INTRODUCTION

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CREATINE PHOSPHOKINASE (CPK, EC 2.7.3.2; ATP: creatine N-phosphotransferase) catalyzes the reversible reaction, PCr + ADP + H⁺  ATP + Cr, where PCr and Cr are phosphocreatine and creatine, respectively. In phosphagen-containing tissues such as skeletal muscles, heart, and brain, the relative concentrations of PCr and Cr are frequently interrogated in vivo using ¹H- and ³¹P-magnetic resonance spectroscopy (MRS). In some cases, ¹H-MRS studies use the methyl protons of total Cr {tCr; the sum of PCr concentration ([PCr]) + Cr concentration [Cr]} as a kind of internal standard against which to evaluate the concentrations of other intermediates, whereas in many studies, the behavior of tCr per se under different metabolic states is the focus of attention (8, 11, 16, 22, 25). To better evaluate such data and because recent ¹H-MRS studies have yielded some unexpected results not necessarily consistent with traditional models of CPK function (12-14, 21), we decided to examine the ¹H- and ³¹P-MRS detectable pool size and behavior of tCr under different metabolic states in human gastrocnemius muscle. The goal of our first study (6) was a careful analysis of the echo time (TE) dependence of the Cr methyl proton signals at 3.02 ppm in human gastrocnemius in the nonwork or rest condition, presumably a highly reproducible metabolic state of the tissue. These studies showed that a two-component decay of the central peak of the dipolar coupled methyl triplet is caused by a rapid (34 ms) dipolar dephasing and by a less-rapid transverse relaxation or T₂ of ~162 ms (6). From these studies, it became evident that the relationships between the in vivo tissue concentration of tCr and the peak intensities of MRS spectra, even in the resting state, may not be simple exponential functions of TE and that detailed T₂ information may be requisite in studies requiring precise tCr concentration ([tCr]) estimates. In the present studies, we extended MRS examination of the human gastrocnemius to include conditions that were expected to perturb the tCr pool. Initial data, described in a preliminary report (21), were obtained for this muscle with and without Cr supplementation both at rest and ischemic fatigue (IF; i.e., in 4 well-defined metabolic states with the tCr pool in widely differing physiological conditions). Our results indicate that the MRS behavior of tCr in the IF state differs substantially from that observed in the resting condition.

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Subjects. Experiments were carried out on 12 healthy power-trained (PT) athletes (O₂ 47.65 ml · kg¹ · min¹) and 12 healthy endurance-trained (ET) athletes (O₂ 65.04 ml · kg¹ · min¹). Cr supplementation was one means we used to modify the tCr pool (27); accordingly, 6 subjects from each group of 12 undertook a Cr-loading regime for 7 days (5-g doses 4 times/day taken with 0.75 l of orange juice). The other 12 subjects took sucrose as a placebo. Exercise was the second means used to perturb the tCr pool. Thus, on the final day of loading, subjects returned to perform an exercise protocol. Follow-up studies were then performed 1) on 11 subjects at rest to evaluate the TE dependence of the methyl triplet [these data are already published (6)], 2) on six additional subjects to assess the TE dependence of ¹H-tCr spectra for muscle at rest compared with IF, and 3) on a final set of six subjects to evaluate PCr utilization during muscle work using a previously published (1) ³¹P-MRS methodology. In each test series, subjects performed a familiarization exercise trial [similar to that previously reported (21)], returning after 24 h for the actual experimental protocol.

Exercise protocol. All subjects completed a plantar-flexion exercise of the right foot against a load of 10 kg with a frequency of 1 stroke/s. The load was increased by 1 kg/min until the subject attained volitional fatigue. A displacement transducer provided for calculation of total work done. Immediately after exhaustion, a pressure cuff was inflated for 5.5 min (>350 mmHg) superior to the knee; this condition of IF allowed collection of spectra before PCr resynthesis (18). On removal of the cuff, recovery of tCr was monitored for 10 min with the same measurement technique as before (data acquired in 2-min blocks).

MRS. The exercise was performed while lying supine in a 3 Tesla superconducting magnet (Magnex Scientific PCI; console from Surrey Medical Imaging Systems, Surrey, UK). The MRS procedures were similar to those already described (6). To briefly reiterate for MRS specialists, experimental data were acquired using a quadrature birdcage resonator for transmission and signal reception, with the right medial gastrocnemius muscle (MGM) centered in the field in a circumscribing radio frequency coil. To minimize susceptibility difference effects, a plastic bag that contained Kaopectate solution was positioned adjacent to the interrogated region of the calf muscle. Typical line widths of the central methyl peak of the tCr in muscle at rest were ~0.05 ppm (~6 Hz). Multislice gradient echo imaging in the transverse, sagittal, and coronal planes was used to register the point resolved spectroscopy (PRESS)-selected volume precisely to the MGM (Fig. 1). In addition to voxel placement, the angular orientation of the tibia relative to the main static magnetic (B_o) field was estimated to measure the relative angular displacement and to minimize the variation in leg placement between subjects. In an initial set of studies, a series of water-suppressed spectra was obtained from 24 subjects at a single TE of 100 ms to enable a comparison to be made of the MRS visible tCr pools at rest and in IF with and without Cr supplementation. After zero filling (to 2,048 data points), filtering (2-Hz exponential), and fast Fourier transform of the time-domain data, the spectra were deconvoluted using a PERCH analysis package in which each peak was filtered to a Gaussian function (software distributed by the PERCH Project, Dept. of Chemistry, Univ. of Kuopio, Finland). The calculated areas (peak intensities) were taken to represent the MRS visible tCr pool size in the different metabolic states, using rest as the reference standard. A further series of water-suppressed spectra was acquired at rest on 11 subjects using a symmetric PRESS-pulse sequence (3, 5) with inversion null water suppression (17) to determine the TE dependence of the methyl triplet (TR = 2 s; TE = 20-300 ms, in 4- to 20-ms increments for a total of 17 TE values; 1,024 data points; 124 averages). Because of the complexity of ¹H-MRS of tCr, the decay characteristics of these spectra were analyzed in detail and published elsewhere (6). However, we wanted to compare these T₂ values in rest (6) to T₂ in IF. To this end, a final series of water-suppressed spectra was acquired on six subjects comparing the above data at rest to data collected during 5.5 min of IF. The TE dependence was determined as before (TR = 2 s; TE = 20, 40, 60, and 80 ms or 20, 50, and 80 ms; 1,024 data points; 128 averages), but the IF data acquisitions were necessarily abbreviated compared with rest, with the 5.5-min acquisition period being governed by the discomfort level tolerable to the subjects. Finally, ³¹P-MRS data were obtained for rest, IF, and recovery using previously described procedures (1).

View larger version (119K): [in this window] [in a new window]   Fig. 1.   Representative magnetic resonance (MR) image of a cross-section of a subject's lower leg showing volume of interest in the medial gastrocnemius. A cross-section of a test tube containing a reference creatine (Cr) solution is shown in the lower left quadrant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

³¹P-MRS measurement of PCr concentrations at rest, in IF, and in recovery. The relative contribution of PCr to tCr in the different metabolic states was monitored with ³¹P-MRS and shown to decrease from the resting state by ~95% during IF. Recovery patterns were similar to, but slower than, those following strenuous exercise without imposed ischemia (see our own data in Ref. 1 for comparison). For the ¹H-MRS studies below, these ³¹P-MRS results indicate that PCr is the major component of tCr in resting muscle, whereas in IF, tCr is largely composed of Cr (Fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   31P-magnetic resonance spectroscopy (MRS) determined change in phosphocreatine (PCr) amplitude in medial gastrocnemius at rest, during 5.5 min of ischemic fatigue (IF), and during recovery using protocols described in previous studies (1, 15). Six subjects were used to generate these representative profiles; means are plotted with bars indicating standard deviation.

Empirical ¹H-MRS measures of tCr. Changes in the tCr methyl peak intensity at TE = 100 ms for several conditions (at rest, in IF, and during 10 min of recovery) are summarized in Table 1 for several conditions for PT subjects pre- and post-Cr loading plus pre- and postsucrose loading, the control condition. An equivalent data set is shown for ET individuals in Table 2. Similar patterns appear both in PT and ET individuals with or without Cr supplementation: an apparent decline in peak intensity in IF (compared with the rest condition) that generally recovers rather slowly (to ~75-80% of resting peak intensities within the first 10 min post-IF). After Cr supplementation, an increase in tCr could be detected in PT individuals both at rest and in IF, whereas in ET individuals, this could only be detected in the IF state. These first experiments were based on the assumption that on transition from rest to IF, the CPK reaction (PCr + ADP + H⁺ ATP + Cr) coupled to actomyosin ATPase (ATP + H₂0  ADP + Pi) leads to a net decline in PCr concentration with a stoichiometric increase in Cr and Pi concentrations (or a net reaction of PCr  Cr + Pi), as is observed in in vivo ³¹P-MRS studies (1, 7, 25). Thus [tCr] should remain the same, and if PCr and Cr made similar contributions to ¹H-MRS spectra, as they do in simple solution, then no changes should occur in the ¹H-MRS visible tCr during an exercise protocol. That is, the intensity and spectral form of the 3.02-ppm peak would remain constant. This result was not observed; so to better evaluate the changes noted in Tables 1 and 2, we examined tCr at varying TE values both at rest and at IF.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Representative total Cr (tCr) 1H-MR spectra at echo time or TE = 40 ms for medial gastrocnemius under rest conditions (A, modified from Ref. 6) and during 5.5 min of IF (B). The methyl triplet at ~3 ppm is clearly resolved in the upper panel (muscle at rest).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   1H-MR spectra for metabolites (tCr at 3.05 ppm; choline and other trimethylamines at ~3.2 ppm) at different TE (different echo times or TE values) for muscle at rest and during IF. Spectra are presented as composite averages from data acquired from 6 different subjects (in arbitrary units standardized to tCr in the resting condition at TE = 40 ms). T2, transverse spin-spin relaxation time.

Cr supplementation. Cr supplementation caused MRS visible increases in tCr peak intensities under two conditions: during IF in PT and ET groups (Tables 1 and 2) but only in PT individuals both under resting and IF conditions (Table 1). The magnitude of increases, where observed, were similar to those previously reported (for example, see Refs. 14 and 27). These metabolic conditions were only assessed at TE = 100 ms.

Global frequency shift on rest-IF transition. A global frequency shift of all MRS signals (10.6 ± 4.4 Hz) was observed when the muscle metabolic state changed from rest to IF with a gradual return to normal during recovery. The time course of these frequency shifts is illustrated in Fig. 5.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Global frequency shift magnitude and time course during transition from rest to IF and to recovery.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

A priori expectations. On transition from rest to IF, the net reaction (PCr  Cr + Pi) would lead to no change in [tCr]. Our original expectation was that the ¹H-MRS 3.02-ppm peak intensity and multiplicity would be the same for muscle in the two states. Our initial spectra, acquired at TE = 100 ms, showed that signal intensity declined on transition from rest to IF and that line width broadened. However, further analysis to account for T₂ changes showed that this apparent drop in intensity was caused by a decrease in the T₂ of the tCr signal during IF compared with the resting state. Moreover, when this change was allowed for, the MRS visible tCr pool size was unchanged (as expected from the above reaction). It is the difference in behaviors of tCr between rest and IF, clearly not predicted and not expected from traditional models of phosphagen and muscle function (1, 2, 7, 15, 16, 25), that we need to address.

Mobility of tCr during IF. In our companion paper (6), we showed that the magnitude of the residual dipolar coupling is dependent not only on the orientation angle with respect to B_o, but also on the distribution of orientations. The changes in tCr triplet in going from rest to the IF state might arise from either a new orientation of the pool of tCr molecules closer to the "magic angle" [where dipolar splittings vanish (13)] or, and perhaps more likely, changes occurring in the space available between actin and myosin chains during IF could affect the distribution of the orientations of the tCr methyls to the B_o field (6, 12, 13). With the use of magnetically oriented liposomes or bicelles containing PCr or Cr, recent studies (27) showed similar results (line broadening and residual dipolar coupling) as a function of phospholipid concentration. These data were interpreted to indicate that the dipolar coupling effects of metabolites such as tCr in skeletal muscle are caused by weak macroscopic ordering induced by liposome alignment parallel to B_o [i.e., by the structural features of the tissue at both the micro- and macroscopic levels (27)]. In the present study, although we have been referring to the total pool (tCr), in fact, we are mainly monitoring change in the observed metabolite from PCr (rest) to Cr (IF). It is therefore conceivable that the differences we observed in tCr in the two states reflected the different local intracellular environments these two metabolites experienced. In going from rest to IF, the space between the actin and myosin chains is sufficiently reduced through the formation of protein-protein and enzyme-substrate complexes that there would be an increase in steric hindrance to motion of tCr molecules (PCr or Cr and their respective hydration spheres). One might expect this hindrance to motion to result in a reduction in the observed T₂ of the respective methyl protons and in the dipolar coupling changes, as observed in vitro (27). Clearly, further studies are required to clarify these observations.

Metabolic state-dependent global frequency shifts. An unexpected observation from these studies was the global frequency shifts of all MRS visible signals during transition from rest to IF and then on to recovery (Fig. 5). This result is suggestive of a susceptibility shift that could result from deoxygenation. During initial rest conditions, myoglobin (Mb) and Hb are mostly saturated with oxygen and thus diamagnetic; but with pressure cuff inflation at the end of exercise (IF), Mb and Hb become largely deoxygenated. A paramagnetic effect of deoxygenated Mb on the proton spectra of metabolites has recently been demonstrated (20). The susceptibility shifts observed among the three different metabolic states could similarly result from such effects of deoxygenated Hb and Mb on neighboring metabolite molecules.

Implications. Over the last 3-4 decades, two general frameworks (we shall term them models 1 and 2) for how muscle metabolism functions and is regulated have dominated thinking in the field (for literature in this area, see Refs. 8, 10, 11, 19, and 27). In fact, these two views are nicely illustrated by the vertebrate phosphagen system. Model 1 assumes that 1) the total acid-extractable pool of Cr + PCr (tCr) occurs in aqueous solution and is fully accessible to CPK, 2) that solution chemistry rules apply globally in muscle cells in vivo {which, it is assumed, is why many ³¹P-MRS studies, including our own (1, 15), consistently show that change in [PCr] accurately reflects change in ATP demand or work rate of skeletal muscles and why in vivo estimates of P/0 ratios (7) can be shown to be close to theoretically expected values}, and 3) that the main CPK-phosphagen function is to "buffer" ATP concentrations during large-scale changes in muscle work and in ATP-turnover rates (2, 8, 16, 25).