This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.
Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120°/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.
The overall goal of this procedure is to induce exertional damage to the quadriceps femoris muscles using voluntary lengthening (eccentric) contractions in human subjects.
Contraction-induced skeletal muscle damage is a common consequence of exercise that is marked by delayed onset muscle soreness1, transient strength loss, and elevated muscle-specific enzymes in the blood2. Exertional muscle damage is most pronounced following exercise to which the subject is unaccustomed, particularly when eccentric contractions are involved3. Exertional muscle damage is typically benign. Soreness subsides, and both serum proteins and strength typically return to pre-damage levels within a few days to weeks after the damaging insult. In extreme cases, exertional muscle damage can lead to a life-threatening syndrome know as rhabdomyolysis. However, exertional muscle damage is usually insufficient to cause clinical rhabdomyolysis in healthy individuals4 in the absence of compounding factors including heat stress, dehydration5, infection6 or rare genetic predispositions7.
Contraction-induced muscle damage is typically less severe than toxin-induced or freezing-induced injury, methods often used in rodent studies8,9. Yet, contraction-induced injury provides a useful method to study the muscle damage response with notable advantages. First, it is a safe and ethical method for use with human subjects1-3. Thus, interspecies translation of the results is not needed as data can be obtained directly from human subjects. Moreover, translating data obtained from rodent studies is very difficult given that the severity of injury seen in the rodent injury models exceeds the level of damage that would be ethical to induce in human subjects. Second, contraction-induced damage is commonly experienced and a natural process of exercise. Therefore, this mode of damage induction is useful for studying muscle damage in the context of exercise, adaptation to exercise as well as overt muscle injury. Here we describe a safe and reliable method to induce and evaluate skeletal muscle damage using lengthening contractions in humans.
The following procedures are in accordance with the standards of the Brigham Young University Institutional review board (IRB).
1. Prepare the Contraction Protocols
NOTE: The following protocol instructions are based on the Biodex Advantage software. Navigating the software and operating the dynamometer will be different if different systems are used.
2. Baseline Measurements
3. Damage Induction
4. Muscle Damage Assessment
Using the methods presented here, baseline soreness, serum creatine kinase activity, and strength (isometric and isokinetic) measurements were taken in 7 untrained young men. The following day, the subjects underwent the muscle damaging eccentric contraction protocol described above. To provide indices of muscle damage, follow up assessments of strength, soreness and serum creatine kinase activity were made. Strength was measured immediately after as well as 24, 48, 72, and 96 hr after exercise. Soreness was measured 24, 48, 72, and 96 hr after exercise. Serum creatine kinase was measured at 48 and 120 hr following exercise. These data were analyzed using a one-way repeated measures analysis of variance with a Dunnett's multiple comparison tests to compare post-exercise values to the pre-exercise value. Compared to pre-exercise, isometric and isokinetic force was decreased out to 24 and 48 hr post-exercise, respectively, and returned similar to pre-exercise values thereafter (Figure 2). Soreness was significantly increased at 24, 48, and 72 hr after exercise (Figure 3). Serum creatine kinase was significantly elevated 48 hr after exercise (Figure 4). Figure 5 shows an atypical isometric force curve in the days following damaging exercise.
Figure 1: The visual analog scale used to quantify muscle soreness of the quadriceps femoris muscles. Subjects are instructed to do two bodyweight squats and then indicate on the line the degree of soreness they felt in the quadriceps muscles during the squat maneuver. The researcher quantifies this by measuring the distance of the mark in mm from the no-soreness end of the scale. Please click here to view a larger version of this figure.
Figure 2: Isometric force (A) and 60o/s isokinetic force (B) of the knee extensor muscles one day before (pre), immediately after (imm post), 24, 48, 72, and 96 hr after a bout of 300 maximal effort lengthening contractions. Data are expressed as a percent of the pre-exercise force value (mean ± SD). Data were analyzed with a one-way repeated measures analysis of variance with a Dunnett's test for multiple comparisons. * indicates significant difference from pre (p < 0.05). This figure is adapted from reference3. Please click here to view a larger version of this figure.
Figure 3: The soreness response curve one day before exercise (pre) as well as at 24, 48, 72, and 96 hr following 300 maximal-effort lengthening contractions. The data are expressed on the log10 scale (mean ± SD). * indicates significant difference from pre exercise (p < 0.05). This figure is adapted from reference3. Please click here to view a larger version of this figure.
Figure 4: Serum creatine kinase (CK) activity (mean ± SD) 24 hours before (pre), 48 and 120 hr after 300 maximal-effort lengthening contractions (LC). Data are presented as the percentage of pre-exercise values on the log10 scale. * Indicates significant difference from pre (p < 0.05), n.s. indicates no significant difference. Please click here to view a larger version of this figure.
Figure 5: Isometric force of the knee extensor muscles of one subject who showed an atypical response. Force measurements immediately after (imm post) 24, 48, 72, and 96 hr after a bout of 300 maximal effort lengthening contractions were not reduced compared to the pre exercise (pre) value in this subject. Please click here to view a larger version of this figure.
Several steps are critical to obtaining the desired results of this protocol. First, subjects must be adequately familiarized to the contraction protocols, particularly the force measurements. Be sure that the subject understands exactly what they are expected to do and give them an opportunity to practice the strength tests prior to data collection. Subjects who are not adequately familiarized with these procedures may show a learning curve over the days following the damage induction. This can be a confounding variable rendering the strength measurements invalid. Figure 5 shows data from an individual who may have not been properly familiarized. This subject showed increasing strength due, perhaps, to learning over the course of the experiment despite the damaging exercise. Second, the selection of the subject population may also be critical to this protocol. Exertional muscle damage may vary greatly from subject to subject depending on many factors, including the trained status of the individual. Individuals accustomed to eccentric exercise will show much less damage compared to unaccustomed individuals. This may be an important consideration when selecting a subject population. If a researcher is interested in observing a large damage response, a population unaccustomed to eccentric exercise will be most likely to provide this outcome. Finally, it is critical that the subject is consistently positioned on the dynamometer over the repeated visits. The researcher should record the position settings for each adjustable component of the dynamometer for each subject and reposition it accordingly for each follow-up test. This will minimize variability due to postural differences.
Many different dynameters are available. While the methods provided here are specific to the Biodex dynamometer and control software, these methods can be adapted for use with other dynamometers after accounting for operating differences. The use of the dynamometer as presented here provides advantages over some other methods used to induce muscle damage in humans. These advantages include accurately quantifying the total work done during the exercise, controlling angular velocity, and accurately measuring strength and strength loss. However, other methods of damage induction can be used in the event that a dynamometer is not available or not preferred. Hubal and co-workers13 used a repeated chair sit and rise method to effectively induced muscle damage. In this study, subjects performed lengthening contractions by lowering into a chair. Stupka and co-workers14 used traditional knee extensor exercise during which subjects performed the lengthening component of the contraction at 120% of concentric one repetition maximum. Downhill running is also an effective stimulus to cause muscle damage15,16. These other methods of damage induction may be preferred if the researcher is interested in observing damage from a more real-world or sport-specific stimulus or from a closed kinetic chain movement. Another modification to this protocol that the researcher may consider is the frequency of blood sampling for creatine kinase measurement. To get the most informative picture of serum creatine kinase post-exercise, the researcher may choose to take blood samples every 24 hr for five to six days after damage. This will ensure that the peak serum creatine kinase values are not missed. Also this will provide better information as to the nature of the changes in serum creatine kinase, such as whether the curve follows a mono or biphasic shape.
One limitation of this protocol is that the strength loss, serum creatine kinase, and soreness, are indirect markers of damage. While direct evidence of damage can be observed by obtaining a muscle sample and using electron microscopy, other histological methods used in rodent studies do not detect voluntary contraction-induced damage in human muscle17,18. Considering the limited methods capable of directly detecting muscle damage in the context of voluntary contraction-induced damage in humans, indirect markers of damage are among the best options10.
Aspects of both data collection and analysis may require troubleshooting. During the muscle damaging protocol, the subject must trigger the movement of the shaft arm for each eccentric contraction. The subject does this by contracting against the stationary arm in an extended knee position until a threshold force value is met which triggers the shaft movement and the eccentric contraction. The threshold force value is 10% of the torque value programed into the contraction protocol (step 1.3.4.2). Subjects may have difficulty meeting the threshold force in the extended knee start position, especially after several sets have been completed. The researcher can help the subject reach the threshold to initiate the contractions by pulling on the shaft arm. Alternatively, the torque value programed into each contraction of the protocol (1.3.4.2) can be reduced so that the threshold is lower. However, the torque value must not be set too low because if the subject exceeds the torque value the shaft arm will stop and the contraction will be interrupted. The nature of the creatine kinase and soreness data may also provide some trouble for the investigator. These data sets are often not normally distributed. Both variables tend to show non-homogeneity of variance such that increased variability occurs with higher mean values. Due to the nature of these variables, a log transformation is often appropriate to normalize the distributions and homogenize the variance prior to statistical analysis. Alternatively, non-parametric tests may be used.
The exercise-induced muscle damage protocol described in this paper may have broad applications. The primary strength of using the protocol is the potential to study the cellular and molecular processes that govern muscle repair, regeneration, and adaptation directly in human subjects. Furthermore, delayed onset muscle soreness and temporary strength losses are often undesired consequences of strenuous, or novel exercise. Using an exercise-based damage protocol such as the one described here, researchers are provided a validated model can test the effectiveness of nutritional, nutraceutical, or pharmacological interventions or other treatments meant to protect against delayed onset muscle soreness following exercise.
In conclusion, this manuscript describes a safe and reliable method to cause and quantify contraction-induced skeletal muscle damage in humans. It uses eccentric contractions of the knee extensors that are controlled by a dynamometer to induce damage. Muscle damage is assessed noninvasively with delayed onset muscle soreness, serum creatine kinase and strength loss. While the protocol is written for a specific dynamometer for damage induction and force measurements, this protocol can be adapted for use with other dynamometers as well as other modes of contraction induced damage all together.
The authors have nothing to disclose.
The authors have no acknowledgements.
Biodex Dynomometer | Biodex Medical Systems | 850-000 | Other models are available and should produce similar results |
Creatine Kinase kit | Sigma-Aldrich | MAK116 | |
Serum Vacutainers | BD Bioscience | 367812 | |
Winged safety push button blood collection set | BD Bioscience | 367338 | |
Cryogenic vials | Sigma-Aldrich | V5007 | We use the 2mL vials to store serum aliquots |