Respiration Physiolog); 89 (1992) 195-207 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0034-5687/92/$05.00
195
RESP 01930
Diaphragmatic fiber type specific adaptation to endurance exercise Scott K. Powers a David Criswell a, Fu-Kong[Lieu a Stephen Dodd a and Harold Silverman ° • Center for Exercise Science. Departments of Exercise Science and Physiology. Unirersity of Florida. Gainesriile. FL and bDepartments of Zoology, and Physiology. Louisiana State University. Baton Rouge. LA. USA
(Accepted 6 April 1992) Abstract. Recent evidence suggests that exercise training results in a significant improvement in the oxidative capacity of the mammalian diaphragm; however, limited data exist concerning which diaphragmatic fiber types are metabolically altered due to training. To test the hypothesis that exercise training increases the oxidative capacity of diaphragmatic type ! and lie fibers only, we examined the effects of endurance training on the fiber type specific changes in oxidative capacity, cross-sectional area, and capillarity of the costal diaphragm. Female Fischer-344 rats (age ca 180 days) were divided into either a sedentary control group (n = 6) or an exercise training group (n = 6). The trained animals exercised for 10 wks on a motor-driven treadmill (60 min.day =1; 5 days.wk ° t) at a work rate equal to ca 55-655~ Vo:.,,,~. Capillaries were identified histologically and fiber types determined using ATPase histochcmistry. Fiber cross-sectional area (CSA) and succinate dehydrogenase (SDH) activity in individual fibers were measured using a computerized image analysis system. Compared to control animals, training did not increase the capillary to fiber ratio in any diaphragm fiber type (P> 0.05); however, training increased capillary density (capillary No./CSA) in type lie fibers due to a reduction in cell CSA (P< 0.05), Further, training resulted in significant (P< 0.05) increases in total diaphragmatic SDH activity (A increase- 17,5°/0) and an increase in SDH activity in both type ! (A increase- 14')~o)and lie fibers (A increase- 17.4%). in contrast, training did not alter (P> 0.05) SDH activity in type lib fibers, These data support the hypothesis that endurance training results in significant improvements in the oxidative capacity of type i and lie fibers in the costal diaphragm of rodents. However, the increase in relative SDH activity and capillary density in type lie fibers is achieved primarily via a reduction in fiber CSA.
Aerobic metabolism, diaphragm, exercise training; Fatigue, diaphragm; Mammals, rat; Respiratory muscles, diaphragm, exercise training
The mammalian diaphragm is the most important muscle of inspiration. At rest, the work performed by the diaphragm in healthy young individuals is generally low and constitutes only 1-2% of the whole body oxygen consumption (Milic-Emili, 1991). However, during muscular exercise, the work and energy cost of breathing is greatly increased (Milic-Emili, 1991). Clearly, this increase in inspiratory work results in an Cor,espondence to: S.K. Powers, Center for Exercise Science, Room 33, FLG, University of Florida, Gainesville, FL 32611, USA.
196
S. K, POWERS et al.
elevated metabolic load on the diaphragm and thus breathing can be considered a form of muscular exercise for the diaphragm. The ability of diaphragm to adapt to changing metabolic demands has man~ theoretical and clinical implications. In this regard, recent studies have demonstrated that regular endurance exercise increases the activity of oxidative (lanuzzo et al., 1982; Moore and Gollnick, 1982; Powers et al., 1990, 1992), beta oxidation (lanuzzo et al., 1982; Powers et ai., 1992), and antioxidant enzymes (Powers et al., 1992) in homogenates prepared from the costal diaphragm of rodents. However, to date, limited data exist concerning which diaphragmatic muscle fibers adapt in response to altered activity patterns. Sleek and Fournier (1989) have proposed that diaphragmatic type I fibers are recruited during light ventilatory behaviors and that both type I and lla diaphragmatic fibers are recruited during heavy ventilatory efforts in cats, Further, these authors argue that diaphragmatic type llb fibers are not recruited during normal ventilatory activities and are reserved for expulsive behaviors only. It follows that while low-to-moderate intensity exercise would increase inspiratory muscle activity, it seems likely that this type ofdiaphragmatic effort would result in the recruitment of type I fibers and IIa fibers only. Hence, we hypothesize that low-to-moderate intensity endurance exercise will result in an improvement in the oxidative capacity of diaphragmatic type I and lla fibers with no improvements in the oxidative capacity of type llb fibers. To test this hypothesis these experiments quantitatively examined the exercise-training-induced changes in diaphragmatic muscle fiber morphometry, oxidative capacity, and capillarity.
Methods Anh~aals attd exerci,~e tra#~h~g. These experiments were approved by the university committee for animal research and followed the guidelines established by the American Physiological Society. Female Fischer-344 rats (age ca 180 days) were divided into 0ither a sedentary control group (n--6) or an exercise training group (n = 6). All animals were fed ad libitum and maintained on a 12-h light photoperiod. The training protocol selected for this study has been shown to improve diaphragmatic oxidative capacity in muscle homogenates (Powers et al,, 1992). The exercise training group ran on a treadmill 5 days,week ° i for 10 weeks; each training session began with a 5-min 'warm-up' at ca 15 re.rain -! (0% grade). On day I (week 1) of training the animals began exercising at 17 m,min =1 (10 rain duration/0% grade) with the duration of exercise being increased by 1-3 rain.day ° i until the animals reached 60 rain of exercise (including the warm-up), The duration of exercise remained at 60 rain for the duration of the study, During weeks 2-6 the treadmill speed was increased gradually until a spell of 20 m, min ~i was reached; the spell remained constant through weeks 6-10. Beginning w ~ k 7, the treadmill grade was increased by 1,5%.week -! until roaching a zenith of a 6 ~ grade during week 10 of traitii,lg, A summary of the training protocol is contained in Table 1,
197
SDH ACTIVITY IN SKELETAL MUSCLE FIBERS TABLE I Training protocol for the exercised trained animals Week No.
(min,day- i)~
(days.wk- i)
Speed (m,min- i)
?"0 Grade
1 2 3 4 5 6 7 8 9 10
15 25 30 45 60 60 60 60 60 60
5 5 5 5 5 5 5 5 5 5
17 18 18 19 19 20 20 20 20 20
0 0 0 0 0 0 1.5 3,0 4,5 6.0
" Total exercise time on the fifth training day of each week.
Tissue removal. Animals were euthanized with an i.p. injection of 90 mg.kg -I sodium pentobarbital and a section of the anterior costal diaphragm and the left plantaris muscle was quickly removed and frozen in isopentane cooled by liquid nitrogen. The muscle samples were stored at -80 °C until assay. SDH activity it) the plantaris muscle. To determine the effects of our exercise training program on locomotor muscles we measured succinate dehydrogenase (SDH) activity in the plantaris muscle. The plantaris muscle was chosen as a representative locomotor 'marker' muscle because it is actively recruited during treadmill exercise in the rat and is a mixed muscle similar to the costal diaphragm (Moore and Gollnick, 1982). All tissue samples were homogenized in cold 100 mM phosphate buffer wit~! 0.05?/o bovine serum albumin (BSA) (1:20 wt/vol; pH ~ 7.4). Whole muscle homogenization included a 15=see treatment with a tissue homogenizer (Uitra=Turrax T25, IKA works, Cincinnati, OH) followed by 10 passes of the homogenate in a tight=fitting PotterEIvehjem homogenizer. We have demonstrated previously that this homogenization technique is proficient in cellular and mitochondrial disruption in the rat diaphragm (Lawler et ai., 1989). Following completion of homogenization, the homogenates were centrifuged (3 °C) for 10 min at 400 x g. The supernatant was then decanted and assayed for SDH activity in all muscle samples using the technique described by Singer (1974). All assays were performed in duplicate at 25 °C and all samples were assayed on the same day to avoid interassay variation. Quantification of s#)glefiber SDH activity: Overview and theory. To determine the SDH activity in individual muscle fibers we employed the quantitative histochemical microphotometric procedure developed by Blanco et al. (1988). Briefly, the theory and procedures involved in this analysis are as follows: SDH is mitochondrial membrane bound and has no cytosolic component; this is an important consideration in his-
198
S.K. POWERS et al.
tochemical reactions since enzymes that are not membrane bound may diffuse out of the section during incubation therefore invalidating a quantitative analysis. The principle of measurement of SDH in this procedure involves the progressive reduction of nitroblue tetrazolium (NBT) to an insoluble colored compound (NBT-diformazan) which is used as a reaction indicator. The reduction of NBT occurs due to a release of H + from the conversion of succinate to fumarate; the transfer of H + to the electrontransport chain is prevented by sodium azide. Methoxyphenazine methosulfate was used as an exogenous electron carrier due to its efficiency and usefulness in preventing non-specific reduction of NBT (Kugler, 1982). In measuring the fiber SDH activity, 2-3 muscle sections were placed on a cover slide and introduced into a Columbia jar (25 ° C) containing the reaction medium (see below). We have demonstrated that the optical density increases as a linear function of the NBT-diformazan (NBT-dfz) and that the SDH reaction is linear with respect to time over a period of 4-5 min. Based on these results, we chose a single end-point time of 3 min for the reaction with the reaction stopped by multiple rinses in deionized water. The coverslips were air dried in the dark and mounted using a glycerin aqueous mounting medium (Aqua Mount, Pittsburgh, PA). The image was immediately digitized and the optical density of individual muscle fibers was determined using a microphotometric procedure implemented on a computer-based image processing system (see details below). The rate of NBT-dfz deposited within a muscle fiber was calculated using the Beer-Lambert equation: [NBT.dfz] = OD/k x L
where O D is the optical density (A.min =i) of the muscle fibermeasured at 570 nm (peak absorbance wavelength for NBT-dfz); k is the molar extinction coefficientfor NBT-dfz (26478 mol=l.cm=1); and L is the path length (12-#m section thickness). The reaction medium c~,n~ained a final concentration of: E D T A = 5 raM; sodium azide --0.75 raM; methox:cphenazine m¢thosulfate (mPMS) = I m M ; N B T = 1.5 m M ; succinate---80 raM; and phosphate buffer(I00 raM, pH = 7.6).These concentrations have been shown to be the optimal incubation medium for measurement of histochemicalS D H activityin skeletalmuscle and the rationalefor the assay has been discussed in detail by Blanco et aL (1988). All chemical reagents were obtained from Sigma (St. Louis, MO).
Applicationofimage analysissyswm. The image of the muscle cross-sectionwas magnifiedusing a compound lightmicroscope (Nikon model Aphaphot 2, Japan) and an image obtained using a video scanner (Truevision,model Targa M8, Indianapolis,IN). The video in,age w~s digitizedinto a matrix of 512 x 512 pixels(Java Video analysis software,Jandel Scientific,Corte Madera, CA) with 256 possiblelevelsofgray and was visualized using a highoresolutiongraphic monitor (Sony Trinitron,Japan). The tungsten light source of the microscope was restrictedto 570_+ 5 nm by interposing a monochromatic filter(Omega Optical, Orlando, FL) between the light source and
SDH ACTIVITY IN SKELETAL MUSCLE FIBERS
199
microscope stage. The image was shade-corrected to reduce errors due to uneven illumination of the image field or by optical aberrations (Blanco et al., 1988). It is noteworthy that the path length for light absorbance through the tissue is of critical importance and was controlled by cutting muscle sections at 12 #m. According to manufacturer specifications the cryostat used in these experiments (Reichert, model Histostat, Buffalo, NY) introduces an error of 1-3 % in the thickness of serial sections. Further, we demonstrated in pilot experiments (data not shown) that the relationship between section thickness and measured optical density is linear. The gray levels of the video system were calibrated for OD units using a series of neutral density filters (Omega Optical, Orlando, FL) ranging from 0.0 to 1.40D units. Optical density measurements in the current experiments ranged from ca 0.25 to 0.85. To determine the mean SDH activity of individual muscle fibers, the fiber boundaries were outlined from the digitized video image of the muscle cross-section by means of a cursor controller to produce a graphic overlay. The mean SDH activity was determined by averaging the OD of all pixels within the outlined fiber. To reduce electronic noise, each digitized image was the average of 10 separate scans. The final estimate of the change in OD was computed by subtracting the blank (fiber without substrate) for the same fiber.
Muscle fiber typing and morphometry. Serial cross sections of each muscle were cut at 12/~m in a cryostat (Reichert, model Histostat, Buffalo, NY) maintained at -20 °C. Muscle fibers were classified as type I, lla, or llb based on their staining for myofibrillar adenosine triphosphate (ATPase) after alkaline (pH = 10.3) and acid (pH --4.5) preincubation using the procedure described by Brooke and Kaiser (1970). The proportions of fiber types were determined from a random sample of 200-300 fibers from across the entire section of each muscle. A~ter complctiol~ of myofibriUar ATPase reaction, the different fiber types wore identifi0d and labeled on a photomicrograph obtained from a computer interfaced video graphic system (Sony model UP-850, Japa1~). The cross-sectional area of each fiber was determined from SDH reacted nondehydrated sections using computerized planimetry with the system being calibrated by a stage micrometer immediately prior to measurement. Computation of muscle fiber SDH activity from histochemical assay. A histochemical estimate of the total SDH activity of each muscle sample was calculated based upon the mean SDH activities of type I, IIa, and lib muscle fibers, the mean cross-sectional area (CSA) of muscle fibers, and their relative proportions using the following formula (Sieck et al., 1989): whole muscle SDH activity=I(% type l to total CSA)×(mean SDH acti,~ity type I)] + [(% type IIa to total CSA) × (mean SDH activity type lla)] + [(% type lib to total CSA) × (mean SDH activity type lib)]
200
S.K. POWERS et al.
Determination of fiber capillarization. Capillaries were identified from 12-#m thick cross-sections of muscle stained with a metachromic basic dye (1% toluidine blue). Fiber t~pcs were determined from the previously mentioned photomicrographs of muscle sections histochemically assayed for ATPase. It is noteworthy that this crosssectional technique does not systematically analyze stereological features of capillary tortuosity. However, Mathieu et al. (1983) have demonstrated that the error of this technique in predicting capillary length in muscle is small and therefore it is unlikely that these stereological factors contributed to significant errors in the present study. For data analysis, capillarization was described as (1)the capillary-to-fiber ratio and as (2) capillary number per cross-sectional area of fiber (capillary density). Statbtical analysis. The data were analyzed using one-way analysis of variance, Statistical significance was established at P
195
RESP 01930
Diaphragmatic fiber type specific adaptation to endurance exercise Scott K. Powers a David Criswell a, Fu-Kong[Lieu a Stephen Dodd a and Harold Silverman ° • Center for Exercise Science. Departments of Exercise Science and Physiology. Unirersity of Florida. Gainesriile. FL and bDepartments of Zoology, and Physiology. Louisiana State University. Baton Rouge. LA. USA
(Accepted 6 April 1992) Abstract. Recent evidence suggests that exercise training results in a significant improvement in the oxidative capacity of the mammalian diaphragm; however, limited data exist concerning which diaphragmatic fiber types are metabolically altered due to training. To test the hypothesis that exercise training increases the oxidative capacity of diaphragmatic type ! and lie fibers only, we examined the effects of endurance training on the fiber type specific changes in oxidative capacity, cross-sectional area, and capillarity of the costal diaphragm. Female Fischer-344 rats (age ca 180 days) were divided into either a sedentary control group (n = 6) or an exercise training group (n = 6). The trained animals exercised for 10 wks on a motor-driven treadmill (60 min.day =1; 5 days.wk ° t) at a work rate equal to ca 55-655~ Vo:.,,,~. Capillaries were identified histologically and fiber types determined using ATPase histochcmistry. Fiber cross-sectional area (CSA) and succinate dehydrogenase (SDH) activity in individual fibers were measured using a computerized image analysis system. Compared to control animals, training did not increase the capillary to fiber ratio in any diaphragm fiber type (P> 0.05); however, training increased capillary density (capillary No./CSA) in type lie fibers due to a reduction in cell CSA (P< 0.05), Further, training resulted in significant (P< 0.05) increases in total diaphragmatic SDH activity (A increase- 17,5°/0) and an increase in SDH activity in both type ! (A increase- 14')~o)and lie fibers (A increase- 17.4%). in contrast, training did not alter (P> 0.05) SDH activity in type lib fibers, These data support the hypothesis that endurance training results in significant improvements in the oxidative capacity of type i and lie fibers in the costal diaphragm of rodents. However, the increase in relative SDH activity and capillary density in type lie fibers is achieved primarily via a reduction in fiber CSA.
Aerobic metabolism, diaphragm, exercise training; Fatigue, diaphragm; Mammals, rat; Respiratory muscles, diaphragm, exercise training
The mammalian diaphragm is the most important muscle of inspiration. At rest, the work performed by the diaphragm in healthy young individuals is generally low and constitutes only 1-2% of the whole body oxygen consumption (Milic-Emili, 1991). However, during muscular exercise, the work and energy cost of breathing is greatly increased (Milic-Emili, 1991). Clearly, this increase in inspiratory work results in an Cor,espondence to: S.K. Powers, Center for Exercise Science, Room 33, FLG, University of Florida, Gainesville, FL 32611, USA.
196
S. K, POWERS et al.
elevated metabolic load on the diaphragm and thus breathing can be considered a form of muscular exercise for the diaphragm. The ability of diaphragm to adapt to changing metabolic demands has man~ theoretical and clinical implications. In this regard, recent studies have demonstrated that regular endurance exercise increases the activity of oxidative (lanuzzo et al., 1982; Moore and Gollnick, 1982; Powers et al., 1990, 1992), beta oxidation (lanuzzo et al., 1982; Powers et ai., 1992), and antioxidant enzymes (Powers et al., 1992) in homogenates prepared from the costal diaphragm of rodents. However, to date, limited data exist concerning which diaphragmatic muscle fibers adapt in response to altered activity patterns. Sleek and Fournier (1989) have proposed that diaphragmatic type I fibers are recruited during light ventilatory behaviors and that both type I and lla diaphragmatic fibers are recruited during heavy ventilatory efforts in cats, Further, these authors argue that diaphragmatic type llb fibers are not recruited during normal ventilatory activities and are reserved for expulsive behaviors only. It follows that while low-to-moderate intensity exercise would increase inspiratory muscle activity, it seems likely that this type ofdiaphragmatic effort would result in the recruitment of type I fibers and IIa fibers only. Hence, we hypothesize that low-to-moderate intensity endurance exercise will result in an improvement in the oxidative capacity of diaphragmatic type I and lla fibers with no improvements in the oxidative capacity of type llb fibers. To test this hypothesis these experiments quantitatively examined the exercise-training-induced changes in diaphragmatic muscle fiber morphometry, oxidative capacity, and capillarity.
Methods Anh~aals attd exerci,~e tra#~h~g. These experiments were approved by the university committee for animal research and followed the guidelines established by the American Physiological Society. Female Fischer-344 rats (age ca 180 days) were divided into 0ither a sedentary control group (n--6) or an exercise training group (n = 6). All animals were fed ad libitum and maintained on a 12-h light photoperiod. The training protocol selected for this study has been shown to improve diaphragmatic oxidative capacity in muscle homogenates (Powers et al,, 1992). The exercise training group ran on a treadmill 5 days,week ° i for 10 weeks; each training session began with a 5-min 'warm-up' at ca 15 re.rain -! (0% grade). On day I (week 1) of training the animals began exercising at 17 m,min =1 (10 rain duration/0% grade) with the duration of exercise being increased by 1-3 rain.day ° i until the animals reached 60 rain of exercise (including the warm-up), The duration of exercise remained at 60 rain for the duration of the study, During weeks 2-6 the treadmill speed was increased gradually until a spell of 20 m, min ~i was reached; the spell remained constant through weeks 6-10. Beginning w ~ k 7, the treadmill grade was increased by 1,5%.week -! until roaching a zenith of a 6 ~ grade during week 10 of traitii,lg, A summary of the training protocol is contained in Table 1,
197
SDH ACTIVITY IN SKELETAL MUSCLE FIBERS TABLE I Training protocol for the exercised trained animals Week No.
(min,day- i)~
(days.wk- i)
Speed (m,min- i)
?"0 Grade
1 2 3 4 5 6 7 8 9 10
15 25 30 45 60 60 60 60 60 60
5 5 5 5 5 5 5 5 5 5
17 18 18 19 19 20 20 20 20 20
0 0 0 0 0 0 1.5 3,0 4,5 6.0
" Total exercise time on the fifth training day of each week.
Tissue removal. Animals were euthanized with an i.p. injection of 90 mg.kg -I sodium pentobarbital and a section of the anterior costal diaphragm and the left plantaris muscle was quickly removed and frozen in isopentane cooled by liquid nitrogen. The muscle samples were stored at -80 °C until assay. SDH activity it) the plantaris muscle. To determine the effects of our exercise training program on locomotor muscles we measured succinate dehydrogenase (SDH) activity in the plantaris muscle. The plantaris muscle was chosen as a representative locomotor 'marker' muscle because it is actively recruited during treadmill exercise in the rat and is a mixed muscle similar to the costal diaphragm (Moore and Gollnick, 1982). All tissue samples were homogenized in cold 100 mM phosphate buffer wit~! 0.05?/o bovine serum albumin (BSA) (1:20 wt/vol; pH ~ 7.4). Whole muscle homogenization included a 15=see treatment with a tissue homogenizer (Uitra=Turrax T25, IKA works, Cincinnati, OH) followed by 10 passes of the homogenate in a tight=fitting PotterEIvehjem homogenizer. We have demonstrated previously that this homogenization technique is proficient in cellular and mitochondrial disruption in the rat diaphragm (Lawler et ai., 1989). Following completion of homogenization, the homogenates were centrifuged (3 °C) for 10 min at 400 x g. The supernatant was then decanted and assayed for SDH activity in all muscle samples using the technique described by Singer (1974). All assays were performed in duplicate at 25 °C and all samples were assayed on the same day to avoid interassay variation. Quantification of s#)glefiber SDH activity: Overview and theory. To determine the SDH activity in individual muscle fibers we employed the quantitative histochemical microphotometric procedure developed by Blanco et al. (1988). Briefly, the theory and procedures involved in this analysis are as follows: SDH is mitochondrial membrane bound and has no cytosolic component; this is an important consideration in his-
198
S.K. POWERS et al.
tochemical reactions since enzymes that are not membrane bound may diffuse out of the section during incubation therefore invalidating a quantitative analysis. The principle of measurement of SDH in this procedure involves the progressive reduction of nitroblue tetrazolium (NBT) to an insoluble colored compound (NBT-diformazan) which is used as a reaction indicator. The reduction of NBT occurs due to a release of H + from the conversion of succinate to fumarate; the transfer of H + to the electrontransport chain is prevented by sodium azide. Methoxyphenazine methosulfate was used as an exogenous electron carrier due to its efficiency and usefulness in preventing non-specific reduction of NBT (Kugler, 1982). In measuring the fiber SDH activity, 2-3 muscle sections were placed on a cover slide and introduced into a Columbia jar (25 ° C) containing the reaction medium (see below). We have demonstrated that the optical density increases as a linear function of the NBT-diformazan (NBT-dfz) and that the SDH reaction is linear with respect to time over a period of 4-5 min. Based on these results, we chose a single end-point time of 3 min for the reaction with the reaction stopped by multiple rinses in deionized water. The coverslips were air dried in the dark and mounted using a glycerin aqueous mounting medium (Aqua Mount, Pittsburgh, PA). The image was immediately digitized and the optical density of individual muscle fibers was determined using a microphotometric procedure implemented on a computer-based image processing system (see details below). The rate of NBT-dfz deposited within a muscle fiber was calculated using the Beer-Lambert equation: [NBT.dfz] = OD/k x L
where O D is the optical density (A.min =i) of the muscle fibermeasured at 570 nm (peak absorbance wavelength for NBT-dfz); k is the molar extinction coefficientfor NBT-dfz (26478 mol=l.cm=1); and L is the path length (12-#m section thickness). The reaction medium c~,n~ained a final concentration of: E D T A = 5 raM; sodium azide --0.75 raM; methox:cphenazine m¢thosulfate (mPMS) = I m M ; N B T = 1.5 m M ; succinate---80 raM; and phosphate buffer(I00 raM, pH = 7.6).These concentrations have been shown to be the optimal incubation medium for measurement of histochemicalS D H activityin skeletalmuscle and the rationalefor the assay has been discussed in detail by Blanco et aL (1988). All chemical reagents were obtained from Sigma (St. Louis, MO).
Applicationofimage analysissyswm. The image of the muscle cross-sectionwas magnifiedusing a compound lightmicroscope (Nikon model Aphaphot 2, Japan) and an image obtained using a video scanner (Truevision,model Targa M8, Indianapolis,IN). The video in,age w~s digitizedinto a matrix of 512 x 512 pixels(Java Video analysis software,Jandel Scientific,Corte Madera, CA) with 256 possiblelevelsofgray and was visualized using a highoresolutiongraphic monitor (Sony Trinitron,Japan). The tungsten light source of the microscope was restrictedto 570_+ 5 nm by interposing a monochromatic filter(Omega Optical, Orlando, FL) between the light source and
SDH ACTIVITY IN SKELETAL MUSCLE FIBERS
199
microscope stage. The image was shade-corrected to reduce errors due to uneven illumination of the image field or by optical aberrations (Blanco et al., 1988). It is noteworthy that the path length for light absorbance through the tissue is of critical importance and was controlled by cutting muscle sections at 12 #m. According to manufacturer specifications the cryostat used in these experiments (Reichert, model Histostat, Buffalo, NY) introduces an error of 1-3 % in the thickness of serial sections. Further, we demonstrated in pilot experiments (data not shown) that the relationship between section thickness and measured optical density is linear. The gray levels of the video system were calibrated for OD units using a series of neutral density filters (Omega Optical, Orlando, FL) ranging from 0.0 to 1.40D units. Optical density measurements in the current experiments ranged from ca 0.25 to 0.85. To determine the mean SDH activity of individual muscle fibers, the fiber boundaries were outlined from the digitized video image of the muscle cross-section by means of a cursor controller to produce a graphic overlay. The mean SDH activity was determined by averaging the OD of all pixels within the outlined fiber. To reduce electronic noise, each digitized image was the average of 10 separate scans. The final estimate of the change in OD was computed by subtracting the blank (fiber without substrate) for the same fiber.
Muscle fiber typing and morphometry. Serial cross sections of each muscle were cut at 12/~m in a cryostat (Reichert, model Histostat, Buffalo, NY) maintained at -20 °C. Muscle fibers were classified as type I, lla, or llb based on their staining for myofibrillar adenosine triphosphate (ATPase) after alkaline (pH = 10.3) and acid (pH --4.5) preincubation using the procedure described by Brooke and Kaiser (1970). The proportions of fiber types were determined from a random sample of 200-300 fibers from across the entire section of each muscle. A~ter complctiol~ of myofibriUar ATPase reaction, the different fiber types wore identifi0d and labeled on a photomicrograph obtained from a computer interfaced video graphic system (Sony model UP-850, Japa1~). The cross-sectional area of each fiber was determined from SDH reacted nondehydrated sections using computerized planimetry with the system being calibrated by a stage micrometer immediately prior to measurement. Computation of muscle fiber SDH activity from histochemical assay. A histochemical estimate of the total SDH activity of each muscle sample was calculated based upon the mean SDH activities of type I, IIa, and lib muscle fibers, the mean cross-sectional area (CSA) of muscle fibers, and their relative proportions using the following formula (Sieck et al., 1989): whole muscle SDH activity=I(% type l to total CSA)×(mean SDH acti,~ity type I)] + [(% type IIa to total CSA) × (mean SDH activity type lla)] + [(% type lib to total CSA) × (mean SDH activity type lib)]
200
S.K. POWERS et al.
Determination of fiber capillarization. Capillaries were identified from 12-#m thick cross-sections of muscle stained with a metachromic basic dye (1% toluidine blue). Fiber t~pcs were determined from the previously mentioned photomicrographs of muscle sections histochemically assayed for ATPase. It is noteworthy that this crosssectional technique does not systematically analyze stereological features of capillary tortuosity. However, Mathieu et al. (1983) have demonstrated that the error of this technique in predicting capillary length in muscle is small and therefore it is unlikely that these stereological factors contributed to significant errors in the present study. For data analysis, capillarization was described as (1)the capillary-to-fiber ratio and as (2) capillary number per cross-sectional area of fiber (capillary density). Statbtical analysis. The data were analyzed using one-way analysis of variance, Statistical significance was established at P
