The etiology of the damage/repair process and alterations in muscle function following unaccustomed, eccentric exercise has been extensively studied [1
]. Disturbances in the regulation and concentration of intracellular Ca2+
and changes in the rate of muscle protein degradation appear to be an integral part of this process [5
]. Enhancing muscle recovery could involve ameliorating the initial insult of injury by improving the Ca2+
handling ability of the muscle and/or increasing the rate of regeneration by augmenting muscle protein synthesis. While other contributing factors, such as post-injury inflammation, and proliferation and differentiation rates of muscle satellite cells and myoblasts, respectively, can modulate regenerative capacity, the ability of muscle to buffer the initial physiological damage signal and induce regenerative activity remain fundamental in achieving complete functional recovery [4
Dietary supplements creatine monohydrate (CR) and whey protein (WP) have previously demonstrated ergogenic roles in exercise performance and recovery, and subsequent adaptations from exercise training [8
]. However, the effects of CR and WP supplementation on indicators of muscle damage and recovery after injury in both human [10
] and animal [26
] models are equivocal. Studies using acute ingestion of CR before (~20 g/day) and/or after exercise (~2–3 g/day) have reported no effect on the extent of muscle damage and/or recovery following a high-force, eccentric exercise [12
] or low force, hypoxic resistance exercise challenge [11
], respectively. Similarly, but utilizing a higher dosage of 40 g/day before and 10 g/day after exercise, CR ingestion had no significant benefit on markers of muscle damage and recovery following eccentric contractions of the elbow flexor muscles [13
]. These observations have also been supported following chemically-induced muscle damage, with CR supplementation ineffective in accelerating the time course of muscle recovery in rodents [27
]. In contrast, Bassit et al. [26
] showed that acute CR supplementation (5 g kg−1
body weight per day) for five days was able to attenuate muscle strength loss and rise in plasma markers of muscle damage (i.e., lactate dehydrogenase (LDH) and creatine kinase (CK)) following electrically-induced muscle damage in rats. Furthermore, the same authors reported potential protective effects in triathletes following an ironman competition, albeit, with a low sample size [26
]. In support of these findings, Cooke et al. [10
] reported higher isometric and isokinetic leg extension strength and lower plasma CK levels when CR was supplemented before (~20 g/day) and during the days after (~7 g/day) an intense resistance exercise session.
The majority of WP supplementation studies have focused on the purported benefits of higher protein intake during periods following damaging exercise to enhance recovery [9
]. White and colleagues [16
] found no significant improvement in the rate of muscle recovery following maximal isokinetic eccentric contractions of the quadriceps when WP (23 g) was ingested pre- or post-exercise. Conversely, Buckley et al. [23
] showed improved muscle strength recovery when WP hydrolysate (25 g) was ingested during the days following an intense exercise bout (i.e., 100 maximal knee extensions of the knee extensors) compared to a placebo. However, given no significant elevation in an indirect marker of muscle damage (i.e., CK) was observed, reduced fatigue, rather than attenuation of muscle damage most likely occurred. Notwithstanding, consumption of WP isolate (~25–30 g, 4 times a day) during the days following an intense resistance exercise session attenuated decrements in muscle strength and rises in LDH [25
A limitation in human studies is the use of proxy measures of muscle damage/recovery. This can introduce confusion regarding whether the benefits of supplementation are beyond the initial fatigue recovery or the result inherent to damage. Moreover, the damage protocols typically used in human studies (i.e., downhill running, isolated eccentric contractions) result in high variability in the magnitude of muscle damage, and consequently the effects from the supplement intervention are often variable. Thus, the use of animal models allows for a more direct and comprehensive analysis of muscle damage and regeneration following injury. The purpose of this study was to examine temporal changes in functional, morphological, and biochemical characteristics of muscle recovery following a controlled, standardized, chemically-induced injury to identify potential myotherapeutic benefits of CR and WP. We hypothesized that CR and WP supplementation would restore injury-induced loss of isometric contractile strength sooner by either blunting the extent of the initial damage and/or improving the rate of fiber regeneration.
Creatine supplementation prior to and following a controlled myotoxic injury episode was more effective at restoring functional strength compared to whey protein and placebo supplementation. This enhanced functional capacity was associated with reduced damage following the initial insult, greater CSA of regenerating fibers during the early stages of recovery, and higher levels of muscle contractile protein content during the later stages of recovery. On the contrary, WP seemed to have delayed effects on muscle recovery, both functionally and structurally, following myotoxic injury. The findings from the present study provide important information on the morphological and biochemical mechanisms by which CR and WP are influencing recovery and elucidate the mechanisms for improvements observed in human studies using similar supplementation protocols.
Numerous studies have confirmed that supplementation with CR in conjunction with programmed resistance training is effective for augmenting gains in body and fat-free mass, and muscular strength in both men and women [42
]. These benefits are possibly due to enhanced training-induced increases in satellite cell number and myonuclei concentration within skeletal muscle fibers [46
]. Furthermore, CR supplementation alone may increase muscle mass, which is likely a reflection of increased water retention, rather than effects on mixed muscle protein synthesis [47
]. In the present study, animals consuming CR gained more body weight (approximately 6 g) than animals consuming standard rat chow over a two-week loading period, with this trend continuing during the 14 days post-surgery period, but to a lesser degree. By day 14 post-injury, both injured and uninjured muscles of the CR-supplemented animals were heavier than WP and CON muscles. The heavier injured muscles of the CR-supplemented animals could be due to faster recovery as evidenced by larger CSA of the regenerating fibers and higher contractile protein levels. Indeed, cell culture studies have shown accelerated differentiation of myoblasts into hypertrophic myotubes as a result of creatine’s ability to offset the inhibition of myogenic differentiation typically caused by high levels of oxidative stress following injury [49
]. The uninjured muscles also displayed higher total protein levels and greater fiber CSA, suggesting CR-induced anabolic growth driven by increased cellular hydration status [52
]. In contrast, WP-supplemented animals displayed similar weight gain compared to the CON animals, and no significant changes in fiber CSA and protein levels.
A primary outcome of this study was to determine whether CR and WP supplementation could enhance the functional recovery of the muscle following myotoxic injury. Absolute forces in the injured muscles of the CR-supplemented animals were closer to full recovery (~76% of uninjured values) compared to injured WP muscles (~71% of uninjured values) and CON muscles (~65% of uninjured values) by day 14 post-injury. Furthermore, both injured and uninjured muscles following CR supplementation generated significantly higher forces than WP- and CON-supplemented muscles. However, when corrections were made for cross-sectional area, specific forces were not significantly different between groups. It should be noted that specific force, an indicator of force output based on a given muscle size, can be influenced during the early stages of muscle regeneration by inflammation and muscle swelling, and thus, may explain the non-significant differences between groups.
Histological analysis revealed a significantly higher proportion of intact (non-damaged) fibers in the regenerating CR muscles compared to CON muscles during the early stages post-injury. In addition, CSA of the regenerating and intact fibers was significantly larger in the CR-supplemented muscles compared to the CON muscles. These observations suggest that CR supplementation is reducing the extent of damage and/or enhancing the growth of the regenerating fibers. Though no corresponding functional enhancement was noted at this time point (i.e., day 7) for CR supplementation, it appears that morphological improvements occurring at this early stage may underpin the benefits observed in the later stages of recovery (i.e., higher absolute forces at day 14).
Originally considered solely as a sports supplement, creatine has shown over the past two decades that its role goes beyond cellular bioenergetics, with pleiotropic effects that converge to maintain cell homeostasis, protect membranes, and reduce oxidative stress and apoptosis [50
]. The reduced magnitude of damage following the initial injury insult could be due to improved calcium (Ca2+
) handling ability of the muscle, and thus, less activation of self-accelerating, degradative pathways that lead to damage and degeneration [55
]. CK isoforms, together with its substrates CR and phosphocreatine (PCr), represent an intricate cellular energy buffering and transport system [56
]. The sarcoendoplasmic reticulum (SR) Ca2+
transport ATPase (SERCA) pump derives its adenosine triphosphate (ATP) preferentially from PCr via SR-bound CK. A high ATP/adenosine diphosphate (ADP) ratio within the vicinity of the SERCA pump allows the pump to function optimally [57
]. During times of severe stress such as post-injury, increased PCr stores following CR supplementation may enhance the function of these pumps, and thus, the Ca2+
handling of the muscle. Furthermore, the antioxidant properties of CR may reduce the reactive oxygen species (ROS)-induced inhibition of SERCA pump function [58
], as well as damage to RNA and inhibition of mitochondrial permeability transition, an early event in apoptosis [59
Although SERCA pump activity was not measured directly in the current study, increased Ca2+
uptake rate by SR vesicles from tibialis anterior muscle following CR supplementation has been shown in a previous study [61
]. In the present study, the rate of muscle relaxation, which is an indirect measurement of Ca2+
handling ability of the muscle, was significantly increased in both the injured and uninjured muscles at day 7 and 14 post-injury following CR supplementation. This indirect measurement of Ca2+
handling ability of the muscle could indicate CR supplementation is enhancing SERCA pump function. ROS levels were not measured in the current study, and therefore, we can only speculate on the role of ROS in the current study findings.
An interesting observation was that CR intake prior to injury and post-injury were lower than the expected target dosages. The conversion calculations to determine the target dosages for CR was based on an average rat weight of ~250 g. For example, given that CR-supplemented rats consumed on average 318 ± 18 mg during the loading phase, and weighed closer to ~200 g, their dosage per kg.bw would be around 1.6 g kg−1 body weight per day. If we divide by the rat conversion factor (6.2), we obtain a value of 0.256 g kg−1 body weight per day, which for a 70 kg individual would equate to ~18 g of CR per day. This is very close to the recommended dosage of ~20 g per day for loading. Despite the lower intakes, it was clear that the beneficial effects of CR supplementation were still apparent in the present study. Lower than expected target dosages were also seen in the WP-supplemented animals post-injury, with supplement intakes lower than the supplement target dosage of 5 g kg−1 body weight per day at day 7 and 14 post-injury. Given similar calculations as described above, our rats were consuming around ~33 g per day, which is lower than the targeted ~60 g per day, and could be contributing to the minimal benefits observed from WP.
A number of limitations exist in the current study. Firstly, intramuscular PCr and CR levels were not measured. Both injured and uninjured EDL muscles were tested for contractile properties, lasting approximately 40 minutes, before they were frozen. Thus, variation in PCr breakdown for each muscle would make it difficult to determine the effectiveness of the loading phase. Notwithstanding, given the changes observed in body weight, muscle mass, and force output, we can assume that levels within the muscles increased by ~20%, as typically reported following a standard loading regimen [62
]. Secondly, myotoxic injury results in a greater magnitude of damage, specifically the extent of muscle necrosis, compared to human models of injury. This should be carefully considered when making comparisons between models. The processes of regeneration are very similar in all models, albeit the absolute values reported on the extent and trajectories of the regenerative process, which may vary considerably [63
]. Finally, rats in the current study consumed their recommended dosage of WP in chow over a period of time rather than in one meal via oral gavage, as performed in other studies [64
]. We chose this route of administration to avoid additional stress on the injured animals. However, we acknowledge that not delivering the WP in one meal/bolus, could be a limitation to our supplementation protocol.