris muscle citrate synthase (CS) activity. Values are means t SE expressed per gram wet weight. Groups are young control (YC), young trained (YT), old control (OC), and old trained (OT). * Trained different from control, P < 0.05. t Young different from old, P < 0.05. FIG.
tion of a specific band (fast vs. slow isoform) was determined by obtaining the total GL of the band (mean band GL X band area) and expressed as a percentage of the total obtained from the two isoforms combined. To correct for background staining, the background GL was subtracted out from the mean band GL. Densitometric analysis of the MHC bands revealed that the staining intensity was within the linear range (R2 = 0.99) for the loading volumes used. The coefficient of variation from repeated measurement of a single band was
BETLACH,
ARTHUR
C. VAILAS,
MARION
L. GREASER,
Biodynamics and Muscle Biology Laboratories, University of Wisconsin, Madison, Wisconsin 53706; and Human Energy Research Laboratory, University of Wyoming, Laramie, Wyoming 82071 GOSSELIN,LucE., MICHAELBETLACH,ARTHURC.VAILAS, MARION L. GREASER,AND D. PAUL THOMAS. Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training. J. Appl. Physiol. 73(4): 1282-1286, 1992.-Increases in aerobic capacity in both young and senescent rats consequent to endurance exercise training are now known to occur not only in locomotor skeletal muscle but also in diaphragm. In the current study the effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Exercise training [treadmill running at 75% maximal oxygen consumption (1 h/day, 5 day/wk, X 10 wk)] resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23 mo) trained animals (P < 0.05). Computerized densitometric image analysis of fast and slow MHC bands revealed the ratio of fast to slow MHC to be significantly higher (P < 0.005) in the crural compared with costal diaphragm region in both age groups. In addition, a significant age-related increase (P < 0.05) in percentage of slow MHC was observed in both diaphragm regions. However, exercise training failed to change the relative proportion of slow MHC in either the costal or crural region. aging; Fischer 344
of plasticity in response to changing physiological demand. These changes can occur not only in young animals but in senescent ones as well (12). Previous studies have reported alterations in the biochemical properties of the diaphragm in response to increased resistive loads (1, 15) and increased metabolic demand resulting from chronic locomotor exercise (12). Endurance exercise training has been associated with changes not only in aerobic enzyme capacity of limb skeletal muscle but also in myosin isoform expression (3, 25). Recent evidence indicates similar alterations can occur in the diaphragm muscle from young rats after endurance swim training (31). It is not known if such changes can occur in the diaphragm from senescent animals. Therefore, the purposes of this study were threefold: 1) to determine if differences in MHC composition exist between costal and crural regions of the rodent diaphragm, 2) to see if aging alters MHC composition in these same two regions of the diaphragm, and 3) to determine if chronic treadmill exercise causes alterations in the relative proportion of fast and slow MHC in both diaphragm regions from young and senescent rats. METHODS
THE MYOSIN HEAVY CHAIN (MHC)
composition of a muscle fiber has been shown to correlate both with its velocity of shortening (7, 24) and peak isometric force (7). In addition, the relative proportions of the various myosin heavy chains in a muscle appear to correlate with the relative area occupied by specific fiber types (17). Hence, regions within a muscle with distinct differences in fiber type composition are expected to exhibit different physiological characteristics as well as MHC composition. The diaphragm has two distinct regions, costal and crural. These two regions have different mechanical actions on the rib cage (6) as well as different biochemical properties (23) and fiber type profile (22). Type II fiber atrophy in senescent skeletal muscle has been observed (l7,18) and results in a reduced contribution by these fibers to the total cross-sectional area. In addition, the relative proportion of fast MHC isoform decreases with age and correlates with the concomitant decrease in relative area occupied by the respective fiber type (17). The extent to which aging influences MHC composition in the diaphragm has not been previously examined. The diaphragm, like other skeletal muscles, is capable 1282
0161-7567192
$2.00
Copyright
Animals. Young adult (2.5 mo) and old (20 mo) specific pathogen-free female Fischer 344 rats were obtained from the National Institute on Aging Colony (Indianapolis, IN). The rats were individually caged and maintained in the small animal laboratory at the Psychology Building, University of Wisconsin, according to University of Wisconsin Research Animal Resource Center guidelines. The animals were maintained on an alternating 12:12-h light-dark photoperiod. Purina Rat Chow and water were provided ad libitum. Training. All animals were familiarized with walking on a motor-driven treadmill (lo-15 m/min, 10 min/day) over a l-wk period. At the end of this period, the animals from each age group were weight matched and randomly assigned to either a sedentary control or exercise training group. Hence, the four groups consisted of 1) young control (YC), 2) young trained (YT), 3) old control (OC), and 4) old trained (OT) rats. The YT and OT rats began progressive treadmill running at increasing grade and speed for 1 h/day, 5 days/wk, for 10 wk. By the end of the training period, the young animals were running at 30 m/min, 15% grade, whereas the old animals were running at 15 m/min, 15% grade. These work loads are rela-
0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/jappl at Georgetown Univ Med Ctr (141.161.091.014) on August 24, 2019.
RAT
DIAPHRAGM
MUSCLE
MYOSIN
tively similar in intensity (-75% of maximal oxygen consumption) for the two age groups (21). Killing of the animals and tissue removal. At the end of the lo-wk training regimen, the animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The soleus (SOL), plantaris and extensor digitorum longus (EDL) muscles were removed and quickly frozen in isopentane precooled with liquid nitrogen. The diaphragm was then exposed, and portions of both the midcostal and crural area were removed and frozen. All tissue was stored in a -70°C freezer. Biochemical analysis. Citrate synthase activity was measured on plantaris muscle to document a peripheral training effect. The plantaris muscle was kept on ice while cleaned of connective tissue and minced with scissors. The minced tissue samples were mixed with homogenizing medium (1:19) and homogenized on ice using a glass pestle driven by a Wheaton stirring motor. The homogenizing medium consisted of (in mM) 50 potassium phosphate (pH 7.4), 1 EDTA, 2 MgCl,, 2 ADP, and 0.5 dithiothreitol. Citrate synthase activity was measured spectrophotometrically at a wavelength of 412 nm,
31°C (30). Gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate MHC isoforms from homogenized whole muscle. Gel and sample preparation were based on a modification of the procedure by Fritz et al. (11) and Carraro and Catani (5). Briefly, -10-20 mg of frozen whole muscle were pulverized in liquid nitrogen and dissolved (1:600) in sample buffer A [8 M urea, 2 M thiourea, 0.05 M tris(hydroxymethyl)aminomethane (pH 6.8), 75 mM dithiothreitol, 3.0% SDS, and 0.05% bromophenol blue]. The mixture was homogenized using a glass pestle driven by a Wheaton stirring motor. The sample was heated to 100°C for 4 min and then allowed to cool to room temperature. After further homogenization, the samples were stored at -7OOC and thawed just before the electrophoresis procedure. A 3.5% acrylamide concentration (pH 6.8) was used in the stacking gel, whereas the resolving gel (8 X 10 cm in size, 0.75 mm thick, Hoefer SE250) consisted of 8% acrylamide concentration (pH 8.8) with 25% (vol/vol) glycerol. The samples were run at constant current (20 mA/gel) until the tracking dye reached the bottom of the gel (- 1.75 h). To optimize resolution of the MHC bands, the sample loads were kept small at -250 ng myosin/ lane. After completion of the gel run, the gels were removed from the plates and silver-stained according to a modification of the procedure of Tunon and Johansson (32). Following staining, the gels were placed on a light box (Fotodyne) and imaged on a computerized image processing system. Briefly, this system consisted of Kohu camera and television zoom lens (18-108 mm, F-stop 2.5) connected to a Sony monitor. This monitor was interfaced with a Magnavox computer for both the processing and storage of images (JAVA software). Obtained images were digitized into an array of pixel elements that had a total of 256 possible gray levels (GL). Identification of the fast and slow isoforms was accomplished by comigration of SOL and EDL muscle samples. These two muscle samples consist of primarily slow and fast MHC isoforms, respectively. Relative contribu-
HEAVY
CHAIN
1283
COMPOSITION
YC
YT
oc
OT
1. Effect of training on plantaris muscle citrate synthase (CS) activity. Values are means t SE expressed per gram wet weight. Groups are young control (YC), young trained (YT), old control (OC), and old trained (OT). * Trained different from control, P < 0.05. t Young different from old, P < 0.05. FIG.
tion of a specific band (fast vs. slow isoform) was determined by obtaining the total GL of the band (mean band GL X band area) and expressed as a percentage of the total obtained from the two isoforms combined. To correct for background staining, the background GL was subtracted out from the mean band GL. Densitometric analysis of the MHC bands revealed that the staining intensity was within the linear range (R2 = 0.99) for the loading volumes used. The coefficient of variation from repeated measurement of a single band was
