Muscle adaptations in trained rats with peripheral arterial insufficiency TRENT P. ERNEY, GREGORY M. MATHIEN, AND RONALD L. TERJUNG Department of Physiology, State University of New York, Health Science Center Syracuse, Syracuse, New York 13210

ERNEY,TRENTP., GREGORYM. MATHIEN,ANDRONALD L. TERJUNG.Muscle adaptations in trained rats with peripheral arteriaZ insufficiency. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H445-H452,1991.-The influence of training adaptations, induced within the active muscles of rats with peripheral arterial insufficiency, was assessed with an isolated hindlimb preparation. Femoral artery-stenosed rats, showing symptoms of intermittent claudication, were trained for 14-20 wk by running at 20-35 m/min up a 15% grade for up to -1 h/day, 5 days/wk. Similar total hindlimb blood flows (12.6 ml/min) at a similar arterial O2 content (20.7 vol/lOO ml) yielded similar blood flows (95-117 mlomin-l . 100 g-‘) and O2 deliveries (9-11 pmol min-’ g-‘) to the contracting muscle of sedentary (n = 10) and trained (n = 10) rats. Ten-minute periods of tetanic contractions (100 ms at 100 Hz each) at 4, 8, 13, 45, 60, and 90 tetani/ min were used. Muscle force development was better maintained (P < 0.001) by the trained group. Higher peak O2 consumption (P < 0.01) of the trained (5.69 t 0.53 pmol. min+ *g-l) compared with the sedentary group (3.66 t 0.26 pm01 . min-l . g-‘) involved a greater O2 extraction, since delivery of 0, was not different between groups. Thus adaptations occurred within trained muscle to enhance performance and peak O2 consumption. Muscle citrate synthase activity, an index of mitochondrial content, was greater (P < 0.005) in the trained group, with the low-oxidative white muscle section exhibiting the greatest, change (-threefold sedentary). Adaptations in this section were probably realized functionally, since improvements in muscle performance were evident early in the contraction sequence. Our results imply that the improved exercise tolerance of the trained rats during treadmill running was due, in part, to local adaptations within the muscle not requiring an increase in muscle blood flow. Similar adaptations could be operative in patients with peripheral arterial insufficiency. l

l

blood flow; microspheres; dication

oxygen

delivery;

intermittent

clau-

A CARDINAL FEATURE of individuals with peripheral arterial insufficiency is an abnormally low exercise tolerance. This is most apparent in patients with intermittent claudication where exercise tolerance is “symptom limited” by ischemia within the active limb (31). Interestingly, increased physical activity is recognized as a valuable treatment in the management of these patients (19) and typically leads to significant improvements in exercise tolerance (5, 7, 16, 17, 20, 28, 34). Affected patients are capable of exercising longer and at a higher intensity after training when compared with before training. 0363-6135/91

$1.50 Copyright

The increase in exercise tolerance after training could be due, in part, to an enhanced oxygen delivery realized by an increased blood flow to the working muscle. Angiographic (lo), hemodynamic (16), and direct blood flow (24) evidence indicates that collateral vessel function to an affected limb can be improved. Furthermore, a redistribution of blood flow within the ischemic limb, to more effectively perfuse the active muscles, could be beneficial in improving exercise tolerance (23, 34). These vascular adaptations, however, may not occur in all patients nor account for the entire benefit accrued by training. Numerous studies have been unable to identify any improvement in blood flow to the ischemic muscle or limb after training (5, 7, 17, 28, 34). Yet, they typically report an improvement in exercise tolerance with training. The basis for the improved exercise tolerance, however, has not been established. The increase in exercise tolerance of claudicants probably represents an increased functional capacity of the muscle, since exercise tolerance is symptom limited by ischemia. Furthermore, some patients are capable of performing exercise at a higher intensity (10, 11, 28). Thus the peak oxygen consumption of the active muscle is expected to be improved. The decreased oxygen content in the venous effluent from the active limb found after training (28, 34) could represent an enhanced oxygen extraction by the active muscle. This implies, in the absence of improved blood flow, that peripheral adaptations within the trained muscle have occurred and are important in improving exercise tolerance. An improved oxygen extraction can be expected, since the peak oxygen extraction by even richly vascularized high-oxidative capacity muscle during contractions is not maximal (12). Peripheral adaptations, induced within the active muscle by exercise training, that could be important to claudicants include an enhanced capillarity and an increased mitochondrial content of (cf. Ref. 27). A greater tissue capillarity should improve blood flow through the exchange network surrounding the active fibers to enhance nutrient exchange. Furthermore, a greater volume density of mitochondria is expected to shorten the average diffusion path length within the active muscle to enhance capillary-tissue exchange of oxygen. These adaptations have been found in naturally developing (cf. Ref. 20) and experimentally induced (25) peripheral arterial insufficiency following training. It had not been established, however, whether these adaptations serve to enhance muscle performance of the affected individuals. Thus the

0 1991 the American

Physiological

Society

H445

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purpose of this study was to establish whether training adaptations, induced within the muscle of limbs rendered ischemic by peripheral arterial insufficiency, serve to enhance performance and lead to a greater oxygen extraction. It is essential in this evaluation that blood flow, and therefore oxygen delivery, to the active muscle be the same in the trained compared with sedentary groups. Rats that had surgically introduced bilateral stenosis of the femoral artery were trained (23, 24), and an isolated perfused hindlimb preparation was employed to satisfy this requirement (9, 13). MATERIALS

AND

METHODS

Animal care. Adult male Sprague-Dawley rats obtained from Taconic Farms, (Germantown, NY) were housed 2 rats/cage in a temperature-controlled room (20-21 “C) with a 12 h-12 h light-dark cycle. Rat chow and tap water were provided ad libitum, except where n.oted below. Surgical procedures. Bilateral stenosis of the femoral artery under ether anesthesia was carried out as previously described (23,24). Briefly, a reproducible reduction in arterial diameter was achieved by tying a ligature around the femoral artery and 0.014-in. stainless steel wire. Removal of the wire restored patency of the artery to the limit of the stenosis. This procedure allowed sufficient blood flow for resting conditions, while limiting hyperemia during contractions in situ (23) or treadmill running (24). Training. Before surgical stenosis, animals were familiarized with our motor-driven rodent treadmill (Quinton model 42-15), set at a 15% grade, by running -5 min/day for 3-4 days at 20 m/min. After surgical stenosis, animals (322 t 3 g) were randomly divided into trained or sedentary groups. Beginning 48 h postsurgery, animals in the trained group were exercised 5 days/wk for 14-20 wk. The exercise intensity and duration of the training program were progressive, beginning with running at 20 m/min. After the initial 15-min period, running speed was increased 5 m/min each additional 15 min as the exercise tolerance of the animals permitted. Animals ran to the point where they would no longer keep pace with the treadmill speed. As noted previously (23, 24), running failure was usually preceded by a characteristic gait change, as evidence by the loss of smooth rhythmic limb movements and the appearance of exaggerated hops. This typically occurred soon after the anim als began running at their highest speed. Animals were then immediately taken off the treadmill. Exercise tolerance of a subgroup of sedentary animals (n = 3) was assessed once per week with the same protocol as used for the training program. No sedentary animal was exercised more than two times during the course of the experiment. As the course of the study progressed, we restricted the food intake of the sedentary animals by -10% in an attempt to maintain weights similar to that of the trained group. This modest food restriction reduces primarily the gain in body fat content and does not alter muscle composition nor activity of mitochondrial enzymes (cf. 32). However, a small weight difference between sedentary and trained groups remained (cf. Table 1).

ARTERIAL

INSUFFICIENCY

1. Animal weight, tissue weight, and initial force development TABLE

Body

Wt,

g Sedentary Trained

458t13 423&11*

Gastrocnemius-PlantarisSoleus Wt,

3.44t0.08 2.94t0.12*

Values are means t SE; n = 10 animals different from sedentary (P < 0.05).

Initial g

N

36.5t1.31 33.Okl.09 for all values.

Tension N/g

10.6kO.37 11.3t0.43 * Significantly

Hindlimb preparation. At the end of the training period, sedentary and trained (second day postexercise) rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and prepared for perfusion of the single hindlimb as described in detail (9). Briefly, the sciatic nerve was isolated, the gastrocnemius-plantaris-soleus muscle group was prepared for measurement of tension development, and the femoral artery and vein were isolated to establish a closed vascul .ar circuit. We wanted to establish a high blood flow to the hindlimb musculature, independent of the resistance due to the femoral stenosis. Connective tissue formation around the stenosis ligature and a marked increase in arterial wall fragility, distal to the stenosis, made removal of the ligature impractical. Thus insertion of the arterial catheter was made at a site just distal to th .e stenosis ligature. The requisite highoxygen de1ivery could then be established to study the effects of the local adaptations within the muscle that were p reduced by the stenosis resistance apparent in vivo. Perfusion and stimulation procedures. After surgical preparation, the animal was transferred to a temperature-controlled (37°C) cabinet for insertion of the catheters (9). Perfusion pressure was continuously monitored with a Statham transducer. Animals were perfused at rest for 20-30 min using a nonlimiting flow rate of 9-10 ml/min (9). Perfusion flow rates were determined by timed collections of the venous outflow. The inflow rate was then gradually increased over the next 10 min to -13 ml/min to achieve a high rate of oxygen delivery for contractions. Hindlimbs of these femoral-stenosed animals often demonstrated an altered vascular responsiveness, not common to normal animals, that caused an elevated blood pressure in a number of animals. While we have no explanation for this response, it could be related to change induced by the chronically lower perfusion pressure distal to the stenosis site found in vivo. If the elevated perfusion pressure was continued, a predictable muscle swelling and exaggerated fatigue developed, resulting in the loss of the animal. The elevated resistance was responsive to adenosine. Thus in those animals that demonstrated an elevated pressure, adenosine was infused at a rate eliciting maximal vasodilation (5.4 nmO1omin-l .g hindlimb-I) and resulted in a 25- to 50-mmHg pressure drop. Adenosine infusion was initiated before muscle contractions and was continued throughout the entire experiment in three trained and eight sedentary animals. The dose was determined in prior experiments, and maximum dilation was verified in each animal. Other experiments demonstrated that adenosine does not alter hindlimb oxygen consumption and muscl.e performance during contractions nor blood

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flow distribution. Furthermore, there was no difference in the response between animals of this study that did or did not receive adenosine within each experimental group. The sciatic nerve was placed over bipolar platinum electrodes for stimulation (Grass S48 Stimulator) of the entire distal hindlimb musculature (13). Tension development of the gastrocnemius-plantaris-soleus muscle group was continuously monitored with a load cell (Statham UM-10) connected to a Gould model 2600 recorder. Isometric tetanic contractions were elicited by supramaximal square wave pulses (0.1 ms, -6 V) delivered in lOO-ms trains at 100 Hz, using successive contraction frequencies of 4, 8, 15, 30, 45, 60, and 90 tetani/min (10 min/train frequency). ,4 lo-min contraction period at each frequency was chose to allow steady-state measurements of oxygen consumption and tension development, evident at the 5th and 10th min. Sequential contraction periods of increasing intensity yield similar muscle performance and oxygen consumption for each train frequency as when done independently (cf. 13, 14). Perfusion medium. The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing washed bovine erythrocytes with a hematocrit of 40-41, 4 g/l00 ml of bovine serum albumin, and 100 pU/ml of bovine insulin. Glucose concentration was maintained at 5 mM. Hemoglobin concentration was 13-15 g/100 ml blood. The perfusion medium (250-300 ml) was recycled after discarding the first 30 ml of venous effluent. Oxygen consumption. Oxygen consumption was calculated as the product of arteriovenous oxygen difference and the perfusion flow rate. The oxygen content of arterial and venous blood was determined using a LexO&on analyzer (Lexington Instruments, Waltham, MA). Resting oxygen consumption was calculated from the mean of three observations during the 20- to 30-min rest period. The increase in oxygen consumption above rest, at each contraction frequency (5th and 10th min values averaged), was taken as the oxygen consumption of the contracting muscle mass. Blood flow distribution. Blood flow to each hindlimb muscle and specific muscle M fiber sections (SW RESULTS) -. was determined at the end of the contraction sequence, as done previously (9, 13). Nine-micron microspheres (-1.25 x 10”) were infused in -0.6 ml of perfusion medium over a -30-s period. Tissues were excised, weighed, and counted (Beckman, Gamma 8000) to a 1% error. Blood flow was calculated as blood flow (ml. min-’ -1 = cpm (tissue) X total flow (ml/min) + cpm (total) + weight (g), where cpm is counts per minute. Biochemical assays. Muscle sections from the contralateral superficial medial gastrocnemius (predominantly fast-twitch white), deep lateral gastrocnemius (predominantly fast-twitch red), soleus (predominantly slowtwitch red), superficial fast-twitch white, and deep fasttwitch red vastus lateralis were frozen and stored at -70°C until analyzed for citrate synthase activity (29), a marker of mitochondrial content. Materials. Radioactive YSr-labeled microspheres (9.24 t 0.34 ,um) with a specific activity of 12.6 mCi/g were obtained from 3M (St. Paul, MN) in a suspension of 10% dextran containing 0.05% Tween 80 surfactant. Bovine

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serum albumin (fraction V), adenosine, and the reagents used for biochemical analysis were obtained from Sigma Chemical (St. Louis, MO). Fresh bovine erythrocytes were prepared by extensive washing (18-24 volumes) of acid-citrate-dextrose blood collected at a local meat packing company. Statistical measures. The data were analyzed using a mixed-design repeated measures analysis of variance (30). Specific mean differences were determined using Tukey’s method. Statistical significance was accepted at P c 0.05. RESULTS Treadmillperformance. Exercise tolerance of sedentary animals remained relatively meager, at -15-20 min run time and unchanged throughout the duration of the study (cf. Fig. 1). In contrast, running performance of the trained animals increased dramatically over the initial 3 wk of training until the animals were running 50-60 min/day. The trained animals were able to run at 35 m/ min, and some even 40 m/min, compared with 25 m/min for th e sedentary animals (Fig. 1). The duration of running remained fairly constant until the 8-9th wk of training, where there was a marked decline in exercise duration to -40 min/day. This decline in exercise tolerante may have been related to the gain in body weight. T o sustain a total run time of -60 min/day, the intensity of the training program was reduced somewhat by initiating running at 15 m/min. Thus during the last 4 wk of the study each 15-min running interval required running at a rate of 5 m/min slower than that illustrated on the right in Fig. 1. Muscle performance in situ. As shown in Table 1, initial isometric force development was similar between sedentary and trained groups. Figure 2 illustrates the decline in tension development during each of the tetanic contraction conditions. Tetanic tension development was better maintained (P < 0.001) in the trained group compared with sedentary group, at all contraction frequencies beyond 4 tetani/min. Trained muscle developed 40~45%

more

tension

than

sedentary

muscle

over

the

range of 30-90 tetani/min.

l

g

)

I I I I I I II wifi O012 3 4 5 6 7 Training

Duration

I III 8

9

10

I 11

12

(Weeks)

1. Daily exercise duration (min/day; mean t SE) and trained animals. Note that running speed (given on also increased as treadmill run time increased. Running reduced during weeks IO-13 by 5 m/min less than given scale to achieve a daily exercise duration of -1 h/day (see FIG.

13

of sedentary right scale), speed was on ordinate text).

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\

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-zixi.ned II

U

L

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n

.

I

0

15

l

I

30

-

I

-

45

I

60

=

I

75

-

I

90

TetaniNKnute FIG.

2. Developed

15'

tension

during

stimulation

in situ.

. 1

I

0

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15

30

45

INSUFFICIENCY

peak of 3.65 * 0.27 ~molomin-’ l g-l at 60 tetani/min. A similar pattern of increased oxygen consumption was found for trained muscle; however, oxygen consumption was greater (P < 0.005) with a peak value of 5.69 t 0.56 pm01 . min *g-l at 90 tetani/min. A significant increase (P < 0.005) in oxygen consumption is also found between trained (6.08 t 0.56 pmol min. g-‘) and sedentary muscle (4.00 t 0.25 prnol min-’ *g-l), when the peak oxygen consumption, irrespective of contraction condition, was taken for each animal. The rate of oxygen consumption per unit of force development was similar between groups. This is evident in Fig. 3 by the superimposed linear relationship (O-60 tetani/min) when the measured oxygen consumption is corrected for the tension loss at each frequency. The slopes represent an oxygen cost of tension development of 16.1 nmol oxygen/N for the sedentary muscle and 16.0 nmol oxygen/N for the trained muscle. Muscle blood flow and oxygen delivery. The greater oxygen consumption observed in the contracting trained muscle occurred even though blood flow (-100 ml min-l JO0 g-l), and therefore oxygen delivery (-9-10 pm01 . min-’ *g-l), was not different from that of contracting sedentary muscle (Table 2). Furthermore, as illustrated in Table 3, blood flows and oxygen deliveries to specific muscle fiber sections were not different between groups. Oxygen deliveries to the high-oxidative red muscle sections were between 18 and 23 prnol. min-’ *g-l, whereas oxygen deliveries to the low-oxidative white muscle sections were only 5-8 prnol. min-‘og-l. Since oxygen deliveries were not different between groups, the greater oxygen consumption of the trained group must have occurred by a greater extraction of oxygen. A greater oxygen extraction by the trained muscle over the range of 45-90 tetani/min is suggested from the results shown in Fig. 4; however, the differences were not statistically significant. Even though total perfusion of each hindlimb was controlled to be similar (12.6 ml/min), blood flow to the contracting muscle of the distal hindlimb was not identical. A slight redistribution of flow to the contracting muscle within the trained hindlimb may have occurred, possibly due to an increased muscle blood flow capacity, especially in the fast-twitch white sections (22). Although oxygen delivery to the contracting muscle was not statistically different between groups (Table 2), the -20% greater apparent blood flow in the trained group could have imparted an advantage. If the criterion for comparison of sedentary and trained responses is an equivalent oxygen delivery to the contracting muscle and not simply equivalent total hindlimb inflows, the benefit of training remains. This criterion was met by sequentially eliminating the highest oxygen delivery animal in the trained group and the lowest oxygen delivery animal in the sedentary group until oxygen deliveries to the contracting muscles of the groups were quantitatively the same. When oxygen delivery to the sedentary (9.74 t 0.60 pm01 . rnirPg g-l; n = 8) and trained (9.63 t 1.33 prnol min-l. g-‘; n = 8) groups were the same (1% difference), the peak oxygen consumption of the trained group was still -40% greater (P < 0.02) than that of the sedentary group (5.64 t 0.47 vs. 4.05 t 0.30 prnol. min-’ g-l, rel

10’ 0

ARTERIAL

60

75

90

Tetani/Minute FIG. 3. Oxygen consumption (open symbols) during contractions in sit,u. Note that since 0, cost of developed tension was not different between groups, increase in expected 0, consumption corrected for tension loss, shown in Fig. 2 (closed symbols), is essentially linear with contraction frequency.

Oxygen consumption. Resting muscle oxygen consumption was similar between sedentary (0.45 t 0.11 pmol. min-l. g--l, n = 10) and trained animals (0.42 t 0.19 pm01 . min. g-l, n = 10). As illustrated in Fig. 3, oxygen consumption of the sedentary muscle increased progressively, as the frequency of contractions increased, to a

l

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TABLE

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Sedentary Trained

TABLE

are means

Perfusion Pressure, mmHg

12.6t0.09 12.6kO.16

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Arterial 0, Content, vol/lOO ml

llOtl1 117tl5

k SE; n = 10 animals

for all values.

Blood Flow,* ml. mine1 . 100 g-’

20.9t0.2 20.6t0.3

* To contracting

Oxygen Delivery,* pm01 . min-’ . g-l

95.0t8.9 117s3.4

8.79t0.80 10.85tl.33

muscles.

3. Tissue blood flow and oxygen delivery Blood

Flow,

ml. min-l

-100

Gastrocnemius Red F-W

Sedentary Trained Values

ARTERIAL

2. Tissue perfusion conditions Total Blood Flow, ml/ min

Values

PERIPHERAL

are means

Gastroc-plantaris-soleus (Mixed)

Soleus (STR)

White (FTW

Oxygen

g-’

236t50

57t9

249t34

94t10

217t50

91-c-29

23lt51

115t23

of: SE; ~2 = 10 animals

for all values.

FTR,

fast-twitch

Delivery,

pmol . mine1 . g-’

Gastrocnemius Red @‘TN 21.9k4.63

5.25kO.79 8.56-t-2.72

18.2-c-4.69 red; FTW,

fast-twitch

Gastroc-plantaris-soleus (Mixed)

Soleus (ST@

White (FTW

23.lt3.10 21.524.88

white;

STR,

slow-twitch

8.6720.94 10.7t2.17

red.

that of the sedentary. The fast-twitch and slow-twitch high-oxidative red fiber sections also exhibited greater values in the trained group but by a much smaller margin compared with the fast-twitch white sections. DISCUSSION

0

0

15

30

45

60

75

90

Tetani/Minute FIG.

4. Oa extraction

during

contractions

in situ.

spectively). Even if the apparent disparity between blood flows to the fast-twitch white section of the gastrocnemius (cf. Table 3) is eliminated by matching these oxygen deliveries, our conclusions remain unchanged. Thus we believe that the increase in oxygen consumption realized by the trained muscle was due to the inherent characteristics of the muscle and not confounded by subtle differences in oxygen delivery. Mitochondrial enzyme activity. As shown in Table 4, the activity of citrate synthase was markedly increased (P < 0.001) in muscle by exercise training. This was most apparent in the fast-twitch white muscle sections of the lower (gastrocnemius) and upper (quadriceps) leg muscles, where the trained muscle was nearly threefold TABLE

The major finding of this study demonstrates that training-induced adaptations, within the active muscle of animals with peripheral arterial insufficiency, enhance muscle performance and increase the metabolic capacity for contractions. This occurred experimentally even though oxygen delivery was no greater than that to the nontrained muscle. The oxygen cost of force development by the gastrocnemius-plantaris-soleus muscle group was similar (-16 nmol/N) between groups. Thus the much improved tension development of the trained group, over the range of moderate to intense contraction conditions, required a greater energy supply to maintain the contractile performance. This was achieved by a markedly increased oxygen consumption, the maximum being approximately 50% greater than that of sedentary muscle. This greater energy flux was, in turn, achieved by a greater extraction of oxygen from the arterial delivery. Thus limb muscle that was ischemic during exercise in vivo (24) adapts to exhibit a vastly improved capillarytissue oxygen exchange after exercise training. The improved muscle performance measured in our experiment represents the response of the gastrocnemius-plantaris-soleus muscle group. This muscle group of the rat is composed of -75% low-oxidative fast-twitch white, l&-20% high-oxidative fast-twitch red, and 510% high-oxidative slow-twitch red muscle (1). These three

4. Citrate synthase activity Quadriceps

Sedentary Trained

Gastrocnemius

Red @‘TN

White (FTW

54.7tl.2

9.5tl.2

77.4t5.7

2l.lt4.1

Values are means -+ SE (pmol. min-l -8-l); n = 8 animals for all values. from corresponding tissue values of the sedentary group. FTR, fast-twitch

Red @‘TW 47.4t2.2 57.1t3.8

All values red; FTW,

White (FTW 12.2t0.9 33.7t2.4

of the trained group are significantly fast-twitch white; STR, slow-twitch

Soleus WW 28.9k4.2 39.2t1.4

different red fiber

(P < 0.001) sections.

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E

17 16 15 14 13

0

Fast-TwitchRed

l rl

E E $ ; O ; Fast-Twitch

White

00 urn

Gl2 -3 11 4 10 0 9

sedentary Trained

FastiTwitchRed

!@h3 WV7

0 -

10

0 q

Trained

3 ca

1

0

15

30

45

60

75

90

Tetani/Minute 5. Estimated cle and high-oxidative situ (see text). FIG.

tension for low-oxidative fast-twitch red muscle

fast-twitch white musduring contractions in

fairly distinct muscle fiber populations have inherently different blood flow capacities (18, Zl), mitochondrial contents (2, 6), and fatigue characteristics (4). Thus the composite force development that we measured must reflect the mass-averaged response of a heterogeneous mass of muscle fibers. While it is difficult to measure the response of individual fiber types during muscle stimulation in situ, it is possible to estimate the probable response of the low-oxidative and high-oxidative muscle components after introducing a couple of assumptions. These predictions may offer insight as to where adaptations may occur within the muscle. As discussed previously (l3), if the initial decline in force development is assigned to the fast-twitch white motor units (4), up to the limit of their mass composition within the mixedfibered whole muscle (I), the performance characterized in Fig. 5 for sedentary and trained groups would be expected.’ Accordingly, the expected response for the fast-twitch white muscle for each group exhibits a more accelerated rate of fatigue when compared with the response for the corresponding mixed-fibered muscles illustrated in Fig. 2. Furthermore, if the response illustrated for fast-twitch white is correct, then the response for the remaining 25% of the muscle mass identified as fast-twitch red2 in Fig. 5 would occur to obtain the measured performance of the entire mixed-fibered muscle mass (Fig. 3). Thus reasonable estimates of the responses of the low- and high-oxidative muscle sections can be made by apportioning the measured response obtained from the entire mixed-fibered muscle group. It is evident from the comparisons illustrated in Fig. 5 that the primary alteration with training is expected to have occurred in fast-twitch white muscle. Expected ’ Assigning the initial fatigue to the fast-twitch white muscle seems justified, since these motor units exhibit exceptionally poor performance characteristics, relative to the high-oxidative motor units, during tetanic contractions (cf. Ref. 4). Thus force development of the fasttwitch white muscle is expected to approach zero (100% fatigue), enveloping all of its muscle mass as fatigue of the whole muscle proceeds from 100 to 25% of initial. ’ For simplicity the small 5-10% mass contribution of the slowtwitch red muscle fiber type is considered to respond similar to that of the high-oxidative fast-twitch red.

.rlE tif El

6 5 4 3 2

Fast-Twitch

0

15

30

45

60

White

75

90

Tetani/Minute FIG. 6. Estimated 0, consumption for white muscle and high-oxidative fast-twitch tractions in situ (see text).

low-oxidative red muscle

fast-twitch during con-

force development is much greater in the trained fasttwitch white muscle compared with that of sedentary. If this characterization of the fiber-specific responses is correct, then the corresponding differences in oxygen consumption illustrated in Fig. 6 are expected between trained and sedentary muscle, since the oxygen cost of force development was not different between groups.” Thus the difference in oxygen consumption between trained and sedentary groups actually measured for the mixed-fibered muscle is predicted to be due to the response of the fast-twitch white muscle mass. Interestingly, the response in the high-oxidative muscle mass is expected to be similar between groups, since our stimulation conditions were not sufficiently strenuous to require an inordinant energy expenditure. We cannot predict whether a functional improvement and increased peak oxygen consumption were introduced in the highoxidative muscle by this training program, since the stimulation protocol did not proceed to a high enough frequency to sufficiently challenge this high-oxidative muscle type. The greater peak oxygen consumption of the trained muscle, when oxygen delivery was the same as that to the sedentary muscle, implies that the blood-tissue exchange characteristics of the muscle were improved by training. Factors that influence blood-tissue oxygen exchange include mitochondrial volume and intracellular distribution, resistance to oxygen diffusion through the tissue, tissue capillarity, and flow dynamics through capillaries. Training is known to alter a number of these factors in a manner expected to provide benefit in oxygen exchange. For example, endurance-type exercise training increases muscle capillarity in normal individuals (cf. 27). Furthermore, this training adaptation appears to be ’ This assumes, of course, that the oxygen cost of contraction measured for the whole mixed-fibered muscle group represents the value for the fast-twitch white and fast-twitch red muscle. This assumption seems reasonable in view of the similar specific activities of myofibrillar ATPase (33) and of sarcoplasmic reticulum Ca”+ uptake (26) for sections of these muscle types in the rat.

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AND

PERIPHERAI

a general phenomenon, even found when peripheral arterial insufficiency is present. While we did not evaluate tissue samples for capillarity changes in this study, we have found that animals trained by the same exercise program, but for a shorter duration of 6-7 wk, exhibit a significantly increased muscle capillarity (25). Capillaryto-fiber ratio and the number of capillary contacts surrounding each fiber increased in the fast-twitch white muscle section of the gastrocnemius. This enrichment in capillary network should improve blood-tissue exchange of oxygen and thereby could have contributed to the observed greater oxygen consumption. In addition, an increase in mitochondrial content could serve to shorten the average diffusion path length within the muscle. The typical training response of an enhanced mitochondrial content found in muscle of normal individuals (15) and those with peripheral arterial insufficiency (5, 8, 20) was found in the present study. Citrate synthase activity, an enzyme marker for mitochondrial capacity, increased to nearly threefold that of sedentary in the fast-twitch white section of the trained gastrocnemius (cf. Table 4). Other increases, but of much smaller magnitude, were found in the high-oxidative fast-twitch and slow-twitch sections of the distal limb musculature. Interestingly, there is an excellent correspondence between where the muscle adaptive changes occurred and the physiological responses in our experiment. As discussed above, the fasttwitch white muscle is expected to contribute most to the observed increase in muscle performance and oxygen consumption. Similarly, the fast-twitch white muscle section exhibited the greatest adaptive response to training (cf. Table 4). Thus these design changes in muscle could have contributed to the improved oxygen consumption demonstrated by the trained muscle. Which factor(s) is responsible for the improved oxygen consumption, however, cannot be determined from the present study. Factors leading to the improved muscle performance and energy flux in our perfused hindlimb experiments, where oxygen delivery was high, were probably also operant within the ischemic muscle in vivo during treadmill running. Exercise tolerance was markedly improved by exercise training. Trained rats were able to run longer and at a higher treadmill speed (cf. Fig. 1). The faster running speed would require a higher rate of oxygen consumption by the working muscle (3). Evidence from indicates that this the perfused hindlimb preparation could have occurred by a greater extraction of oxygen from the arterial delivery. In addition, muscle performance could be even better supported if, as recently found (24), there was a redistribution of the limited flow within the limb to better perfuse the active muscle. If these adaptations are induced in patients that are physically active, they could account for the greater oxygen extraction observed across the working limb after training (28, 34). The greater oxygen consumption of the active muscle could, in turn, contribute to the enhanced exercise tolerance, even in the absence of any increase in total blood flow. The results of the present study indicate that peripheral adaptations, induced within the active muscle by exercise training, serve to increase aerobic capacity and

ARTERIAL

H451

INSUFFICIENCY

enhance muscle function. The data provide additional evidence in support of the recommendation that physical activity represents an important adjunct in the management of patients with peripheral arterial insufficiency (W . We gratefully acknowledge the excellent technical assistance of Judy Freshour and David Barrett. This study was supported by National Heart, Lung, and Blood Institute Grant HL-37387. Address for reprint requests: R. L. Terjung, Dept. of Physiology, SUNY Health Science Center, 766 Irving Ave., Syracuse, NY 13210. Received

23 April

1990; accepted

in final

form

1 October

1990.

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