Muscle creatine uptake and creatine transporter expression in response to creatine supplementation and depletion.

The total creatine pool size [Cr(total); creatine (Cr) + phosphocreatine (PCr)] is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The Cr(total) likely is controlled by long-term modulation of Cr uptake via the sodium-dependent Cr transporter (CrT). To test this hypothesis, adult male Sprague-Dawley rats were fed 1% Cr, their muscle Cr(total) was reduced by approximately 85% [1% beta-guanidinoproprionic acid (beta-GPA)], or their muscle Cr(total) was repleted (1% Cr after beta-GPA depletion). Cr uptake was assessed by skeletal muscle (14)C-Cr accumulation to Cr and PCr by using hindlimb perfusion, and CrT protein content was assessed by Western blot. Cr uptake rate decreased with dietary Cr supplementation in the white gastrocnemius (WG; 45%) only. Depletion of muscle Cr(total) to approximately 15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (22%), corresponding to 70-150% increases in muscle CrT content. In contrast, the inherently lower Cr uptake rate in the WG was unchanged with depletion of muscle Cr(total) even though CrT band density was increased by 230%. Thus there was no direct relationship between apparent muscle CrT abundance and Cr uptake rates. However, Cr uptake rates scaled inversely with decreases in muscle Cr(total) in the high-oxidative muscle types but not in the WG. This implies that factors controlling Cr uptake are different among fiber types. These observations may help explain the influence of initial muscle Cr(total), time dependency, and variations in muscle Cr(total) accumulation during Cr supplementation.     

Muscle creatine kinase isoenzyme expression in adult human brain.

Previous studies have suggested that MM creatine kinase is a muscle-specific protein and is not present in adult brain tissue. We have isolated a protein from human brain with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is identical to the muscle M creatine kinase isoenzyme subunit at all 30 sequenced amino acid residues and possesses creatine kinase enzymatic activity following nondenaturing agarose-gel electrophoresis. Immunohistochemistry localizes M creatine kinase to discrete areas of adult human brain. Northern blot analysis of both total and poly(A)-selected RNA isolated from brain did not detect M creatine kinase mRNA. However, polymerase chain reaction amplification of cDNA synthesized from human placenta, heart, and brain mRNA detected M creatine kinase message in both heart and brain but not placenta which contains no detectable M creatine kinase protein. N1E115 and NS20Y, mouse neuroblastoma cell lines which have been used as models of neural cell differentiation, were found also to express MM creatine kinase. Moreover, a transiently transfected reporter gene with 4,800 base pairs of M creatine kinase upstream region fused to chloramphenicol acetyltransferase was expressed during differentiation of these neural cell lines. In summary, MM creatine kinase is present in human brain and we suggest the M creatine kinase upstream region is sufficient to modulate M creatine kinase expression in certain neuronal cells and may be regulated independently from other muscle genes.     

Effect of creatine loading on neuromuscular fatigue threshold.

The purpose of this investigation was to determine the effect of creatine (Cr) loading on the onset of neuromuscular fatigue by monitoring electromyographic fatigue curves from the vastus lateralis muscle using the physical working capacity at the fatigue threshold (PWC(FT)) test. Using a double-blind random design, 15 women athletes [mean age 19.0 +/- 2.0 (SD) yr] from the university crew team received a placebo (n = 8; 20 g glucose) or Cr (n = 7; 5 g Cr monohydrate + 20 g glucose) four times per day for 5 consecutive days. Analysis of covariance was used to analyze the data (covaried for presupplementation PWC(FT) values). The adjusted mean postsupplementation PWC(FT) value for the Cr group (mean = 186 W) was significantly (P < 0.05) higher than that of the placebo group (mean = 155 W). These findings suggest that Cr loading may delay the onset of neuromuscular fatigue.     

Shortening of muscle relaxation time after creatine loading.

The effect of creatine (Cr) supplementation on muscle isometric torque generation and relaxation was investigated in healthy male volunteers. Maximal torque (Tmax), contraction time (CT) from 0.25 to 0.75 of Tmax, and relaxation time (RT) from 0.75 to 0.25 of Tmax were measured during 12 maximal isometric 3-s elbow flexions interspersed by 10-s rest intervals. Between the pretest and the posttest, subjects ingested Cr monohydrate (4 x 5 g/day; n = 8) or placebo (n = 8) for 5 days. Pretest Tmax, CT, and RT were similar in Cr and placebo groups. Also in the posttest, Tmax and CT were similar between groups. However, posttest RT was decreased consistently by approximately 20% (P < 0.05) in the Cr group from the first to the last of the 12 contractions. In addition, the mean decrease in RT after Cr loading was positively correlated with pretest RT (r = 0.82). It is concluded that Cr loading facilitates the rate of muscle relaxation during brief isometric muscle contractions without affecting torque production.     

Effects of 30 days of creatine ingestion in older men

In this investigation we evaluated the effects of oral creatine (Cr) supplementation on body composition, strength of the elbow flexors, and fatigue of the knee extensors in 20 males aged 60-82 years who were randomly administered Cr or placebo (P) in a double-blind fashion. Subjects ingested either 20 g of Cr or P for 10 days, followed by either 4 g of Cr or P, respectively, for 20 days. Tests were conducted pre-supplementation and following 10 and 30 days of supplementation. Leg fatigue was determined using an isokinetic dynamometer; subjects performed 5 sets of 30 maximal voluntary contractions at 180 degrees x s(-1), with 1 min of recovery between sets. The strength of the elbow flexors was assessed using a modified preacher bench attached to a strain gauge. There was a significant interaction (P < 0.05; group x time) in leg fatigue following supplementation. However, this interaction appears to have resulted from a combination of the improved fatigue score by the Cr-supplemented group and the decreased fatigue score by the P-supplemented group, because when the simple main effects were analyzed for the groups individually, there was no significant difference over time for either of the groups. There were no significant differences in body mass, body density, or fat-free mass as assessed by hydrostatic weighing, or strength between the Cr-supplemented or P-supplemented groups. These data suggest that 30 days of Cr-supplementation may have a beneficial effect on reducing muscle fatigue in men over the age of 60 years, but it does not affect body composition or strength.     

Effect of creatine phosphate supplementation on anaerobic working capacity and body weight after two and six days of loading in men and women

The purpose of this study was to determine the effects of 2 and 6 days of creatine phosphate loading on anaerobic working capacity (AWC) and body weight (BW) in men and women. Sixty-one men (n = 31) and women (n = 30) randomly received 1 of 3 treatments (4 x 5 g.d(-1) x 6 days) using a double blind design: (a) 18 g dextrose as placebo (PL); (b) 5.0 g Cr + 20 g dextrose (Cr); or (c) 5.0 g Cr + 18 g dextrose + 4 g of sodium and potassium phosphates (CrP). AWC was determined at baseline and following 2 and 6 days of supplementation using the Critical Power Test. BW increased significantly over time, and the mean value for the men was significantly greater compared to that for women, but there were no interactions (p > 0.05). There were gender-specific responses for AWC expressed in both absolute values (kJ) and relative to BW (kJ. kg(-1)), with the women demonstrating no significant interactions. For the men, CrP loading significantly increased AWC following 2 days (23.8%) and 6 days (49.8%) of supplementation vs. PL (kJ and kJ.kg(-1)). Cr supplementation increased AWC 13-15% in both genders compared to PL (1.1%- 3.0% decline); although this result was not statistically significant, it may have some practical significance.     

Muscle power after glucose-potassium loading in undernourished patients.

The force-frequency characteristics and maximal relaxation rate of the adductor pollicis muscle were measured before and after 48 hours of intravenous loading with glucose (104.5 kJ (25 kcal)/kg/24 h) and potassium (20 mmol(mEq)/500 ml glucose) in eight undernourished patients about to undergo surgery. Both variables of skeletal muscle performance, which were depressed when compared with data from 100 healthy volunteers, improved significantly after glucose-potassium loading. The improvement was accompanied by restoration of muscle glycogen values and return of respiratory exchange ratios towards unity. These results imply that if muscle power is a yardstick for preoperative nutritional rehabilitation then a simple regimen of energy-electrolyte repletion may be cost effective in preparing undernourished patients for major surgery.     

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.     

Muscle function in men and women during maximal eccentric exercise

This study assessed muscle fatigue patterns of the elbow flexors in untrained men and women to determine if sex differences exist during acute maximal eccentric exercise. High-intensity eccentric exercise is often used by athletes to elicit gains in muscle strength and size gains. Development of fatigue during this type of exercise can increase risk of injury; therefore, it is important to understand fatigue patterns during eccentric exercise to minimize injury risk exposure while still promoting training effects. While many isometric exercise studies have demonstrated that women show less fatigue, the patterns of fatigue during purely eccentric exercise have not been assessed in men and women. Based on the lack of sex differences in overall strength loss immediately post-eccentric exercise, it was hypothesized that women and men would have similar relative fatigue pattern responses (i.e., change from baseline) during a single bout of maximal eccentric exercise. Forty-six subjects (24 women and 22 men) completed 5 sets of 10 maximal eccentric contractions on an isokinetic dynamometer. Maximal voluntary isometric contraction strength was assessed at baseline and immediately following each exercise set. Maximal eccentric torque and contractile properties (i.e., contraction time, work, half relaxation time, and maximal rate of torque development) were calculated for each contraction. Men and women demonstrated similar relative isometric (32% for men and 39% for women) and eccentric (32% for men and 39% for women) fatigue as well as similar deficits in work done and rates of torque development and relaxation during exercise (p > 0.05). Untrained men and women displayed similar relative responses in all measures of muscle function during a single bout of maximal eccentric exercise of the elbow flexors. Thus, there is no reason to suspect that women may be more vulnerable to fatigue-related injury.     

Muscle endurance is greater for old men compared with strength-matched young men.

The purpose was to compare the time to task failure for a sustained isometric contraction performed at a submaximal intensity with the elbow flexor muscles by young and old men who were matched for strength. Eight young men (18-31 yr) and eight old men (67-76 yr) sustained an isometric contraction at 20% of maximal voluntary contraction (MVC) torque until the target torque could no longer be achieved for at least 5 s. The maximal torque exerted at the wrist was similar for the young and old men before the fatiguing task (65.9 +/- 8.0 vs. 65.4 +/- 8.7 N x m; P > 0.05), and they experienced similar reductions in MVC torque after the fatiguing contraction (31.4 +/- 10.6%; P < 0.05). The time to task failure was longer for the old men (22.6 +/- 7.4 min) compared with the strength-matched young men (13.0 +/- 5.2 min; P < 0.05), despite each group sustaining a similar torque during the fatiguing contraction (P > 0.05). The increases in torque fluctuations, electromyographic (EMG) bursting activity, and heart rate were greater for young men compared with the old men, and they were less at task failure for the old men (P < 0.05). Mean arterial pressure increased at a similar rate for both groups of men (P > 0.05), whereas the averaged EMG activity and rating of perceived exertion reached similar values at task failure for the young and old men (P > 0.05). These findings indicate that the longer time to task failure for the old men when performing the submaximal contraction was not due the absolute target torque exerted during the contraction.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.