Journal of Applied Physiology
Vol. 83, No. 1, pp. 67-73, July 1997
EXERCISE AND MUSCLE

Functional deficits in medial gastrocnemius grafts in rats: relation to muscle metabolism and -AR regulation

Lisa M. Larkin^1,2, John A. Faulkner², Richard T. Hinkle², Cheryl A. Hassett², Mark A. Supiano^1,2,3, and Jeffrey B. Halter^1,2,3

¹ Division of Geriatric Medicine, Department of Internal Medicine, and ² Institute of Gerontology, University of Michigan, Ann Arbor 48105; and ³ Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48109

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Larkin, Lisa M., John A. Faulkner, Richard T. Hinkle, Cheryl A. Hassett, Mark A. Supiano, and Jeffrey B. Halter. Functional deficits in medial gastrocnemius grafts in rats: relation to muscle metabolism and -AR regulation. J. Appl. Physiol. 83(1): 67-73, 1997.This study tested the hypothesis that alterations in the metabolic integrity of grafted muscle contribute to its diminished ability to sustain power. Compared with control muscles, muscles studied 120 days after the grafting procedure had lower specific force and sustained power. The sustained power protocol resulted in a depletion of muscle glycogen in control (83%) and grafted (85%) animals. Grafts had lower pre- and poststimulation glycogen, diminished citrate synthase activity, and greater hexokinase activity. No differences were observed in phosphofructokinase activity, glucose transporter GLUT-4 content, fiber type, -adrenergic-receptor (-AR) density, or binding affinity. Isoproterenol-stimulated adenylyl cyclase activity was lower in grafted vs. control muscle, suggesting an uncoupling of the -AR-effector complex. Thus the diminished ability of the grafted muscle to sustain power may be explained, in part, by a decrease in energy available from glycogen stores and/or a decrease in oxidative capacity.

sustained power; hexokinase; citrate synthase; glycogen; GLUT-4; fiber type; -adrenergic-receptor function

INTRODUCTION

GRAFTING OF WHOLE SKELETAL MUSCLES is used clinically to correct dysfunction or repair injuries to skeletal muscle. Dysfunction may arise from inherited or acquired diseases or from denervation resulting from direct trauma (19, 21). Although many of the morphological and physiological characteristics return to control values after standard (7, 10), standard with nerve implant (26), or nerve intact (6, 7, 10) models for grafting of whole skeletal muscles in rats, a deficit in the development of specific force and increased fatigability are observed regardless of the procedure used for grafting. Several alterations in the delivery and utilization of substrate in the muscle could explain the inability of grafted muscles to maintain levels of sustained power comparable to those of control muscles. A decrease in blood flow to grafted muscle could affect delivery of substrate needed for muscle activity. Despite the attractiveness of this hypothesis, blood flow studies have not demonstrated significant differences in the blood flow normalized by estimates of the viable muscle mass for grafted compared with control muscles (5).

Another mechanism that, if altered, could explain diminished metabolic capacity of skeletal muscle is decreased transport of glucose into the muscle. Decreased hexokinase activity and/or glucose transporter GLUT-4 content could lower glucose uptake and diminish substrate available to working muscle. In addition, decreases in initial intracellular energy stores of muscles such as ATP, creatine phosphate, and glycogen could limit the length of time a muscle is able to sustain power. An important mechanism for activation of muscle glycogenolysis during exercise is stimulation of muscle -adrenergic receptors (-ARs) (13). Thus diminished -AR responsiveness could limit work capacity in contracting skeletal muscle. Changes in the glycolytic and/or oxidative capacity of the muscle could also lead to diminished ability to produce energy needed to sustain power via the glycolytic pathway and tricarboxylic acid cycle.

In this study, we investigated several metabolic mechanisms for the diminished ability of grafted skeletal muscle to sustain power. We tested the hypothesis that the impaired ability of stabilized grafts of medial gastrocnemius muscles (MGN) to develop and sustain power would be associated with a diminished glucose transporter content, glycogen stores, -AR function, or activity of enzymes of both glycolysis and the tricarboxylic acid cycle.

METHODS

Animals and animal care. Twenty male Fischer 344 rats, 2 mo of age, were obtained from the National Institute on Aging animal colony maintained by Harlan Sprague-Dawley Laboratory (Indianapolis, IN). All 20 rats underwent the grafting procedure in which their left leg was used; the right leg remained unaltered and was later used as a paired control for the grafted leg for metabolic measures. Because of the inability to obtain viable contractile data from the contralateral leg, 14 age-matched animals (i.e., 6 mo old) were also obtained from the National Institute on Aging animal colony maintained by Harlan Sprague-Dawley Laboratory and used as controls for contractile measures. At 120 days after surgery, 10 of the grafted animals were used to determine contractile properties, whereas the remaining 10 grafted animals were used for determining metabolic properties. After the recovery period, three of the grafted muscles did not respond to stimulation, decreasing the number of animals for contractile properties in the grafted group to seven. Because of the duration of the contractile testing, reliable normalized power and sustained power data could not be obtained from grafted MGN muscle of all rats, decreasing the number of grafted animals for these measurements to eight in the control group and to six in the grafted group. Rats were acclimated to our colony conditions, i.e., light cycle and temperature, for 1 wk before surgical procedure. Rats were housed individually in hanging plastic cages (28 × 56 cm) and kept on a 12:12-h light-dark cycle at a temperature of 20-22°C. The rats were fed Purina laboratory rodent chow 5001 and were given water ad libitum.

Grafting procedure. All surgical procedures were performed in an aseptic environment in accordance with the "Guide for the Care and Use of Laboratory Animals," [DHEW Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205]. As previously described by Miller et al. (23), the tendon and nerve of the left MGN muscle were severed, and the muscle was grafted orthotopically with repair of the nerve and tendon. Briefly, animals were anesthetized with pentobarbital sodium (65 mg/kg). The left MGN was isolated from surrounding muscle and connective tissue, the distal and proximal tendons were severed, and the muscle was removed from its bed. The muscle was then placed back into its original position and repaired by using 8-0 Ethilon suture. The branch of the tibial nerve innervating the MGN was isolated, severed, and then repaired by using epineurial sutures of 11-0 Ethilon suture. Care was taken to leave the blood supply intact. The incision was closed in layers by using 4-0 Ethilon suture. The animals were allowed to recover for 120 days before contractile and metabolic properties were measured.

Surgical preparation for contractile measurements. After the 120-day recovery period, the animals were again anesthetized with pentobarbital sodium (65 mg/kg). Supplemental doses were administered as required to maintain an adequate depth of anesthesia. The MGN was isolated from surrounding muscle and connective tissue. A 0-0 silk suture was tied around the distal tendon, and then the tendon was severed. The distal tendons of the soleus, lateral gastrocnemius, and plantaris muscles were also severed to minimize their effect on subsequent force and power measurements. The animal was then placed on a Plexiglas platform, and body temperature was maintained with a heating pad. The hindlimb was secured by pinning the femur near the origin of the MGN and clamping the foot to the platform. The distal tendon of the MGN was then tied to a lever arm of a servomotor (Cambridge Technology, model 6650). The servomotor was used to measure force and also served to shorten the muscle during power measurements. Once the MGN muscle was secured to the platform, the tibial nerve was exposed and stimulated with a bipolar electrode (Harvard Apparatus) connected to a Grass model S88 stimulator. Data were viewed on an oscilloscope and analyzed using ASYST computer software (Keithley Instruments).

Contractile measurements. The distal tendon of the MGN was tied to the servomotor lever arm, and the muscle length was adjusted to the optimum length for development of maximum isometric force (2) . The optimum length was used to calculate the optimum fiber length (L_f). Maximum isometric tetanic force was determined by subjecting the muscle to periods of stimulation at increasing frequencies.

All power measurements were made during isovelocity shortening contractions through 10% of L_f, as previously described by Brooks and Faulkner (4). Briefly, contractions were initiated at 105% of L_f, and muscle was shortened to 95% of L_f. Stimulation of the nerve and initiation of the shortening ramp occurred simultaneously, and stimulation was terminated at the end of the shortening ramp. The integrated area under the force curve during only the shortening ramp was used to determine the average force developed during a contraction, and the power was calculated as the product of the average force during the shortening and the velocity of shortening (4). An increase in the shortening velocity increased the power until maximum was reached. The shortening velocity that resulted in maximum power was defined as the optimal shortening velocity.

Maximum sustained power was determined by using repeated isovelocity shortening contractions and gradually increasing the number of contractions per unit time (train rate) at a constant train duration. Duty cycle is defined as the product of the train rate (number of contractions per unit time in Hz) and train duration (in s). A stimulation frequency (100 Hz) and train duration (67 ms), which produced ~75% of maximum power in both the grafted and control muscle, was used for all tests of sustained power. Because the train duration was held constant at 67 ms for both the grafted and control muscles at each duty cycle, the train rate (number of contractions per unit time in Hz) was also identical for grafted and control muscles at each duty cycle. Sustained power was calculated as the product of the shortening velocity, the average force developed during shortening contractions at a given duty cycle, and the duty cycle. The train rate and, therefore, the duty cycle were increased every 5 min until the maximum sustained power was reached.

Histology. Muscle biopsies were taken from the middle of the MGNs from each of the animals used to measure contractile properties. Fourteen-micrometer-thick sections were cut in a cryostat and incubated at varying pH conditions to measure myofibrillar adenosinetriphosphatase (ATPase) activity. Muscle fibers were classified as type I and type II on the basis of myofibrillar ATPase activity at pH 9.4, as previously described by Brooke and Kaiser (1). Type II fibers were further subdivided into type IIa and IIb on the basis of myofibrillar ATPase activity at pH 4.6. The entire cross-sectional area of each MGN was digitized and each fiber type classified and its area calculated by using a microcomputer imaging device (Imaging Research, St. Catharines, Ontario).

Analytic procedures. Control and grafted MGN samples were analyzed for glycogen content (µmol/g muscle) via the method of Hassid and Abraham (12). Muscle homogenates (from a 700-g supernatant) were used to determine citrate synthase activity (µmol · µg protein¹ · min¹) by using the method of Serre (27). Hexokinase activity (units/mg protein) was measured by using the method of Easterby and Qadri (9). Phosphofructokinase activity (µmol · min¹ · mg protein¹) was measured by using the method of Mansour et al. (20). Protein content (mg/g tissue) was determined with a Bradford protein assay kit from Bio-Rad (Richmond, CA).

The protocol used for isolation of total membranes has been described previously by Klip et al. (18). The procedure used for Western analysis of GLUT-4 glucose transporter was a modified version of a method previously described by Kahn et al. (14). Modifications were as follows: duplicate samples from each animal were separated on a 10% polyacrylamide resolving gel and then eletrophoretically transferred onto Immobilon polyvinyl difluoride (PVDF) membrane (Millipore, Milford, MA). Immobilon PVDF membranes were then blocked for 60 min with tris(hydroxymethyl)aminomethane (Tris)-buffered saline with Tween-20 (100 mM Tris, 0.9% NaCl, 2.0 ml Tween-20, pH 7.5) and 5% Carnation nonfat dry milk. Immunoblotting was performed by using a rabbit polyclonal antibody against insulin-regulatable glucose transporter GLUT-4 protein (East Acres Biologicals, Southbridge, MA), followed by ¹²⁵I-labeled anti-rabbit immunoglobulin (Amersham, Arlington Heights, IL) and exposure of the transfer membrane to Kodak X-OMAT film at 80°C for 24 h. ¹²⁵I-labeled transfer membranes were then cut and counted on a gamma counter (Tm Analytic, Elk Village, IL). Total counts per minute were corrected for background, and then specific counts were normalized for the internal standard.

Norepinephrine content was determined by high-performance liquid chromatography with electrochemical detection, as described by Karlsson et al. (15). Alumina extracts were injected onto the column, and peak heights for norepinephrine (ng/g muscle) were compared with those produced by the internal standard 3,4-dihydroxybenzylamine.

Membrane preparations were analyzed for -AR density and antagonist binding affinity by using Scatchard transformation of [¹²⁵I]iodocyanopindolol (ICYP) -specific binding data. Briefly, 100-µg aliquots of membrane homogenates and seven concentrations of [¹²⁵I]ICYP (sp act 2,200 Ci/mmol; New England Nuclear, Boston, MA) spanning a range of 5-150 pM were incubated with and without 1 µM propranolol (to define nonspecific binding) for 90 min at 25°C in a total volume of 200 µl. Nonspecific binding averaged 25.04% of total binding, and linear Scatchard curves with r values >0.85 were obtained. Additional samples were then stored at 20°C until assayed for adenosine 3,5-cyclic monophosphate (cAMP) concentration by using a radioimmunoassay, as has been previously described (28).

Statistics. Values are presented as means ± SE. Statistical analysis was performed by using Statview 4.01 (Abacus Concepts, Berkeley, CA). One-way analysis of variance (ANOVA) (contractile properties) and Student's paired (metabolic properties) t-test were used to compare differences between grafted and control groups. The mixed procedure test was used when all data points could not be paired using the Student's t-test. A repeated-measures ANOVA was used to determine the effect of isoproterenol (ISP) dose on cAMP production. Differences were considered significant at P  0.05.

RESULTS

Contractile properties. Muscle mass (g) and cross-sectional area of the grafted muscle were not significantly different from control muscles (Table 1). Measurements of twitch force (N), and specific force (N/cm²) were significantly lower in the grafted muscles compared with values for control muscles (Table 1). In addition, power normalized for muscle mass (W/kg) and maximum normalized sustained power (W/kg) were significantly less in the grafted compared with control muscles (Table 1). At each duty cycle, normalized sustained power of the grafted was significantly less compared with values for control muscles (Fig. 1).

Table  1.   Contractile properties of grafted and control medial gastrocnemius muscle in 6-mo-old male Fischer 344 rats Control Grafted P Value %Control Body mass, g 371 ± 9  380 ± 8  0.5074 102 (n = 14) (n = 7) Muscle mass, mg 716 ± 14  720 ± 23  0.8794 101 (n = 14) (n = 7) CSA, mm2 55 ± 1  56 ± 2  0.6403 102 (n = 14) (n = 7) Maximum twitch 3.53 ± 0.2  2.34 ± 0.2  0.0009 66   tension, N (n = 14) (n = 7) Maximum tetanic 13.7 ± 0.2 10.6 ± 0.5 0.0001 77   tension, N (n = 13) (n = 7) Specific force, N/cm2 25.3 ± 0.2  19.0 ± 0.7  0.0001 75 (n = 13) (n = 7) Normalized power, 182 ± 3  110 ± 7  0.0001 60   W/kg (n = 12) (n = 6) Maximum normalized 4.8 ± 0.1  3.7 ± 0.3  0.0015 77   sustained power, (n = 8) (n = 6)   W/kg Values are means ± SE; n, no. of rats. Total cross-sectional area (CSA) of muscle fibers was estimated by dividing muscle mass by product of fiber length and 1.06 mg/mm3, the density of mammalian skeletal muscle.

Metabolic characteristics. MGN weight (g) and protein content (mg protein/g muscle) of the muscles used for studying metabolic parameters did not differ between the experimental groups (Table 2). Phosphofructokinase activity (µmol · min¹ · mg protein¹) and GLUT-4 content (%control) did not differ between experimental groups. Hexokinase activity (units/mg protein) was significantly greater (P = 0.0003), whereas citrate synthase activity (µmol · min¹ · mg protein¹) was significantly lower in the grafted compared with control muscles (P = 0.02; Table 2). Glycogen concentration (expressed as µmol/g muscle) was lower in both control (83%) and grafted (85%) muscles after stimulation with the sustained power protocol (Fig. 2; P = 0.0001). Both pre- and poststimulation glycogen concentrations were lower in the grafted compared with control muscles (P = 0.01).

Table  2.   Metabolic characteristics of grafted and control medial gastrocnemius muscle in 6-mo-old male Fischer 344 rats Control Grafted P Value %Control Muscle mass, g 715 ± 36  659 ± 21  0.1905 92 (n = 10) (n = 10) Protein content, mg 213 ± 11  221 ± 12  0.4921 104   protein/g tissue (n = 10) (n = 8) Hexokinase, units/mg 24.1 ± 4.7  58.8 ± 5.4  0.0003 244   protein (n = 8) (n = 7) GLUT-4 content, 100.0 ± 4.2  100.6 ± 4.8  0.9271 101   %control (n = 10) (n = 10) Phosphofructokinase, 1.70 ± 0.4  1.65 ± 0.4  0.4299 97   µmol · min1 · mg protein1 (n = 8) (n = 5) Citrate synthase, 181 ± 14  139 ± 8  0.0290 77   µmol · µg protein1 · min1 (n = 8) (n = 7) Values are means ± SE; n, no. of rats.

Histochemistry. The fiber type population distribution of the MGN did not differ significantly between the grafted and control muscles. The predominant fiber type was the type IIb fiber representing 76.5 ± 1.4 and 79.2 ± 0.2% of the total population of fibers, in control vs. grafted muscle, respectively. The remaining population consisted of type IIa fibers 14.4 ± 1.4 and 12.1 ± 0.7% and type I fibers 9.0 ± 0.3 and 8.7 ± 0.5%, in control vs. grafted muscle, respectively.

Adrenergic function. Norepinephrine content (ng/g muscle) was significantly lower in the grafted muscle compared with the control muscle (control muscles, 32.1 ± 3.4; grafts, 23.5 ± 2.1 ng/g muscle; P = 0.0005). Equilibrium binding studies using [¹²⁵I]ICYP in the presence and absence of 1.0 µM propranolol yielded saturable binding. Scatchard transformation of the specific binding data yielded linear plots from which receptor density and binding affinity were determined. Neither -AR density (control, 31.9 ± 2.4; grafted, 35.0 ± 2.6 fmol/mg protein) nor antagonist binding affinity (control, 15.5 ± 1.0; grafted, 15.1 ± 1.0 pM) was different in the grafted compared with control muscles.

By repeated-measures ANOVA, there was a significant dose-dependent increase in ISP-stimulated adenylyl cyclase activity in both the grafted and control muscles (P < 0.0001), indicating an appropriate response to -AR stimulation. Despite the similarity in the response to increasing dose, as shown in Fig. 3, there was a significant effect of grafting on ISP-stimulated adenylyl cyclase activity, with the grafted muscle having lower activity at all doses of ISP compared with control muscle (P = 0.04; by repeated-measures ANOVA). In addition, there was a significant interaction between the response to increasing ISP dose and the surgical treatment (P = 0.03); as the dose of ISP increased, the percent difference in adenylyl cyclase activity between the grafted and control muscles increased. Consequently, at the maximal dose of ISP (10⁴ M), adenylyl cyclase activity was 65% lower in the grafted compared with control group (Fig. 3). Neither basal (unstimulated) nor adenylyl cyclase activity stimulated via sodium fluoride activation of G_s protein revealed any significant differences between the grafted and control muscles (Table 3). Direct stimulation of adenylyl cyclase activity with forskolin tended to cause a greater increase in adenylyl cyclase activity in grafted compared with control muscles, although the increase was of borderline statistical significance (P = 0.055; Table 3).

8

6

4

Table  3.   Adenylyl cyclase activity of grafted and control medial gastrocnemius muscles in 6-mo-old male Fischer 344 rats Control Grafted P Value %Control Basal, pmol/mg protein 665 ± 34  576 ± 48  0.1478 87 NaF, %AC stim above basal 353 ± 23  311 ± 13  0.1178 88 Fsk, %AC stim above basal 321 ± 17  372 ± 18  0.0550 116 Values are means ± SE; n = 9 rats in each group. AC stim, adenylyl cyclase stimulation; Fsk, forskolin.

DISCUSSION

In accordance with previous studies in the rat (6, 7, 10, 26), the present study on stabilized MGN muscle grafts demonstrated a 25% decrease in specific force and a 23% decrease in normalized sustained power compared with control muscle. The present study extends these previous observations by assessing metabolic characteristics in the grafted MGN muscle, which could contribute to the diminished ability of the grafted muscle to sustain power compared with control muscle. Because of limited availability of muscle tissue, this study limited its findings to carbohydrate metabolism. Hexokinase activity was significantly increased, and GLUT-4 glucose transporter content was not significantly altered by the grafting procedure, suggesting that reduced glucose uptake or metabolism may not be a limiting factor in muscle function in these studies. Grafted muscles had a lower prestimulation glycogen concentration, suggesting possible impaired storage of glycogen and decreased energy available from glycogen during sustained power. The diminished ability of the grafted muscle to develop force and sustain power may also be explained, in part, by a shift to a more anaerobic (glycolytic) metabolism. We observed a significant decrease in citrate synthase activity of the grafted muscle, which is compatible with a reduction in oxidative capacity. In addition, we observed no significant alteration in the key regulatory enzyme of glycolysis, phosphofructokinase. Because during a sustained power protocol the muscle depends on a combination of aerobic and anaerobic metabolism, any decrease in aerobic capacity would decrease the ability of the muscle to sustain power.

Previous studies have shown that the recovery of muscle functionality in grafted muscle is associated with the degree of reinnervation and revascularization of the muscle after the grafting procedure (11). Incomplete revascularization cannot explain the lower resistance to fatigue in the grafted MGN muscle, since the vasculature was left intact during the surgical procedure. On the other hand, the nerve to the MGN muscle was severed and repaired during the grafting procedure, so denervation could be a contributing factor. One study has shown that disuse of skeletal muscle due to denervation leads to a decrease in the oxidative capacity of skeletal muscle (25). We found that citrate synthase activity, an indicator of oxidative capacity in skeletal muscle, was 23% lower in the grafted compared with control muscle. Therefore, the diminished ability of the grafted muscle to resist fatigue and sustain power may be due, in part, to incomplete reinnervation of the MGN muscle, leading to disuse and subsequent decreased activity of enzymes associated with oxidative metabolism. Further studies are needed to determine the completeness of the reinnervation of grafted MGN muscles and to test this hypothesis.

In physiological conditions in which low oxidative capacity exists, such as disuse atrophy after denervation, skeletal muscle will utilize glycogen stores as the major fuel for energy (22). However, glycogen stores become rapidly depleted and are thus unsuitable for sustained contractile activity. Chronic stimulation of untrained skeletal muscle leads to a transient increase of glucose uptake and glycolysis to compensate for the rapid depletion of available glycogen stores. To compensate for the increased need for glycolysis from free glucose, the muscle transiently increases the content and activity of hexokinase (30). With recovery from denervation, oxidative enzymes increase and the muscle shifts to a more oxidative energy metabolism. In the present study, both glycogen content and oxidative capacity were significantly decreased and hexokinase activity was increased 2.4-fold in the grafted muscle compared with control. GLUT-4 protein content, which has been shown to be a predominant determinant of the capacity for glucose uptake in skeletal muscle (17), did not significantly differ in grafted compared with control muscles. In addition, the activity of phosphofructokinase, the key regulatory enzyme for glycolysis, was unchanged after the grafting procedure, suggesting no impairment in the glycolytic pathway. These data suggest that the stabilized grafted MGN muscle has shifted toward glycolytic metabolism and is likely relying on glucose uptake from the circulation as well as on its glycogen stores as the major sources of energy. The similarity of these metabolic findings to those found in atrophied muscle suggests that disuse of the grafted muscle may contribute to the functional changes observed.

In accordance with these data, a study done by Cote et al. (8) demonstrated that oxidative capacity was decreased in nerve-reimplanted grafts in soleus muscle. In this muscle, however, glycogen stores were not different, and there was no observable difference in resistance to fatigue (8). The differences between these two studies may be explained by the difference in predominant fiber type in the two muscles studied. The MGN is a muscle with predominately fast-glycolytic (type IIb) fibers (Table 3), whereas the soleus is predominately a slow-oxidative muscle (type I) fibers. The ability to sustain power is greatest in muscles having the greatest percentage of fast-oxidative glycolytic (type IIa) fibers, followed by slow-oxidative (type I), followed by fast-glycolytic (type IIb) fibers (3). In addition, slow-oxidative fibers have a greater ability to spare glycogen during a metabolic challenge than fast-glycolytic fibers. Therefore, the soleus muscle having the greatest ability to sustain power and spare glycogen before grafting appears to be less affected by the surgical procedure and retains the ability to sustain power and spare glycogen after the grafting procedure.

The recovery of a muscle's ability to produce specific force is directly related to the number of innervated muscle fibers (11). Therefore, it may be possible to use the percentage decrease in specific force generated by the grafted muscle as a way of estimating the percentage decrease in innervated muscle fibers. This estimation may be used to correct other contractile data for the decrease in innervated muscle fibers. For example, since the specific force of the grafted muscle is 75% of the specific force produced by the control muscle, we could estimate a 25% decrease in innervated fibers in grafted muscle. Sustained power in the grafted muscle was decreased by 23% in the grafted compared with control muscle. Thus the deficit in the ability to sustain power may be explained entirely by a 25% decrease in the percentage of innervated fibers after the grafting procedure. Because normalized power, on the other hand, decreased by 40% in the grafted compared with the control muscle, correction for the number of innervated muscle fibers does not entirely explain the observed deficit. However, this approach to estimation of the potential impact of decreased innervation on metabolic parameters is problematic. There may be a population of noninnervated fibers that do not contribute to the development of specific force but will still contribute to the overall metabolic properties of the muscle. Further studies need to be done to determine whether the metabolic status of completely denervated MGN muscle fibers after this grafting protocol can fully explain the metabolic changes in a regenerated muscle based on the percentage of innervated vs. denervated muscle fibers.

Sustained power, which depends mainly on aerobic metabolism, relies on the usage of intracellular glycogen stores for energy. The sustained power protocol substantially depleted MGN glycogen content. Therefore, a change in the regulation of glycogenolysis might also contribute to the decreased ability to sustain power in the grafted muscle. In this study, there was significantly less -AR-stimulated adenylyl cyclase activity in the grafted compared with control muscle. In contrast, there was no significant difference in the ability of sodium fluoride to increase the accumulation of cAMP via G_s-protein stimulation of adenylyl cyclase between grafted and control muscle. These data suggest that the -AR is uncoupled from its effector enzyme, adenylyl cyclase, in the grafted muscle. Such uncoupling could possibly have contributed to diminished utilization of glycogen. However, since both the grafted and control muscles depleted their glycogen stores by approximately the same percentage during the sustained power protocol, it does not appear that the degree of uncoupling observed in the grafted muscles affected the muscles' ability to utilize the available glycogen stores during the sustained-power protocol.

Desensitization of the -AR-effector complex due to chronic exposure to increased norepinephrine concentrations is one possible explanation for the -AR findings in our study (29). Norepinephrine content in the grafted MGN muscle was decreased rather than increased. One explanation for diminished norepinephrine content of the grafted MGN tissue could be an increase in the rate of norepinephrine release and a depletion of norepinephrine stores in peripheral nerve terminals (16, 24). Because norepinephrine is stored in the nerve terminals, the observed decrease in norepinephrine content may also indicate incomplete reinnervation of the grafted MGN muscle and an increase in percentage of denervated fibers. Again, further studies are needed to determine the completeness of the reinnervation of grafted MGN muscles as well as the dynamics of norepinephrine metabolism.

In conclusion, the results of this study suggest that the deficits in the contractile properties of the grafted muscle cannot be explained by a decrease in muscle mass or by changes in the distribution of muscle fiber type, which did not significantly differ between the grafted and control muscles. We hypothesize that diminished muscle glycogen stores and/or a decrease in enzymes of oxidative metabolism may contribute to the diminished ability of grafted muscle to produce force, power, and sustained power.

ACKNOWLEDGEMENTS

The authors thank Eric Leiendecker, Bahaa Qasawa, and Marla Smith for technical assistance and Andrzej Galecki for his statistical advice.

FOOTNOTES

Address for reprint requests: L. M. Larkin, Geriatrics Center, MSRB3, 6301, 1150 West Medical Center Dr., Univ. of Michigan, Ann Arbor, MI 48109-0642.

Received 11 October 1996; accepted in final form 11 March 1997.

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