Variation and limitations in fiber enzymatic responses in hypertrophied muscle GORDON

R. CHALMERS,

Brain Research Institute Los Angeles, California

ROLAND

and Department

R. ROY,

AND

V. REGGIE

EDGERTON

of Physiological Science, University of California,

90024- 1527

CHALMERS,GORDONR., ROLANDR. ROY, AND V. REGGIE light ATPase fibers, particularly EDGERTON.Variation and limitations in fiber enzymatic and the muscle (4, 15, 16, 22, 24). size responses in hypertrophied muscle. J. Appl. Physiol. 73(Z): 631-641,1992.-The present study was designed to determine whether the degree and kind of adaptation of a muscle fiber to a functional overload (FO) are determined by properties that are intrinsic to that fiber. The study also addresses the question of the capability of fibers to maintain a normal level of coordination of proteins per fiber as fiber volume changes dramatically. The plantaris muscle of six adult female cats was overloaded for 12 wk by bilateral synergist removal. Plantaris muscle fiber mean size doubled after FO, although some very small fibers that stained dark for adenosinetriphosphatase (ATPase) were observed in some of the FO muscles. There appeared to be no change in total succinate dehydrogenase activity per fiber. A reduction in succinate dehydrogenase activity per unit volume was observed in a substantial number of fibers, reflecting a disproportionate increase in fiber volume relative to mitochondrial volume. In contrast, total a-glycerophosphate dehydrogenase activity and actomyosin ATPase activity increased as fiber size increased, whereas there was no change in a-glycerophosphate dehydrogenase and ATPase activities per unit volume. Control and FO muscle fibers generally expressed either a fast or slow myosin heavy chain type, but in some cases FO muscle fibers expressed both fast and slow myosin heavy chains. The persistence of variability in fiber sizes and enzyme activities in fibers of overloaded muscles suggests a wide range in the adaptive potential of individual fibers to FO. These data indicate that a severalfold increase in cell size may occur without significant qualitative changes in the coordination of protein regulation associated with metabolic pathways and ATP utilization. quantitative histochemistry; succinate dehydrogenase activity; cu-glycerophosphate dehydrogenase activity; muscle fiber morphology; compensatory hypertrophy; cat

muscle functional overload (FO) via synergist removal results in marked morphological and biochemical adaptations in the overloaded muscle. In studies with a duration of ~30 days, the mass of the overloaded rat plantaris increases by 80-100% (l&16,18,22, 24, 28, 30). Similarly, the size of fibers that stain light (type I) and dark (type II) for myosin adenosinetriphosphatase (ATPase) at an alkaline preincubation increases by 60-100% and 40-73%, respectively (16, 24, 25). Further increases in size occur with endurance training (25), indicating some importance in the amount of loading on the hypertrophic response. These adaptations in size are usually accompanied by an increase in the percentage of INTHERAT,

and size

in the deep portion

of

Variable effects of FO on muscle enzyme activities have been reported. The oxidative capacity of whole muscle homogenates is either reduced (5, 18) or unchanged (7,15,16,25) after 30-100 days of FO. However, endurance training can either return the oxidative capacity to normal levels (3) or increase it above control levels (2, 25). The glycolytic capacity of the rat plantaris after FO is reported to be unchanged (5, 15, 16) or decreased (4). A decrease in glycolytic capacity appears to be accompanied by a reduction in myofibril ATPase activity (2,4), reflecting some degree of coordination of these two enzyme systems (4). Previous attempts to functionally overload the cat plantaris have been largely unsuccessful compared with attempts in rats. For example, although there was a significant increase in the percentage of type I (light ATPase) fibers, Wetzel et al. (33) observed no change in fiber size after 6 wk of plantaris FO induced by synergist denervation. Similarly, the plantaris weight was unchanged in nonexercised cats in which the plantaris was overloaded by synergist removal (T. Cope, personal communication, and Roy and Edgerton, unpublished results). A moderate increase to a doubling of plantaris mass was observed, however, in those FO cats that were walked on a treadmill for as little as 2-3 min/day, three to four times/wk (Roy and Edgerton, unpublished results). Available data do not provide any insight into the interrelationships between the size and enzymatic activities within single fibers of muscles induced to hypertrophy. Muscle fibers of hypertrophied muscles may have an unchanged total oxidative capacity per fiber yet demonstrate a reduced oxidative capacity per unit of fiber because of disproportionate changes in mitochondrial and cell volume (2, 7). The study of single fibers permits an examination of this possibility and of other issues such as whether light and dark ATPase fibers respond differently to FO. Accordingly, the present study was undertaken to define the degree to which the interrelationships between cell size and the metabolic enzyme activities in normal plantaris muscle fibers persist after hypertrophy. METHODS Animal preparation. Six adult female cats (body weight 3.2-3.5 kg) were deeply anesthetized using pentobarbital

0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society

631

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

632

CAT

PLANTARIS

MUSCLE

FIBERS

sodium, sufficient to keep withdrawal and eye blink responses suppressed. Under aseptic conditions skin incisions were made to expose the dorsal region of both hindlimbs from the popliteal area to the calcaneus. The plantaris muscle was functionally isolated by removing its major synergists as follows. After ligating the blood supply and cutting the nerve branches to the soleus and medial gastrocnemius, these muscles were completely excised from both legs. The lateral gastrocnemius then was also removed, except for a small fleshy portion which remained attached near the proximal end of the plantaris. Care was taken to avoid injury to the plantaris muscle, nerve, and blood supply. The overlying fascia and skin were sutured in layers and a topical antiseptic (Furacin) was applied. The cats were placed in an incubator for postsurgical recovery and then were transferred to large vivarium cages (2 X 1.2 X 3 m). The FO cats were started on an exercise program -4 wk postsurgery, when it was evident that digitigrade locomotor capabilities had recovered. The exercise program started with the FO cats walking around a room for up to 15 min/day, 6 days/wk. Progressively, the exercise duration was increased to 25 min/day, and the intensity was increased by eliciting highly active running and jumping activity by throwing table tennis balls around the room in which the cats played. Twelve weeks after the initial surgery, each FO cat was anesthetized as described above and the right plantaris muscle was injected with a solution of horseradish peroxidase (HRP) for a separate study. The incisions then were closed as described above and the cats were returned to their cages for 96 h. During this period the FO cats were included in the daily exercise session only if fully recovered from surgery. After 96 h each cat was deeply anesthetized and perfused through the left ventricle with 0.5 liter of phosphate-buffered saline as part of the HRP study. The right and left plantaris muscles from each of the cats were removed bilaterally, cleaned of fat and connective tissue, and weighed wet. Two l&mm blocks from the midbelly of each left plantaris muscle were quickly frozen in isopentane cooled by liquid nitrogen and stored at -7O*C until processed. Six control adult female cats (body weight 2.6-3.9 kg) were housed in 0.6 X 0.5 X 0.8-m cages and kept in the vivarium for the same duration as the FO group. Injection of HRP, survival times, and terminal experimental procedures were the same as described for the FO group. All procedures followed the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals at and were approved by the Animal Use Committee UCLA. Histochemical procedures. Serial cross sections were cut in a cryostat at -20°C. Two lo-pm-thick sections were stained for actomyosin ATPase at an alkaline (pH 9.5) and an acid (pH 4.35) preincubation according to the technique described by Nwoye et al. (23), and each fiber examined was classified as either a light or dark ATPase fiber based on the staining density. Succinate dehydrogenase (SDH) activity, an oxidative marker enzyme, and a-glycerophosphate dehydrogenase (GPD) activity, a glycolytic marker enzyme, were determined histochemically

AFTER

FUNCTIONAL

OVERLOAD

as described by Martin et al. (21). Briefly, SDH activity was determined in lo-pm-thick sections in a medium containing (in mM) 100 phosphate buffer (pH 7.6), 0.75 sodium azide, 1 1-methoxyphenazine methylsulfate, 1.5 nitro blue tetrazolium, 5.5 EDTA-disodium salt, and 48 succinate disodium salt. The reaction was run at room temperature for 8 min and then stopped by repeated washes with distilled water. GPD activity was determined in 14-pm-thick sections in a medium containing (in mM) 100 phosphate buffer (pH 7.4), 0.2 l-methoxyphenazine methylsulfate, 1.2 nitro blue tetrazolium, 0.1 azide, and 6.2 glycerophosphate. The GPD reaction was run at 37*C for 19 min and then washed with distilled water several times. For each assay, three sections were incubated in medium containing substrate, and three were incubated in identical medium lacking substrate. The latter sections were used as tissue blanks to correct for nonspecific staining that occurred during the reaction. Actomyosin ATPase activity was determined by a technique described by Jiang et al. (17). Briefly, three tissue sections (10 pm thick) were incubated for 16 min in a 37°C pH 8.6 medium containing (in mM) 18 calcium chloride, 20 barbital sodium, 3 ATP, 1 magnesium chloride, 5 sodium azide, 0.2 ouabain, 0.1 calmodulin, and 1% gelatin. The remainder of the procedure was carried out at room temperature. The tissue sections were then washed three times for 3 min each in 1.5% CaCl,, the first wash solution containing 5 mM sodium azide and 0.2 mM ouabain. After a 5-min rinse in distilled water, the tissue sections were incubated in 150 mM CoCl, for 5 min. This was followed by rinsing again in distilled water, three times for 3 min each and then immersing in 1% ammonium sulfide for 20 s. After rinsing with distilled water, the coverslips were mounted on glass slides with aqamount (Bioimedical Specialties, Santa Monica, CA). The reaction rate for this procedure is linear for up to 16 min (17). Three serial sections incubated in medium lacking ATP substrate were used as tissue blanks to correct for nonspecific staining that occurred during the reaction. Tissue analysis. In each tissue section a region of fibers in the deep portion of the muscle (close to the bone) was digitized as a grey level image using a computer image processing system (PSICOM 327, Perceptive Systems, Houston, TX) as described elsewhere (21). Briefly, with the use of a Xl0 objective, each pixel within the picture was digitized as one of 256 grey levels and then converted to an optical density (OD). Each fiber of interest was then identified within the digitized picture, and the mean OD of all the pixels within the fiber was determined. For each fiber the average of the three sections without substrate was subtracted from the average of the three sections with substrate. The difference between these averages, i.e., the OD due to enzyme-specific staining, was divided by the reaction time to give a mean reaction rate per pixel. Because a pixel represents a unit volume of tissue, the measure is an estimate of the concentration of the enzyme. For the measurement of SDH and GPD activities, the reaction rate, in OD/min, was converted to enzyme activities with the equations described by Martin et al. (21). Fiber cross-sectional area was determined

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

CAT PLANTARIS

MUSCLE

FIBERS

AFTER

FUNCTIONAL

OVERLOAD

633

1. Mean CSA and enzymatic activities of dark- and light-staining ATPase fibers in plantaris of control and FO cats

TABLE

Control

No. of fibers CSA, pm2 SDH, nmol/min X 10e3 GPD, nmol/min ATPase, ODlmin X 10m3 Total SDH, nmol min-’ prnW2 Total GPD, nmol min-’ prnm2 X 10m3 Total ATPase, OD min-’ prnW2 l

l

l

l

l

l

FO

Light

Dark

Light

Dark

95 1,943*543 266.6t47.3 18.1t17.5 12.9t3.4 510t141 38.2t46.7 25.7k10.5

205 2,429-t867 199.2t82.3 57.4t26.6 21.425.4 444k153 151.1t108.8 53.4k26.5

115 4,361+2,152* 199.2*46.2* 27.2d4.4 15.8t3.4 88Ok523 98.lt39.0* 70.0t38.0*

140 6,608+4,060* 144.3t48.7* 49.7+19.4 25.4z7.0 983+710 318.7&194.9* 175.50~130.1*

Values are means & SD. Fibers are classified as dark or light staining for myosin ATPase, alkaline preincubation. Number of cats, 6 in each group (-50 fibers analyzed in each muscle). Total SDH, total GPD, and total ATPase values are calculated with individual fiber data rather than mean data. FO, functional overload; CSA, cross-sectional area; ATPase, myosin adenosinetriphosphatase activity; SDH, succinate dehydrogenase activity; GPD, a-glycerophosphate dehydrogenase activity; total SDH = SDH X CSA; total GPD = GPD X CSA; total ATPase = ATPase X CSA. * Significantly different from control (P < 0.05).

from the fibers outlined in the digitized pictures of the SDH tissue sections reacted with substrate. To estimate the total enzymatic activity in a section of a fiber, the product of fiber size and enzymatic activity was calculated for each fiber, i.e., total SDH, total GPD, and total ATPase. This measure indicates the total enzymatic capacity of the muscle fiber segment within the tissue section, i.e., the product of the number of enzyme molec ules and their specific activity (19, 20). One section stained for SDH activity (with substrate) from each cat was digitized using a ~25 objective. The distribution of SDH staining within individual fibers was determined using a “pixel peeling” procedure. This procedure involv ved outlini ng individual fibers within the digitized image and then determining the mean OD of the

A30

outermost ring of pixels within a fiber outline. The mean OD of the next inner ring of pixels was then determined, and the procedure was repeated until the ring of measurement closed upon itself at the center of the fiber. The mean OD for each pixel peel ring for each fiber was then plotted. In this procedure there was no subtraction of corresponding blank tissue OD values because it was not possible to reproduce identical pixel peel rings in a serial blank section. Thus, the pixel peel OD measurement is not a quantitative measure of SDH activity. This procedure does, however, measure qualitatively the SDH activity distribution from the periphery to the central regions of individual fibers. Immunohistochemistry. Serial 14-pm-thick sections were cut from frozen muscle blocks of two representative control and FO cats and were stained for myosin ATPase acid or alkaline preincubation as described above or for

25

IIL-

620 5 r $0

0i

B45 40 35-

0

B35 30

630$5-

25

s20w * u15LL JO- , 50,.

8

n

t z 2o

--.

ILIJ

5 a l5 E 10

26.5

ATPase

(OD/min

x

45 10-s)

1. Frequency distribution of actomyosin ATPase activities of light (Cl) and dark (m) ATPase fibers in plantaris muscle of control (A) and functional overload (FO) (B) cats. Note wide range of ATPase activities of fibers classified as either light or dark ATPase fibers. Overlap in ATPase activities between light and dark fibers is due to cat-tocat variability (see Fig. - 9). FIG.

II

20

5 0

i 0

-

CROSS-SECTIONAL

10000

AREA

20000 (ym2)

FIG. 2. Frequency distribution of fiber cross-sectional areas of light (Cl) and dark (m) ATPase fibers in plantaris muscle of control (A) and FO (23) cats. Note general increase in fiber area and appearance of very small dark ATPase fibers in FO cats.

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

634

CAT PLANTARIS

MUSCLE

FIBERS AFTJZR FUNCTIONAL

OVERLOAD

immunocytochemical myosin heavy chain (MHC) isoform expression. For the immunocytochemical determinations, frozen sections were air dried for 30 min, and all subsequent reactions were performed at room temperature. The sections were incubated for 30 min in a blocking solution containing 2% bovine serum albumin and 2% rabbit serum in phosphate-buffered saline, pH 7.4. One section from each cat was incubated for 2 h with a monoclonal antibody (S58, provided by Dr. F. Stockdale, Stanford University) specific for slow MHC. The slow MHC antibody was produced in the mouse using embryonic chick hindlimb muscle as the immunogen (8). After extensive washing in phosphate-buffered saline, primary antibody/antigen complexes were detected by incubating for 2 h with rhodamine-conjugated goat antibody to mouse immunogobulins (tetramethylrhodamine isothio-

0

A 0

CROSS-SECTIONAL

20000

10000

AREA

(Frn2)

FIG. 4. Frequency distribution of fiber cross-sectional areas of light (Cl) and dark (m) ATPase fibers in plantaris muscle of FO cnt G (A) and cat W(B). Note differences in response of 2 muscles to FO compared with control data shown in Fig. 2A.

cyanate, Accurate Scientific, New York, NY). The sections were then washed extensively with phosphate-buffered saline, dipped in 75 and 95% ethanol and mounted with Krystalon (Scientific Products, Baxter, McGraw Park, IL). The final section examined for each cat was incubated with a fast MHC antibody (MY-32, Sigma Chemical, St. Louis, MO), followed by incubation with a fluorescein-conjugated goat anti-mouse IgG antibody (fluorescein isothiocyanate, Sigma Chemical) with similar procedures as described for the slow MHC reaction. Three-dinwasionul reconstruction of fibers. Twentynine muscle fibers in one FO muscle were reconstructed over an 8.4-mm length. The tissue was cut into lo-pmthick sections, with a section being saved every 90 pm. These sections were stained for actomyosin ATPase at an alkaline preincubation as described above, and in 11 representative sections the fibers were digitized with a serial section three-dimensional reconstruction system (Eutectic Electronics, Raleigh, NC). Surface rendering of the three-dimensional image was performed using a graphic workstation (Stardent Computer, Newton, MA). Statistical analysis. A one-way nested analysis of variance, cat nested within experiment, was used for overall group comparisons. The degrees of freedom were determined by the number of cats. A x2 analysis was used to determine any significant differences in the proportion of light and dark ATPase fibers between the two experimental groups. RESULTS FIG. 3. Plantaris cross section, stained for actomyosin ATPaee activitv. alkaline oreincubation (DH 9.5). from FO cats C (A) and W (El) , I and a control cat (C). In comparison to control note occurrence of very large dark and light ATPase fibers in A and B and very small dark ATPase fibers in A. Bar. 200 urn. _I

. .

,,

,

I

Control and FO cats had similar body weights [3.2 f 0.2 vs. 3.4 f 0.4 (SD) kg], but the mean plantaris muscle mass of the FO cats was more than twice that of the control cats (right side: 11.8 + 1.0 vs. 5.0 f 0.8 g, P c 0.01 and left side: 10.6 + 1.5 vs. 4.5 + 0.5 g, P < 0.01).

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

CAT PLANTABIS

MUSCLE

FIBERS AFTER

FUNCTIONAL

0

635

OVERLOAD

5000

10000

CROSS-SECTIONAL

16000

AREA

(pm

H)

0000

FIG. 6. Belationship between succinate dehydrogenase (SDH) activity and muscle fiber cross-sectional area in control (0) and FO (+) cat plantaria fibers staining dark (A) and light (B) for actomyosin ATPase, alkaline preincubation. Each eymbol represents single muscle fiber. Compared with control values, note loss of high SDH activity fibers and increase in fiber size after FO in both dark and light ATPase fibers.

and dark ATPase fibers of the FO cats, GPD activities were unchanged, whereas total GPD activity increased significantly (Table 1). Within control cats, the GPD activity of the dark ATPase fibers was approximately three times higher than that in the light ATPase fibers. The GPD activities of both the light and dark ATPase fibers were unchanged in

FIG. 5. Three-dimensional reconstruction of plantaris muscle fibers from FO cat G. A: all fibers reconstructed are shown. B and C: some of outermost fibers have been removed to make inner fibers visible. Note relatively consistent size and shape of all muscle fibers over 8.4 mm of reconstruction. Dark and light caps on top of each muscle fiber indicate dark or light staining for actomyoain ATPase, alkaline preincubation, respectively. Scale cube in A is 66 pm along each edge. Scale along length of fibers is compressed in comparison with scale across fiber cross sections. Tapering of fibers toward bottom is not a narrowing of fibers but is due to perspective from position of view.

Fiber-type composition. An average of 50 and 43 fibers were examined in the deep region of the plantaris of the control and FO cats, respectively. There was a significant increase (P < 0.01) in the proportion of fibers staining lightly for myosin ATPase, alkaline preincubation, i.e., 32 and 45% in control and FO cats. Enzymutic adaptations. The SDH activities of the light and dark ATPase fibers were significantly lower (-25%) in the FO than in control cats (Table 1). This reduction, however, was not accompanied by a significant change in the total SDH activity of the fibers (Table 1). In the light

0

5000

CROSS-SECTIONAL

10000

15000

AREA

20000

(pm2)

FIG. 7. Relationship between a-glycerophosphate dehydrogenase (GPD) activity and muscle fiber cross-sectional area in control (0) and FO (+) cat plantaria fibers staining dark (A) and light (B) for actomyosin ATPase, alkaline preincubation. Each symbol represents single muscle fiber.

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

636

CAT

PLANTARIS

MUSCLE

FIBERS

+ /

0

5000

CROSS-SECTIONAL

10000

15000

AREA

20000

(pm2)

FIG. 8. Relationship between total SDH activity and muscle cross-sectional area in control (0) and FO (+) cat plantaris staining dark (A) and light (B) for actomyosin ATPase, alkaline cubation. Each symbol represents single muscle fiber. A: control = 0.36; FO data, r = 0.86. B: control data, r = 0.81; FO data, r Note large number of fibers in FO cats that have elevated total activities compared with controls.

fiber fibers preindata, r = 0.92. SDH

the FO cats (Table l), although the difference between the light and dark ATPase fibers was reduced approximately twofold. Total GPD activity increased significantly in both light and dark ATPase fibers in the FO cats (Table 1). ATPase activity was unchanged after FO, whereas total ATPase activity was significantly increased (Table 1). Although the ATPase activity was unchanged after FO (Table l), some light and dark ATPase fibers had higher values in the FO cats than in the controls (Fig. 1). The overlap in ATPase activities ‘between the light and dark ATPase fibers seen in Fig. 1 was due to variability between cats, because there was no, or minimal, overlap within an individual cat (see Fig. 9, B and D). Fiber morphology. Mean fiber size of light and dark ATPase fibers was 125 and 172% larger, respectively, in the FO than control cats (Table 1). Figure 2 clearly shows that this was due to an increase in the size of both fiber types. Figure 2B, however, also reveals the appearance of very small dark ATPase fibers in some FO cats, i.e., dark fibers that were smaller than those observed in controls. For example, in cat G a large number of the very small dark ATPase fibers were distributed throughout the muscle cross section (Fig. 3A), and the fiber sizes for the dark ATPase fibers were bimodally distributed (Fig. 4A ). No control cats demonstrated a bimodal distribution of fiber sizes when plotted on an expanded scale (data not shown). In two other FO cats, small dark ATPase fibers were limited to regions in the periphery of the muscle. More commonly, the FO muscles appeared as shown in cat IV (Fig. 3B), demonstrating extreme fiber hypertrophy (Fig. 4B) and a moderate increase in the propor-

AFTER

FUNCTIONAL

OVERLOAD

tion of light ATPase fibers. Note that the large shift in fiber sizes in cat VV(Fig. 4B) resulted in the smallest light and dark ATPase fibers being larger than almost any fiber observed in the control cats (cf. Fig. 4B with Fig. 2A). Muscle fibers in the control cat plantaris have a mean length of -19 mm (29). Twenty-nine fibers in FO cat G, of varying sizes and of both light and dark ATPase types, were reconstructed over a distance of 8.4 mm to examine their morphological characteristics. Although there were some minor changes in fiber shape and position, all 29 fibers examined, including the ones with the smallest cross-sectional areas, maintained a relatively constant size and shape for at least the 8.4 mm that was reconstructed (Fig. 5). Interdependence of fiber size and enzyme activity. The reduction in SDH activity and the increase in fiber crosssectional area in FO cats (Table 1) are reflected in a large downward and rightward shift in the SDH activity vs. area relationship in FO compared with control cats for both dark and light ATPase fibers (Fig. 6). On the other hand, plots of GPD activity vs. area (Fig. 7) and ATPase activity vs. area (not shown) exhibit only a rightward shift for both dark and light ATPase fibers, because only fiber area was altered by FO. Although the average total SDH activity was the same in the control and FO groups (Table l), the total SDH activity vs. area relationship for individual fibers was shifted rightward and upward in the FO cats (Fig. 8), indicating elevated total SDH activities. Plots of total GPD activity vs. area and total ATPase activity vs. area (not shown) exhibited a similar shift. The interrelationship among fiber SDH, GPD, and ATPase activities for each of the six control cats is shown in Fig. 9A. The fibers of each cat are represented by a specific symbol. Each vertical line and attached symbol represents a single fiber, with the size of the symbol being proportional to the size of the fiber within that group. Clusters of fibers are obvious, with most of the clusters dominated by a single symbol (i.e., fibers from an individual cat). These data suggest that the clustering and the diversity of observations in Fig. 9A may be largely due to variability among animals. To illustrate this, SDH, GPD, and ATPase activities were expressed as a percentage of the mean for each measure within each cat (Fig. 9B). These data reveal a consistent relationship among the variables examined within each of the control cats. For example, there is a continuum for each of the three enzyme activities measured with no grouping, except for the division between light and dark ATPase fibers indicated by the arrow. ATPase staining at an alkaline preincubation was dark in all fibers to the right of the arrow, and light in all fibers, except three, to the left of the arrow in Fig. 9B. * In FO cats (Fig. SC) there was a wide range in enzyme activities and a grouping of observations from individual cats similar to that observed in controls (cf. Fig. 9C with 9A). Normalization of the enzyme activities (Fig. 9D) reveals two groups of fibers within each cat. The division coincides quite well with the division on the basis of ATPase staining. ATPase staining was dark in all FO fibers to the right of the arrow and light in all fibers, except eight, to the left of the arrow in Fig. 9D. Among the dark ATP-

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

CAT

PLANTARIS

MUSCLE

FIBERS

AFTER

FUNCTIONAL

637

OVERLOAD

ATPase

ATPase 1 SDH %

12 D

SDH % 186

137

88

1’ 39

f 5 I

1' I

H6

121 ATPase

/

305

SY

I ; 0

I

9 I

12-I ATPase

%

I

%

I

I/ /

GPO % -‘) d13 c,

I&i

FIG. 9. Relationship among SDH, GPD, and actomyosin ATPase activities for control (A and B) and FO (C and D) cats. Each vertical line represents single muscle fiber with different symbol shapes representing different cats within experimental group. Size of symbol is proportional to fiber cross-sectional area within group; i.e., largest symbol indicates largest fiber in that experimental group. A and C: absolute enzyme activities are plotted for SDH (nmol/min x 10-3), GPD ( nmol/min), and ATPase (OD/min X 10e3). Note clusters of fibers, largely due to grouping of observations from individual cats and wide variability among cats in both groups. Effect of FO is reflected in reduced SDH activity. B and D: enzyme activities of each fiber are normalized by expressing as percentage of mean of enzyme activity within each cat. Most light- and dark-staining actomyosin ATPase fibers, alkaline preincubation, are to Leftand right, respectively, of arrows in B and D. This normalization procedure indicates that within each cat there is consistent relationship among SDH, GPD, and ATPase activities and fiber size.

ase fibers of the FO cats (Fig. 9D, right of arrow) there was a greater scattering of enzyme activities than in the equivalent fibers of controls (Fig. 9B). Note that fiber sizes represented by the size of the plotting symbols are scaled within the experimental group, i.e., the largest symbols in the control and FO plots of Fig. 9 mark the largest fiber within each group and thus do not represent fibers of similar sizes (see Fig. 2). Pixel peeling of SDH-stained fibers. The intracellular localization of SDH activity was examined in 33 control and 25 FO muscle fibers. Representative results from 16 control and 13 FO muscle fibers are shown in Fig. 10. The range in OD values observed between fibers was greater in control (Fig. 10, A and B) than in FO fibers (Fig. 10, C and D). All fibers, including the largest FO fibers, had a rather consistent OD from the periphery to the center (Fig. 10, C and D). Immunohistochemistry. Muscle fibers in control cats expressed exclusively either fast or slow MHC. The fibers expressing fast MHC stained dark and light for myosin ATPase at an alkaline and acid preincubation, respectively. Fibers expressing slow MHC stained dark and light for myosin ATPase at an acid and alkaline

preincubation, respectively (Fig. 11, A-D). Although the majority of fibers in the FO cats expressed only fast or only slow MHC, a few expressed both. This observation was made in FO cats that demonstrated fiber hypertrophy (e.g., FO cat VV in Fig. 11, E-H, double-staining fibers not shown), as well as in FO cat G that had small as well as hypertrophied fibers (Fig. 11, L-L). In all cases the small dark ATPase fibers, alkaline preincubation, in FO cat G (Figs. 4A and 11, I-L), were found to express fast MHC and, in a few cases, both fast and slow MHC. DISCUSSION

Fiber-type composition. After FO in the cat, an increased percentage of light ATPase fibers (alkaline preincubation) was observed in the plantaris (33), but not in the medial gastrocnemius (32). In the former study, the walking and jumping capabilities of the cats were assessed 2-3 days/wk, thus providing some form of regular exercise. In the latter study, in contrast, there was no imposed exercise. In addition, a larger percentage of the hindlimb extensor mass remains when the medial gastrocnemius compared with the plantaris muscle is

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

638

CAT

PLANTARIS

MUSCLE

FIBERS

AFTER

FUNCTIONAL

OVERLOAD

C

A 0.351 0.30 0.25 0.20 0.15

FIG. 10. Relationship between optical density of SDH activity and pixel peel ring, i.e., concentric ring between periphery and center of muscle fiber in which SDH staining was measured for control dark (A) and light (B) and FO dark (C) and light (D) actomyosin ATPase fibers, alkaline preincubation. Each line represents single muscle fiber, and each symbol represents mean value of ring of pixels within fiber. Number of concentric rings reflects fiber diameter.

0.10 0.05 I.

1.

1’1

-

1’1’1

D 0.30 0.25 0.20 0.15

0.05

-1 1

0

lo

30

20

40

50

60

PIXEL

700

PEEL

10

’

I

’

20

l

’

I

40

’

I

50

‘I

-

60

1

70

RING

overloaded, resulting in a smaller overload stimulus to the muscle fibers. The results of these and the present studies indicate that some minimum amount of overload must be imposed on an FO muscle to increase the size of muscle fibers, whereas a high overload for a more prolonged period is necessary to increase the percentage of light ATPase fibers in the cat. Enzymatic adaptations. A reduction in SDH activity per unit of fiber in the present study (Table 1) is consistent with the previously reported decreases in whole muscle oxidative capacity in rat FO muscles (5, 18). The fact that the daily exercise imposed in the present study did not elevate the mean oxidative capacity of a population of fibers in the FO muscles (2, 3, 25) probably reflects differences in the exercise regimens and species differences in the responsiveness to different levels of exercise. High-intensity but brief exercise, such as the repeated bouts of running and jumping interspersed with rest periods used in the present study, can induce fiber hypertrophy with minimal effect on the oxidative capacity (14). In contrast, the treadmill training used by Baldwin and co-workers (2, 3) was a continuous activity of moderate intensity, which often results in an increased muscle oxidative capacity without a change in fiber size in rats (14). It has been suggested that the reduced whole muscle oxidative capacity associated with FO may be due to a dilution of the existing mitochondria into a larger muscle mass (2, 7). This is supported by an unchanged monoamine oxidase activity per muscle (an estimation of mitochondrial content) in FO soleus and plantaris muscles of rats (7) and by an unchanged citrate synthase activity in FO rat plantaris muscles when the results are expressed as pmol min-l muscle-l (2). The latter measure, reflecting the total oxidative capacity of the muscle, l

I

30

is analogous to the total SDH activity per fiber as calculated in the present study. The maintenance of normal levels of total SDH activity per fiber in some fibers and an increase in others after FO (Table 1, Fig. 8), when combined with the overall reduction in the SDH activity per unit of fiber, support the idea that there was a disproportionate increase in fiber volume compared with SDH activity in some fibers rather than a downregulation of mitochondria. The percentage increase in mean fiber size, however, was far greater than the decrease in SDH activity. In addition, a large number of light and dark ATPase fibers in the FO cats had total SDH activities that were much higher than any fiber in the control group (Fig. 8), indicating a net increase of SDH activity. Fiber morphology. The doubling of the plantaris mass (wet muscle wt) and the 125 and 172% increase in the size of the light and dark ATPase fibers, respectively, were far greater than that reported in previous cat FO studies (32, 33) and larger than that reported in many rat FO studies (16, 24, 25, 27). Although it is not possible to separate the effects of increased muscle activation (EMG) and force production on the hypertrophic response, there is evidence that fiber size is influenced more by the latter (26, 31). Thus the large increase in muscle mass and mean fiber size observed indicates that the minimal hypertrophic response in earlier cat FO studies (32,33) was likely due to an insufficient level of overloading of the muscle, i.e., the chronic overload was minimal because of the large muscle mass remaining (32) or the low intensity and/or short duration of exercise (33). Combined, these data suggest that the pattern and/or threshold of the overload differs substantially in the cat compared with the rat, perhaps reflecting the less active behavior of the cat. Although there is considerable evidence that hyperpla-

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

CAT PLANTARIS I

MtJSCLE

FIBERS AFTER

FUNCTIONAL

OVERLOAD

639

---1.v

FIO. 11. Actomyosin ATPase and MHC staining in control cat (A-D) and in FO cots W (E-H) and G (I-L). A, E, and I: actomyosin ATPase, alkaline preincubation (pH 9.5); B, F, and J: actomyosin ATPase, acid preincubation (pH 4.35); C, G, and K fluorescein-labeled fast MHC antibody; D, H, and L: rhodamine-labeled slow MHC antibody staining. Control fibers react exclusively for either fast or slow MHC antibodies, whereas some hypertrophied FO fibers, marked with a star, express both fast and slow MHC. To left of arrow in K are 3 very small fibers expressing fast . MHC. Two of these fibers also express slow MHC (IL). Bar, 100 pm.

sia may not play a role in muscle hypertrophy in the rat (12), the presence of small fibers in some cats in the FO group is consistent with results in cats (10) and rats (13) after weight lifting and in the rat plantaris after FO (34). It is interesting that the population of small fibers observed by Yamada et al. (34) expressed only embryonic or small amounts of neonatal myosin, which the authors (34) interpreted as suggesting that these were newly formed fibers. It is curious that, in the present study, some fibers remained very small, whereas others hypertrophied after 12 wk of FO. It cannot be determined from the present data whether the small fibers were exposed to the same loading forces as the fibers that hypertrophied or whether there was a difference in their intrinsic ability to sufficiently upregulate protein metabolism to support a larger volume of cytoplasm. The large number of small fibers observed in FO cat G (Fig. 4A) was not likely due to denervation atrophy. First, if these small fibers were de-

nervated, then the plantaris mass of FO cat G would be expected to be small relative to other FO, cats, unless the remaining innervated fibers hypertrophied to a greater degree than in other FO cats. The plantaris mass of cat G, however, was similar to other FO cats, and the fibers were not excessively hypertrophied (see Figs.. 3 and 4A). Second, 12 wk after FO surgery any denervated fibers would be expected to have been innervated by axonal sprouts from neighboring intact axons. Third, denervated fibers usually demonstrate very dark staining for oxidative marker enzymes, such as NADH-tetrazolium reductase (1). All fibers in cat G that were smaller than any control fibers had SDH activities that were lower than the mean activity of control fibers. Interdependence of fiber size, rnyosin tupc?, and enzyme activities. No dark ATPase fibers in the FO cats had SDH activities in the upper range exhibited.in the control cats, i.e., >325 nmol/min X lo-’ (Fig. 6A). It is possible that the fibers with high SDH activities possessed the great-

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

640

CAT

PLANTARIS

MUSCLE

FIBERS

est potential for hypertrophy, resulting in a decrease in their oxidative enzyme activity. The lack of hypertrophied fibers with relatively high SDH activities (Fig. 6A) indicates that there may have been an upper limit to the combination of fiber size and oxidative capacity which an FO fiber can sustain. The relationship between fiber SDH activity and cross-sectional area in the light ATPase fibers of the FO cats (Fig. 6B) exhibited a pattern similar to that observed in the dark ATPase fibers. The light ATPase fibers, however, did not show the magnitude of hypertrophy demonstrated by the dark ATPase fibers (Table 1, Fig. 2). The reasons for these fiber-type differences in responsiveness to overload are unknown. It is known, however, that the basal rate of protein turnover is more rapid in muscles largely comprised of light, compared with dark, ATPase fibers (11). In addition, a muscle fiber may have some upper limits to,its metabolic capacity to maintain protein components, and this capacity may be more limiting in light than dark ATPase fibers, perhaps because of the difference in protein turnover rate. The twofold higher nucleus-to-cytoplasm ratio in light compared with dark ATPase fibers (6) is consistent with this hypothesis. The interrelationships of SDH, GPD, and ATPase activities of single fibers among control cats are shown in Fig. 9A. Large variability among cats is demonstrated by the control cat marked with the boxes, in which the fibers with the lowest ATPase activities had values as high as the highest ATPase activities observed in the cats marked with circles and spades. When absolute values were normalized per cat (Fig. 9B), a consistent relative distribution in enzyme activities emerged. For example, fibers with low GPD and ATPase activities had high SDH activities and were generally stained lightly for myosin ATPase, alkaline preincubation. In fibers in which the GPD and ATPase activities were high, the SDH activity was generally low. Despite the large increase in fiber size after FO, these same interrelationships were maintained qualitatively (Fig. 9, C and D). Not only did the hypertrophied muscle fibers maintain relatively (although not absolutely) normal enzymatic profiles, the intracellular organization of the cells seemed to be maintained. For example, the SDH activity throughout the cross section of even the largest FO fibers was relatively consistent (Fig. 10). That is, the peripheral regions of the fiber formed during hypertrophy seemed to be metabolically similar to the central regions from which they may have grown (9). Furthermore, these data indicate that oxygen utilization potential throughout the cross section of a fiber was similar even with the longer diffusion distances. Unchanged ATPase activities per unit of fiber in the hypertrophied fibers also suggests that the newly assembled myofibrils had the normal capacity to hydrolyze ATP. Thus, in general, it appears that the control mechanisms that regulate the potential of myofibrils to utilize energy and the relative dependence on metabolic pathways to support these energy needs remain qualitatively unchanged within a given fiber, even when the fiber volume changes dramatically. We thank Alan Hirahara for the surface rendering of the three-dimensional images. We gratefully acknowledge Drs. Frank Stockdale and Jeffrey Feldman, Stanford University, for supplying the slow MHC

AFTER

FUNCTIONAL

OVERLOAD

antibodies and for their expertise and assistance in the immunohistochemical procedures. This work was supported, in part, by National Institute of Neurological Disorders and Stroke Grant NS-16333. Address for reprint requests: V. R. Edgerton, Dept. of Physiological Science, 1804 Life Sciences, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-1527. Received

24 May

1991; accepted

in final

form

26 February

1992.

REFERENCES 1. ARMBRUSTMACHER, V. M. Skeletal muscle in denervation. Pat/&. Annu. 13: l-33, 1978. 2. BALDWIN, K. M., W. G. CHEADLE, 0. M. MARTINEZ, AND D. A. COOKE. Effect of functional overload on enzyme levels in different types of skeletal muscle. J. Appl. Physiol. 42: 312-317, 1977. 3. BALDWIN, K. M., 0. M. MARTINEZ, AND W. G. CHEADLE. Enzymatic changes in hypertrophied fast-twitch skeletal muscle. Pfluegers Arch. 364: 229-234, 1976. 4. BALDWIN, K. M., V. VALDEZ, R. E. HERRICK, A. M. MACINTOSH, AND R. R. ROY. Biochemical properties of overloaded fast-twitch skeletal muscle. J. Appl. Physiol. 52: 467-472, 1982. 5. BALDWIN, K. M., V. VALDEZ, L. F. SCHRADER, AND R. E. HERRICK. Effect of functional overload on substrate oxidation capacity of skeletal muscle. J. Appl. Physiol. 50: 1272-1276, 1981. 6. BURLEIGH, I. G. Observations on the number of nuclei within the fibers of some red and white muscles. J. Cell Sci. 23: 269-284, 1977. 7. CARLO, J. W., S. R. MAX, AND D. H. RIFENBERICK. Oxidative metabolism of hypertrophic skeletal muscle in the rat. Exp. Neurol. 48: 222-230, 1975. 8. CROW, M. T., AND F. E. STOCKDALE. Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Deu. Biol. 113: 238-254, 1986. 9. EPSTEIN, H. F., AND D. A. FISCHMAN. Molecular analysis of protein assembly in muscle development. Science Wash. DC 251: 10391044, 1991. 10. GIDDINGS, C. J., W. B. NEAVES, AND W. J. GONYEA. Muscle fiber necrosis and regeneration induced by prolonged weight-lifting exercise in the cat. Anat. Rec. 211: 133-141, 1985. 11. GOLDBERG, A. L., C. JABLECKI, AND J. B. LI. Effects of use and disuse on amino acid transport and protein turnover in muscle. Ann. NY Acad. Sci. 228: 190-201, 1974. 12. GOLLNICK, P. D., B. F. TIMSON, R. L. MOORE, AND M. RIEDY. Muscular enlargement and number of fibers in skeletal muscles of rats. J. Appl. Physiol. 50: 936-943, 1981. 13. Ho, K. W., R. R. ROY, C. D. TWEEDLE, W. W. HEUSNER, W. D. VAN Huss, AND R. E. CARROW. Skeletal muscle fiber splitting with weight-lifting exercise in rats. Am. J. Amt. 157: 433-440, 1980. 14. HOPPELER, H. Exercise-induced structural changes of skeletal muscle. ISI Atlas Sci. Biochem. 1: 247-255, 1988. 15. IANUZZO, C. D., AND D. CHEN. Metabolic character of hypertrophied rat muscle. J. Appl. Physiol. 46: 738-742, 1979. 16. IANUZZO, C. D., V. CHEN, R. B. ARMSTRONG, B. DABROWSKI, AND E. NOBLE. An experimental model to study chronically hypertrophied skeletal muscle. Adv. Physiol. Sci. 24: 279-289, 1981. 17. JIANG, B., R. R. ROY, AND V. R. EDGERTON. Expression of a fast fiber enzyme profile in the cat soleus after spinalization. Muscle Nerve 13: 1037-1049, 1990. 18. KUHN, F. E., AND S. R. MAX. Testosterone and muscle hypertrophy in female rats. J. Appl. Physiol. 59: 24-27, 1985. 19. MANCHESTER, J. K., M. M.-Y. CHI, B. NORRIS, B. FERRIER, I. KRASNOV, P. M. NEMETH, D. B. MCDOUGAL, JR., AND 0. H. LOWRY. Effect of microgravity on metabolic enzymes of individual muscle fibers. FASEB J. 4: 55-63, 1990. 20. MARTIN, T. P., V. R. EDGERTON, AND R. E. GRINDELAND. Influence of spaceflight on rat skeletal muscle. J. Appl. Physiol. 65: 2318-2325,1988. 21. MARTIN, T. P., A. C. VAILAS, J. B. DURIVAGE, V. R. EDGERTON, AND K. R. CASTLEMAN. Quantitative histochemical determination of muscle enzymes: biochemical verification. J. Histochem. Cytothem. 33: 1053-1059, 1985. 22. NOBLE, E. G., Q. TANG, AND P. B. TAYLOR. Protein synthesis in compensatory hypertrophy of rat plantaris. Can. J. Physiol. Pharmacol. 62: 1178-1182, 1984. 23. NWOYE, L., W. F. H. M. MOMMAERTS, D. R. SIMPSON, K. SERAY-

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.

CAT PLANTARIS

MUSCLE

FIBERS

DARIAN, AND M. MARUSICH. Evidence for a direct action of thyroid hormone in specifying muscle properties. Am. J. Physiol. 242 (Regulatory Integrative Comp. Physiol. 11): R401-R408, 1982. 24. OLHA, A. E., B. J. JASMIN, R. N. MICHEL, AND P. F. GARDINER. Physiological responses of rat plantaris motor units to overload induced by surgical removal of its synergists. J. Neurophysiol. 60: 2138-2151,

1988.

25. RIEDY, M., R. L. MOORE, AND P. D. GOLLNICK. Adaptive response of hypertrophied skeletal muscle to endurance training. J. Appl. Physiol. 59: 127-131, 1985. 26. ROY, R. R., K. M. BALDWIN, AND V. R. EDGERTON. The plasticity of skeletal muscle: effects of neuromuscular activity. Exercise Sport Sci. Reu. 19: 269-312, 1991. 27. ROY, R. R., K. M. BALDWIN, T. P. MARTIN, AND V. R. EDGERTON. Biochemical and physiological changes in overloaded rat fast and slow twitch ankle extensors. J. AppZ. Physiol. 59: 639-646, 1985. 28. ROY, R. R., I. D. MEADOWS, K. M. BALDWIN, AND V. R. EDGERTON. Functional significance of compensatory overloaded rat fast muscle. J. AppZ. Physiol. 52: 473-478, 1982.

AFTER

FUNCTIONAL

OVERLOAD

641

29. SACKS, R. D., AND R. R. ROY. Architecture of the hind limb muscles of cats: functional significance. J. Morphol. 173: 185-195, 1982. 30. SALLEO, A., G. LA SPADA, G. FALZEA, M. G. DENARO, AND R. CICCIARELLO. Response of satellite cells and muscle fibers to longterm compensatory hypertrophy. J. Submicrosc. CytoZ.15: 929-940, 1983. 31. THOMASON, D. B., AND F. W. BOOTH. Atrophy of the soleus muscle by hindlimb unweighting. J. AppZ. Physiol. 68: l-12, 1990. 32. WALSH, J. V., R. E. BURKE, W. Z. RYMER, AND P. TSAIRIS. Effect of compensatory hypertrophy studied in individual motor units in medial gastrocnemius muscle of the cat. J. Neurophysiol. 41: 496508,1978. 33. WETZEL, M. C., R. L. GERLACH, L. 2. STERN, AND L. K. HANNAPEL. Behavior and histochemistry of functionally isolated cat ankle extensors. Exp. NeuroZ. 39: 223-233, 1973. 34. YAMADA, S., N. BUFFINGER, J. DIMARIO, AND R. C. STROHMAN. Fibroblast growth factor is stored in fiber extracellular matrix and plays a role in regulating muscle hypertrophy. Med. Sci. Sports Exercise 21: S173-S180, 1989.

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.