Muscle energetics in immunosuppressed patients

Muscle Energetics in Immunosuppressed Patients M. Francaux, L. Versieux, P. Van Hecke, and P. Cordier

D

ECREASE in plasma phosphorus content is widely recognized as a secondary effect of immunosuppressive drug therapy administered after transplantation. Clinical observations and experiments have suggested that hypophosphoremia can be associated with skeletal muscle dysfunction, such as cramps, muscle weakness, or even myalgia and rhabdomyolysis.1 Because phosphate plays a key role in many biochemical processes, a decrease in plasma phosphate concentration could lead to a reduction of phosphate content in the cell and could therefore impair muscle function. Moreover, a number of drugs, such as cyclosporine, seem to alter aerobic metabolism through its deleterious effects on enzymes of the respiratory chain and on the internal membrane of mitochondria.2 The first goal of this study was to compare the muscle energetics in immunosuppressed patients and in healthy subjects. Then, the same group of sedentary transplanted patients was compared with a group of immunosuppressed patients undertaking regular physical activity.

MATERIALS AND METHODS Nineteen transplanted male patients (nine kidneys, six livers, two hearts, and two pancreas/kidneys) were divided into two groups (TSed, n ⫽ 9; TSpo, n ⫽ 10) following level of physical activity assessment, as estimated by Baecke questionnaire.3 The patients of the TSpo group participated in the 11th World Transplant Games in Sydney, Australia, in 1997. The control group (Ctrl) consisted of seven healthy subjects of similar age. They did not participate in any regular physical activity. The main characteristics and the immunotherapy of the patients are reported in Table 1. To analyze the muscle phosphate content, 31PNMR spectra were acquired from the calf muscle of the right leg of every patient, at rest, during exercise, and during recovery, using a Bruker Biospec

From the Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium (M.F., L.V., P.C.); and Katholieke Universiteit Leuven, Leuven, Belgium (P.V.H.). Supported by Pfizer Corp. Address reprint requests to Dr M. Francaux, 1 Place Pierre de Coubertin B-1348, Louvain-La-Neuve, Belgium.

Table 1. Main Characteristics of Patients and Effects of Immunosuppressive Drug Therapy and Physical Activity on Muscle Bioenergetics

Age (y) BMI (kg/m2) Time after transplant (y) Baecke score Treatment Azathioprine Prednisolone Cyclosporine Tacrolimus Mycofenolate mofetil Methylprednisolone Phosphoremia (mmol/L) [Pi] (mmol/kg) [PCr] (mmol/kg) ⌬[PCr] (%) t1/2 (s) Rate of PCr breakdown (mmol/kg 䡠 min) Rate of PCr repletion (mmol/kg 䡠 min)

Ctrl (n ⫽ 7)

TSed (n ⫽ 9)

TSpo (n ⫽ 10)

46.8 ⫾ 6.72 26.3 ⫾ 3.10 — 7.68 ⫾ 1.39

48.2 ⫾ 6.87 25.8 ⫾ 3.56 5.94 ⫾ 3.19 5.99 ⫾ 2.19

46.4 ⫾ 9.31 24.6 ⫾ 3.10 7.28 ⫾ 4.23 8.69 ⫾ 1.34

— — — — — — 1.16 ⫾ 0.10 2.67 ⫾ 0.73 22.2 ⫾ 1.95 ⫺68.5 ⫾ 19.3 52.9 ⫾ 18.3 7.99 ⫾ 3.22

8 7 8 1 1 0 0.83 ⫾ 0.21 3.47 ⫾ 1.24 22.7 ⫾ 3.85 ⫺62.8 ⫾ 17.2 47.3 ⫾ 23.7 7.56 ⫾ 2.82

8 7 8 1 2 1 0.96 ⫾ 0.21 2.49 ⫾ 0.61 21.7 ⫾ 2.66 ⫺50.7 ⫾ 23.4 47.3 ⫾ 19.7 5.45 ⫾ 2.70

8.17 ⫾ 3.08

7.42 ⫾ 4.63

6.98 ⫾ 4.10

P (Ctrl N TSed)

P (TSed N TSpo)

NS NS — NS

NS NS NS ⬍.01

⬍.01 NS NS NS NS NS

NS NS NS NS NS NS

NS

NS

Ctrl, control group composed of seven healthy, sedentary subjects; Tsed, group composed of nine transplanted, sedentary patients; TSpo, group composed of ten transplanted, physically active patients; BMI, body mass index; t1/2, half-repletion time of PCr after exercise.

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spectrometer (horizontal bore) operating at 4.7 T and 81 MHz. The leg was positioned in the center of the magnet and firmly immobilized over a 50-mm-diameter surface coil by means of a specially designed set-up allowing study of mainly the gastrocnemius muscle, and also some neighboring muscles. The right foot was placed on a fixed pedal that contained a force transducer measuring the torque exerted by each isometric contraction. Prior to the NMR experiment the force generated by maximal voluntary contraction (MVC) was measured. After fine tuning of the probe, with the subject properly positioned, the magnetic field was homogenized on freeinduction decay of water protons. An initial 31P-NMR spectrum was recorded at rest. The signal was acquired on 2048 points (spectral width 3333 Hz) after a 70-microsecond excitation pulse, corresponding to an angle of about 50° in the center of the coil. To improve the signal/noise ratio, 15 free induction decays (FIDs) were accumulated with a repetition time of 20 seconds, so that the recorded spectrum was fully relaxed. Afterwards, the exercise protocol was initiated. It consisted of applying to the pedal a force corresponding to ⬃80% of MVC during 2 minutes. NMR spectra were recorded continuously during a period of 8 minutes (1 minute at rest prior to exercise, during the exercise itself, and during the initial 5 minutes of recovery). A spectrum was acquired every 10 seconds (8 FIDs accumulated with a 1.25-second repetition time). The Fourier transform was calculated after exponential multiplication of the FID (line broadening 5 Hz). The integration of the areas under the peaks was done manually in the frequency domain using the standard software provided with the spectrometer. Because the area under the [␤-P]ATP peaks at rest were not different between the groups, and since it has been proven from measurements and from calculations based on thermodynamic properties of creatine kinase that muscle [ATP] is very constant even when [PCr] is reduced,4 the area under the [␤-P]ATP peak at rest was used as the internal reference and was set to 5.5 mmol/kg wet muscle.5 The mean [PCr] of the six last spectra recorded at rest before exercise was assumed to be equal to the [PCr] measured by the fully relaxed spectrum acquired prior to the exercise protocol. The [PCr] during exercise and recovery was calculated comparing the PCr peak areas to this mean. The rate of PCr breakdown during exercise (millimoles per kilogram per minute) was calculated from the difference between [PCr] at rest and at the end of the exercise. The rate of PCr resynthesis (millimoles per kilogram per minute) was calculated assuming a linear increase of [PCr] during the first minute of recovery. The time of half-PCr repletion (seconds), which was independent of the concentration, was calculated using a monoexponential model:6

[PCr]t ⫽ 共关PCr]rest (1 ⫺ exp(ln 0.5䡠t/t1/2兲兲) ⫹ 关PCr]ex where [PCr]rest is the PCr concentration at rest or at the end of the recovery period, [PCr]ex is the PCr concentration at the end of the exercise, and t1/2 is the time of half-PCr repletion (time zero being the end of the exercise). Intracellular pH (pHi) was calculated from the difference between the frequencies of the Pi and PCr peaks (␦ in parts per million) using the following equation:

pHi ⫽ 6.66 ⫹ log[(␦ ⫺ 3.079)/(5.57 ⫺ ␦)] (see ref 7) pHi was measured at rest and at the end of the exercise. Prior to the NMR experiment, a venous blood sample was taken in each subject to determine plasma phosphorus concentration. Plasma was prepared on EDTA. Inorganic phosphorus concentration was determined using a standard spectrophotometric method (molybdate reaction) purchased from Boehringer-Mannheim.

Fig 1. Mean time course of PCr concentration during an isometric contraction of the gastrocnemius muscle (80% of MVC). The standard deviations are typically 3 mmol/kg. (⌬) Control group (n ⫽ 7); (X) transplanted sedentary patients (n ⫽ 9); (⫹) transplanted, physically active patients (n ⫽ 10). The results are expressed as mean ⫾ standard deviation. The statistical significance of differences observed between means was assessed by parametric statistics after verifying the normality of the distribution and the lack of difference between variances. P ⬍ .05 was considered statistically significant. All statistical analyses were performed using SYSTAT software (Systat Inc, Evanston, Ill).

RESULTS

The main results are reported in Table 1. The level of physical activity estimated by Baecke’s questionnaire was clearly higher in the TSpo than in the TSed group (P ⬍ .001). This score was particularly low in the TSed group (5.99 ⫾ 2.19), although no statistical difference was found between the two groups of sedentary individuals (TSed and Ctrl). Plasma phosphorus concentration was lower in immunosuppressed patients than in healthy subjects (P ⬍ .001), but no difference was observed between the two groups of transplanted patients (TSed and TSpo). In these groups, the mean plasma phosphorus concentration remained above the value of 0.77 mmol/L, defined as the limit of hypophosphoremia. Nevertheless, five patients, three from the TSed group and two from the TSpo group, had plasma phosphorus concentrations below this value. At rest, pH was 7.06 ⫾ 0.04. It decreased by 0.09 ⫾ 0.12 unit of pH during exercise. No difference in pH was ever noted among groups. Before exercise, the [PCr] and [Pi] were ⬃22 mmol/kg and ⬃3 mmol/kg, respectively, in all groups (Table 1). The mean time course of muscle [PCr] during exercise and recovery is presented in Fig 1. Although the reduction of [PCr] observed after exercise was slightly less in physically active patients (TSpo ⫺ 50.7 ⫾ 23.4%) than in the two other groups (Ctrl ⫺ 68.5 ⫾ 19.3%, TSed ⫺ 62.8 ⫾ 17.2%), this difference did not reach the threshold of statistical significance. Thus, there was no difference in [PCr] measured at the end of the isometric contraction, nor in the rate of PCr breakdown. During the recovery period, PCr was resynthesized with a time of half-repletion (t1/2) rather less than 1

MUSCLE ENERGETICS

minute and was similar in all groups. The rate of PCr repletion (millimoles per kilogram per minute) was calculated from the linear increase of [PCr] during the first minute of recovery. As for the other variables related to PCr concentration and kinetics, the rate of repletion after exercise was not different between groups. DISCUSSION

Reduction in plasma phosphate concentration is a wellknown secondary effect of immunosuppressive drug therapy after transplantation. Usually, hypophosphoremia is more pronounced during the 18 months following transplantation. Afterwards, the plasma phosphorus level returns to a normal value.8 In this study, which was performed about 6 years after transplantation, the mean phosphoremia values (Table 1) observed in the transplanted patients were close to, but above, the critical value of 0.77 mmol/L defining hypophosphoremia. Phosphorus plasma concentration was clearly lower in immunosuppressed patients than in healthy subjects (P ⬍ .001). Only five patients presented hypophosphoremia, which was slight. The first purpose of this study was to examine if this reduction in plasma phosphate content alters the muscle bioenergetics. 31P-NMR results showed that muscle Pi and PCr concentrations were not affected at rest. It was seen that phosphate penetrates into the muscle cell against its concentration gradient by means of an active Na⫹-dependent transporter, the stoichiometry of which is one Na⫹ for one Pi. The rate of Pi transport is largely dependent on the transmembrane Na⫹ gradient. In myoblasts, the uptake is half-maximal at 0.2 to 0.3 mmol/L [Pi] and at 25 to 40 mmol/L [Na⫹].9 These properties are consistent with the preservation of an intracellular [Pi] relatively independent of the extracellular Pi. In other words, the Pi content of the cell seems well protected against slight reduction of plasma phosphate concentration, or even against the slight hypophosphatemia observed in our patients. Indeed, the five hypophosphatemia patients showed no significant reduction of muscle Pi and PCr content at rest. Similarly, no difference could be observed between groups in the rate of PCr breakdown as well as in the rate of resynthesis. The rate constant of creatine phosphate repletion was calculated following a monoexponential model,6 and expressed as half-repletion time (t1/2). This parameter is independent of the concentration and is related to the rate of local mitochondrial respiration.6 Thus, observation of [PCr] repletion after exercise allows for testing of the eventual deleterious effects of immunosuppressive ther-

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apy and the influence of training activity on mitochondrial function. The results show that t1/2 was not different between groups. If immunotherapy damaging to the mitochondrial membrane and the enzymes of the respiratory chain, it does not affect the respiration sufficiently to reduce the [PCr] repletion. On the other hand, the physically active transplanted patients did not show a faster [PCr] repletion than the sedentary subjects. Two hypotheses could be formulated to explain this result: (1) immunotherapy reduces the effect of physical training on aerobic metabolism; or (2) the physical activity of the TSpo group is not sufficient to induce a large change in the rate of mitochondrial respiration. The level of physical activity was estimated by Baecke’s questionnaire. The results of the sedentary healthy subjects are in good agreement with the values reported elsewhere,10 but are not different from those registered in transplanted physically active patients (Table 1). This means that the physical activity of the so-called “sportstransplanted” individuals remained low. Moreover, sedentary transplanted subjects presented a Baeckl score still lower, which highlights a definitive lack of physical activity. CONCLUSIONS

In spite of the reduction of plasma phosphorus concentration, long-term immunotherapy affected neither muscle content in energy-rich phosphates nor muscle aerobic metabolism in the patients that participated in this study. REFERENCES 1. Arellano F, Krupp P: Lancet 337:915, 1991 2. Hokanson JF, Mercier JG, Brooks GA: Am J Respir Crit Care Med 151:1848, 1995 3. Baecke JA, Burema J, Frijters JE: Am J Clin Nutr 36:936, 1982 4. Goudemant JF, Francaux M, Mottet I, et al: Magn Res Med 37:744, 1997 5. Harris RC, Hultman M, Nodesjo ¨ LO: Scand J Clin Lab Invest 33:109, 1974 6. Meyer RA: Am J Physiol 254:C548, 1988 7. Malloy CR, Cunningham CC, Radda GK: Biochim Biophys Acta 885:1, 1986 8. Walker GS, Peacock M, Marshall DH, et al: Nephron 26:225, 1980 9. Kemp GJ, Polgreen KE, Radda GK: Biochim Biophys Acta 1137:10, 1992 10. Jacobs DR, Ainsworth BE, Hartman TJ, et al: Med Sci Sports Exerc 25:81, 1993