Effects of intermittent ischemia on contractile properties and myosin isoforms of skeletal muscle


Medicine & Science in Sports & Exercise:
Basic Sciences: Original Investigations

Effects of intermittent ischemia on contractile properties and myosin isoforms of skeletal muscle. Med. Sci. Sports Exerc., Vol. 30, No. 6, pp. 850-855, 1998.

Purpose: This study determined the effects of intermittent ischemia on the contractile properties, fatigue (Tf), and myosin heavy chain composition (MHC) in the rat gastrocnemius-plantarissoleus muscle (GPS) complex.

Methods: Fifty rats were divided into four groups: control (C, N = 12), severed (femoral artery) (S, N = 12), exercise (E, N = 13), and severed/exercise (SE, N = 13). Ischemia was elicited only in the SE group by daily exercise and the other groups served as controls. Exercise in the E and SE groups consisted of running on a treadmill ∼ 35 min·d−1, 5 d·wk−1 for 7 wk.

Results: Body weight, muscle weight, and absolute force were less in the SE group compared with those in C (12, 18, and 12% respectively). However, relative force (N·g−1 of muscle) was greater in the SE group compared with that in C (8%). Maximal shortening velocity (Vmax) was lower in the SE group compared with that in all others (10-14%). Tf was less in the S group compared with that in C and E (28 and 30%, respectively). Type IIx MHC increased and type IIb decreased in gastrocnemius and plantaris muscles in SE compared with those in C.

Conclusions: These data indicate that intermittent ischemia caused a decrease in muscle mass, maximal force development, and Vmax, but had no effect on Tf. The decrease in Vmax may have been related to myosin alterations in the muscles.

Author Information

Department of Exercise and Sports Sciences, University of Florida, Gainesville, FL

Submitted for publication April 1997.

Accepted for publication December 1997.

Address for correspondence: Stephen L. Dodd, Ph.D., Department of Exercise and Sports Sciences, #118 FLG, University of Florida, Gainesville, FL 32611.

Article Outline

Several cardiovascular diseases such as peripheral vascular disease (7) and chronic heart failure (10) may cause intermittent ischemia of skeletal muscle during contractions. A substantial amount of literature describes the vascular and metabolic adaptations that take place to allow skeletal muscle to cope with this alteration in blood supply (1,7,8,14). However, little information exists that describes the extent of adaptation of muscle contractile elements to intermittent ischemia. This is important since chronic ischemia causes significant weakness and debilitation.

Indeed, the functional consequences of ischemia are significant. Although the decrease in endurance of ischemic muscle has generally been attributed to vascular and metabolic changes in the muscle, it is conceivable that the loss of muscle mass and total force generating capacity plays a role as well. Lipkin et al. (19) have shown that chronic heart failure patients had quadriceps force development that was 55% of that predicted for body weight. This loss of absolute force generating capacity would cause the muscle to operate nearer to its maximal force-generating capacity for a given absolute power output. Consequently, the changes in skeletal muscle metabolism that are associated with fatigue might be expected to occur at lower absolute workloads and thus limit endurance of the muscle.

The possibility that muscle protein turnover may be altered is suggested by the findings of several studies that have shown a decrease in the size of both type I and type IIb fibers during chronic ischemia (7,8,30). Whether intermittent ischemia causes the same adaptation is unknown. Interestingly, in studies using patients with unilateral occlusions (7,8), it has been shown that fiber area was decreased in ischemic muscle when compared with that in the nonischemic contralateral muscle. This suggests that ischemia per se, and not disuse, is the major factor in causing the fiber atrophy.

There are conflicting reports as to the possibility of fiber type changes resulting from ischemia (7,8,14,27,30). Much of the confusion is likely because these investigations have used qualitative methods to classify fibers and, in addition, were not able to detect the most recently isolated fiber type, the IIx fiber (23,24). A newer method of classification is based upon the use of gel electrophoresis to separate the myosin heavy chain (MHC) isoforms in muscle. This technique now provides three subclassifications of type II fibers (type IIa, IIx, and IIb) (23,24). The implication of changes in fiber type is important since the type IIx fiber has been shown to have functional characteristics that lie between those of type IIa and IIb fibers (23). Therefore, if there is an altered distribution within the subclassifications of type II fibers, there may also be a corresponding alteration in the speed of shortening of muscle.

Therefore, the purpose of this study was to determine the effects of intermittent ischemia on functional properties and myosin isoforms of limb muscles. To accomplish this goal, we used a rat model which creates ischemia to lower limb muscles during exercise but not at rest.

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Male Sprague-Dawley rats (120 d old) were fed rat chow (Purina, St. Louis, MO) and maintained on a 12-h light/dark photoperiod for 7 d before the beginning of these experiments. During this 7-d period, animals were handled daily to prevent a stress hormone induced reduction in body weight at the beginning of the experiments. These experiments were approved by the university's animal review board and conformed to the policy statement of the American College of Sports Medicine on research with experimental animals.

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Experimental Design

After 7 d of handling, the rats were randomly assigned to a control (C, N = 12), severed (S, N = 12), exercise (E, N = 13), or severed/exercise (SE, N = 13) group for 7 wk of study. The C rats received a sham operation and were confined to cages and given water and rat chow ad libitum. The S and SE groups had the left femoral artery double-ligated and severed. The E rats received a sham operation and, after a 48 h recovery period, the E and SE groups began a program of running on a treadmill (∼35 min·d−1, 5 d·wk−1 for 7 wk). The exercise program began at a low work intensity (15 m·min−1/level treadmill) and the intensity was increased over 2 wk until the animals could maintain an intensity that has been shown to induce training adaptations in limb muscle {25 m·min−1/15° treadmill grade} (18). The E group began and progressed at the same exercise frequency, intensity, and duration as the SE group. Body weights of the animals were measured daily.

The rationale for the experimental design is as follows: The treatments were designed to cause intermittent ischemia in the SE group only. There is substantial evidence that severing the femoral artery does not cause ischemia at rest in these muscles. First, several studies (6,16,25) have shown that this procedure does not cause a significant decrease in blood flow at rest because a significant number of collateral and re-entrant vessels exist. Second, we collected pilot data before these experiments that confirmed that resting blood flow was unaltered.

Only during the increased demand for blood flow during exercise is the muscle ischemic. Thus, only the SE group experienced the intermittent ischemia during these experiments. During exercise of the magnitude used in these experiments where the demand for blood flow is great, femoral artery ligation causes a reduction in muscle blood flow (∼60%) and PO2 (∼50%) in the rat hindlimb for 7-10 wk (1,22).

The intent of this study was not to describe the adaptations to various levels of ischemia but to examine the qualitative changes in the affected muscles. Evidence that muscles were ischemic during training in the SE animals is shown by the fact that muscle adaptations occurred in the SE group that were unique compared with those in the C, S, and E groups.

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Surgical Preparation

After pentobarbital anesthesia, a small incision was made just distal to the inguinal ligament and the left femoral artery was isolated by blunt dissection. Two ligatures were placed tightly around the vessel and it was cut between the ties. Topical antibiotic powder was placed on the wound before closure with sutures. The C and E animals underwent a "sham" surgery with the femoral artery left intact. All animals survived this procedure (N = 25) and recovered with no side effects within 24 h.

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Muscle Preparation

After the treatment period, the animals were anesthetized with 30 mg·kg−1 sodium pentobarbital intraperitoneally, and after a surgical plane of anesthesia was reached, the animals were ventilated through an endotracheal tube with room air. The GPS muscle complex of the left hindlimb was prepared in situ for stimulation.

The muscle group was dissected free from overlying muscles and surrounding connective tissue. Bone pins were put into the tibia and femur and fastened to a metal post attached to a myograph to prevent movement. The calcaneal tendon was attached to an isotonic force transducer (Cambridge model 310, Watertown, MA) with a lightweight chain. The transducer output was amplified and differentiated by operational amplifiers and underwent A/D conversion for analysis with a computer based data acquisition system (GW Instruments, Somerville, MA). The sciatic nerve was carefully placed in a bipolar electrode connected to a square wave stimulator (Grass Instruments, model S48, Quincy, MA). The preparation was moistened with saline and covered with saline-soaked gauze and plastic wrap. The temperature of the preparation was monitored throughout the experiment with a mercury thermometer and maintained near 37°C by means of a heating lamp.

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Measurement of Contractile Properties

After 30 min of equilibration, the muscle complex was stimulated using a protocol that had been determined in a previous study (9) to elicit maximal force (6V/0.2 ms duration/100 ms train/100 Hz). The muscle was repeatedly stretched and stimulated until the maximal isometric tetanic force was obtained (Lo), and the muscle was maintained at this length throughout the experiment. This was done to standardize the data collection procedure and was not meant to represent Lo of each muscle. An afterload resistance was placed on the ergometer at a minimum of eight different loads (over the range of approximately 2-100% of maximal tetanic force) and the muscle stimulated to contract at each load. Force production and velocity of shortening were determined at each load. Velocity of shortening was computed as a function of the distance shortened by the muscle over time at each load. An iterative routine using a least squares technique was used to fit the Hill equation (15) to the data points determined for each animal. Maximal shortening velocity (Vmax) was determined for each animal by solving the equation for velocity when force equaled zero.

After measurement of the isotonic contractile properties, the rate of fatigue was determined for each group. Using the aforementtioned stimulation parameters, the muscle was stimulated at a rate of 1 contraction per second. The muscle complex used in these experiments is composed of predominantly fast, fatigable fibers, and contractions at maximal power output causes fatigue in < 30 s. Thus, the afterload on the muscle was set to elicit approximately 50% of maximal power output (∼30% of Po). Maximal power output was determined using the data from the force/velocity curve. Therefore, the test was designed for each muscle to work at a given percentage of its maximal force generating capability. Fatigue was defined as the time taken to reach 50% of the initial power output obtained.

Following the contractile measurements, animals were euthanized by an overdose of pentobarbital. The GPS complex was removed and each muscle trimmed of excess fat and connective tissue, weighed, and frozen at −80°C until assayed.

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Enzyme Assay

The entire gastrocnemius muscle was assayed for activity of citrate synthase (CS). This enzyme activity was interpreted as a relative measure of the capacity of the TCA cycle.

Before homogenization, excised muscle was quickly inspected for excess fat and tendon in ice-cold rat Ringer solutions and minced. Homogenization included a 15-s treatment with a tissue homogenizer (Ultra-Turrax T25, IKA Works, Cincinnati, OH) followed by 10 passes of the homogenate in a tight-fitting Potter-Elvehjem homogenizer at 0-3°C. After completion of homogenization, the homogenates were centrifuged (3°C) for 10 min at 400g. The supernatant was decanted and assayed to determine CS. All tissue samples were homogenized in cold 100 mM phosphate buffer with 0.05% bovine serum albumin (1:20 wt·vol−1; pH 7.4). CS activity was determined as described by Srere (26). All samples were assayed in duplicate at 25°C. To reduce interassay variability, an equal number of samples from all experimental groups were assayed on the same day.

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Myofibrillar isolation technique. Approximately 100-200 mg of tissue was minced on a chilled petri dish. Precisely 100 mg of tissue was added to 2 mL of chilled solution containing 250 mM sucrose, 100 mM KCL, 5 mM EDTA, and 20 mM Tris at pH 6.8. The tissue was then homogenized and centrifuged (10 min at approximately 1000 × g) and the supernatant removed. The pellet was resuspended and this procedure performed two additional times using 2 mL of a solution containing 175 mM KCL, 0.5% Triton-X, and 20 mM Tris at pH 6.8. Two final spins were made with a solution containing 175 mM KCL and 20 mM Tris at pH 7.0. After the final spin, the pellet was suspended in 500 μL of the solution containing 175 mM KCL and 20 mM Tris at pH 7.0 and yielded a myofibrillar protein pellet of sufficient purity for quantitative assessment of myofibrillar concentration (4).

Myosin heavy chains. Myosin heavy chain (MHC) composition in the gastrocnemius, plantaris, and soleus muscles was determined using one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-Page) as described by Talmadge and Roy (28). Briefly, after determination of protein content of the myofibrillar homogenate using the Bradford method (2), the homogenate was diluted (∼0.25 mg·mL−1) in sample buffer (62.5 mM tris, 1.0% SDS, 0.01% bromophenol blue, 15% glycerol, 5% §-mercaptoethanol, pH = 6.8). One to 3 mg of protein were loaded onto 22-cm long vertical gels (Biorad Protean IIxi) and electrophoresed for 20 h at ∼ 5°C using SDS-Page (4% stacking - 8% separating gel). Gels were stained with Coomassie blue R-250 and destained with a 30%/7% methanol and glacial acetic acid solution. The relative concentrations of myosin heavy chains were determined by scanning the gels using a computerized image analysis system. In our laboratory, the coefficient of variation for repeated measurement of relative MHC composition of a given sample is < 3%.

Statistical analysis. Separate one-way ANOVA's were used to determine differences among groups for each of the variables measured. Post-hoc differences were determined with Newman-Keuls test. Significance was established at the P < 0.05 level.

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Table 1 illustrates the changes in body and muscle weights with each of the treatments. Since animals were randomly assigned to groups, the body weights were not different among groups at the beginning of the study. At the end of the study, the mean body weight for the SE and E groups were significantly less than C (12 and 9%, respectively) while S was not different from C. The muscle weights for the SE group were 18% less than those in the C animals. In addition, it is noteworthy that muscle weights in the SE group were significantly less than those in the E group.

Absolute force developed in the SE group was 12% less than in C while S and E were not different from C (Table 2). It is noteworthy that force developed in the SE group was 13% less than in the E group. However, relative force (force per gram of muscle mass) in the SE group increased 8% compared with that in the C, S, and E groups. Maximal shortening velocity (Vmax) in the SE group was significantly decreased compared with that in C (12%) while S and E were not different from C. Time to fatigue (Tf) was 28% less in the S group compared with that in C while SE was not different from C. Fatigue time in the E group was significantly greater than both C and S. The CS activity in the E and SE groups was significantly higher than either the C or S groups.

As shown in Table 3A, composition of type IIx MHC in the gastrocnemius muscle was 32% greater in the SE group than in C while S and E were not different from C. Concomitantly, type IIb MHC composition in the SE group decreased significantly compared with that in C. There were no differences in type I or IIa MHC composition between groups. The same changes were seen in the plantaris muscle between SE and C in the IIx and IIb fiber distribution (Table 3B). The soleus muscle exhibited a 7% increase in type I MHC composition with a concomitant decrease in type IIa in the SE group compared with that in the C and E groups (Table 3C).

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The findings of this study hinge on two aspects of the model. First, ligation of the femoral artery must not produce ischemia at rest in the muscles studies (S and SE groups). In that regard, our pilot data and several previous studies (6,16,25) clearly indicate that resting blood flow is not changed. In addition, our observations of the animals in the S group indicated that there were no signs of tissue discoloration or hampered movement in the limbs with ligated arteries. Finally, the S group showed no alterations in contractile properties or MHC isoform composition as was found in the SE group. Thus, it is clear that the S group was in no way different from the C group.

Second, ischemia must be produced in the group of exercising muscles that have severed femoral arteries (SE group). Two points can be made in support of this condition. First, several studies have shown that femoral artery ligation, like that used in this study (1,22), or femoral artery stenosis (20) causes muscle ischemia during exercise (∼60% reduction in blood flow). Second, the SE group experienced adaptations to muscle (a decrease in muscle weight, force production, Vmax, and type IIb and IIx MHC) that did not occur in the S or E groups. This indicates a fundamental difference in the adaptation stimulus between these groups which, by design, could only have been ischemia. Therefore, it is clear that blood flow limitation occurred only during exercise in the SE group.

Several findings of this study are unique and add to our knowledge of how muscle adapts to intermittent ischemia. Although the maximal force production by skeletal muscle is reduced after intermittent ischemia, the relatively greater reduction in muscle mass dictates that the force per gram of muscle actually increased. The finding that intermittent ischemia causes a decrease in Vmax suggests that alterations may occur in the contractile elements of muscle. This is supported by the alterations in MHC isoform composition.

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Muscle Mass and Force Production

Our finding of lower muscle weights in the SE group compared with those in all other groups is supported by other studies that have shown whole muscle (30), as well as individual fibers (8,19,27,30), atrophy during ischemia. The cause of muscle atrophy and weakness during chronic ischemia has been postulated to result from the chronic disuse of muscle (5,31). While it is clear that disuse of muscle results in an increased turnover of protein and atrophy (29), several lines of evidence support the involvement of other mechanisms during ischemia. First, two studies (7,8) have examined muscle from both legs in humans with unilateral ischemia and found that ischemic muscle exhibits greater atrophy of fibers than the contralateral, normal leg. This finding suggests that disuse of muscle is not the sole determinant of muscle fiber adaptive response. The findings of the present study also support the notion that the reduced blood flow per se is responsible, in part, for the adaptive response of muscle to ischemia.

How could ischemia result in increased protein turnover in muscle? Potentially, ischemia could alter protein turnover by altering either protein synthesis, protein degradation, or both. One of the most noted effects of ischemia, or more specifically hypoxia, is its effect on the energy state of muscle. Protein synthesis has been shown to be related to the energy state of muscle (3,11,13). Thus, if muscle is ischemic and deficient in ATP production, protein turnover may be increased. The finding of a lack of increase in body weight in the E and S/E groups is likely a function of the generally accepted effects of exercise training on body weight (17).

The unique findings in this study are those related to the changes in contractile properties of muscle. The finding that the maximal force production in the S/E group was significantly less than that in the other groups can be attributed to the reduction in muscle mass. Indeed, it is generally accepted that force is determined by the number of activated cross-bridges in parallel and, therefore, is less in an atrophied muscle than in controls. The fact that muscle weight decreased to a greater extent than did force production resulted in the muscle producing a greater relative force (force per gram of muscle) in the SE group compared with that in C.

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Maximal Shortening Velocity and Myosin Isoforms

The possibility that ischemia causes adaptations in muscle that may affect shortening velocity is suggested by the findings of several studies that have shown an alteration in muscle fiber type resulting from chronic ischemia (14,27). However, the effects on shortening velocity have not been previously examined. The data from this study indicate that the Vmax of muscle is decreased with intermittent ischemia. The most likely explanation for this change is an adaptation in the fiber type of the muscle. In that regard, the findings of this study are most similar to the findings of Hammersten et al. (14) who used patients with intermittent claudication to show a shift from type IIb toward more type IIa fibers. Since we have used a different technique to classify the fibers, we have shown that the shift away from the type IIb fibers in the gastrocnemius and plantaris (∼95% of the muscle mass of the complex studied) results in an increase in the IIx fiber which is undetectable with the methods used by Hammersten et al. (14). This fiber is intermediate in function between the IIa and IIb fibers (23,24). In addition, the shift in soleus from type IIa to type I MHC was significant and likely contributes to a slowing of Vmax. Thus, the alterations in contractile characteristics shown in this study are likely determined by the fiber type changes in these muscles.

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The effects of intermittent ischemia on fatigue are varied and probably depend on type of muscle involved, severity of ischemia, and amount and intensity of use of the muscles. The finding of an increased fatigability of the muscles in the S group was not surprising. Maximal blood flow during exercise, or during maximal stimulation as used in this study, does not return to normal (1,22). Because they were kept sedentary, these muscles did not have the stimulus to adapt to exercise and/or ischemia as did the other experimental groups.

In contrast, the E group, which had unrestricted flow, adapted to the increase demands of exercise by increasing resistance to fatigue. This was also expected based on numerous studies that have shown an increase in endurance with exercise training (12,20,21,31).

The finding that fatigue of the SE group was not different from that in the C group supports the findings of several studies showing that intermittent ischemia results in vascular and histological changes in muscle that act to increase endurance (20,31).

In support of the fatigue data are the changes found in the relative activity of CS. CS is an important oxidative enzyme of the tricarboxylic acid cycle and has been shown to be indicative of the adaptive response to both chronic ischemia (31) and exercise training (21) in the rat. With exercise training, the increased oxidative capacity of muscle (increased CS activity) has been associated with an increase in the muscle's ability to use fat as a substrate, thereby sparing glycogen and delaying fatigue (12).

It is not surprising that CS did not change in the S group. Although maximal blood flow has been shown to be restricted in this condition (6,16,25), this group remained at rest throughout the 7 wk of the study and resting flow has been shown to be sufficient for tissue demands (6,16,25). Therefore, there was not sufficient oxidative stress to cause the muscle to adapt (e.g., increase CS activity). Likewise, it was not surprising that CS increased in the E and SE groups (21,31).

In summary, the findings of this study indicate that intermittent ischemia results in atrophy of skeletal muscle and a decrease in absolute force production. This alteration appears to result from ischemia and not simply from disuse of the muscle. The finding that intermittent ischemia causes a decrease in Vmax suggests that contractile elements within the muscle are altered. The shift from type IIb fibers to type IIx fibers supports this finding. Thus, the combination of a decrease in force and speed of shortening dictate that maximal power output of the muscle is greatly reduced by intermittent ischemia. These findings suggest that much of the debilitation to muscle under these conditions results from ischemia-induced intrinsic changes to the contractile apparatus.

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