Morphology and Anabolic Response of Skeletal Muscles Subjected to Eccentrically or Concentrically Biased Exercise

We obtained and studied 40 healthy adult male (age = 16 weeks, mass = 452.1 ± 35.2 g) Long-Evans rats (Envigo Laboratories, Indianapolis, IN) with the approval of the Institutional Animal Care and Use Committee at the University of Connecticut. All animals were housed in individual cages within the vivarium and allowed to consume food and water ad libitum for the duration of the study.

We used a randomized controlled laboratory study design. Experiments were conducted using a constrained rodent model, and rats were randomly assigned to an eccentric exercise (downhill walking, n = 16), concentric exercise (uphill walking, n = 16), or control (no exercise, n = 8) group. After a 1-week laboratory acclimatization period, rats began their respective exercise protocol based on group randomization. Rats in the exercise groups walked on a motor-driven treadmill (model EXER 3/6 treadmill; Columbus Instruments, Columbus, OH) at 16 m/min on a decline of 16° (eccentric exercise group) or on an incline of 16° (concentric exercise group).²⁰  The exercise comprised three 5-minute bouts of continuous walking with 2 minutes of rest between bouts, for a total treadmill time of 19 minutes. After the exercise, the rats were returned to their individual cages. They were euthanized at 6 or 24 hours postexercise (n = 8 allocated to each time), consistent with reports that these postexercise times are the peak hours of protein synthesis and fiber damage.^21–23  The vastus lateralis (VL) muscle was harvested bilaterally and immediately flash frozen in liquid nitrogen, stored in dry ice, and subsequently stored at −80°C. One VL muscle from each animal was then used for protein-synthesis analysis, and the other VL muscle was used for fiber-damage analysis.

We used traditional Western blot procedures²⁴  to detect activation of the mammalian target of rapamycin (mTOR) pathway via the downstream target of phosphorylated p70^S6k. This measure of protein synthesis, elevated phosphorylated p70^S6k relative to total p70^S6k, was selected because it is associated with greater muscle growth in both animals^21,25,26  and humans.^27,28  The central third of the VL muscle tissue (approximately 20 mg) was sectioned and homogenized in a radioimmunoprecipitation assay buffer (product R0278; Sigma-Aldrich, Inc, St Louis, MO) with a Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100X; Thermo Fisher Scientific, Waltham, MA). After homogenization, the samples were centrifuged at 5000 rpm at 4°C for 5 minutes. The supernatant was removed, and total protein concentration (in mg/mL) was determined using a bicinchoninic acid protein assay kit (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific). Next, 30 μg of the supernatant was calculated for each sample, diluted 1 : 1 with 450 μL of a 2× Laemmli sample buffer and 50 μL of a 1-M solution of dithiothreitol (products 161-0737 and 1610610, respectively; Bio-Rad Laboratories, Inc, Hercules, CA), and boiled with a heating block at approximately 95°C for 5 minutes. Samples were loaded into precast gels (10% Criterion Tris-HCL Protein Gel, 18-well, 30 μL; Bio-Rad Laboratories, Inc) alongside a protein ladder (500 μL Precision Plus Protein Dual Color Standards; Bio-Rad Laboratories, Inc) and were electrophoretically resolved and subsequently transferred to a polyvinylidene difluoride membrane activated via 100% methanol membrane (product 162-0177; Bio-Rad Laboratories, Inc). After transfer, the membrane was incubated in Ponceau S solution (Sigma-Aldrich, Inc) for visualization of the protein and assurance of equal loading in all lanes. Membranes were washed with tris-buffered saline and 0.1% Tween 20 (products BP2471500 and BP337100, respectively; Thermo Fisher Scientific) and subsequently incubated for 1 hour at room temperature in a blocking buffer (tris-buffered saline and Odyssey Blocking Buffer [product 927; LI-COR Inc, Lincoln, NE]). After 1 hour, the blocking solution was poured off, and the membranes were incubated overnight at 4°C in the primary antibodies in 1 : 1000 dilutions of phosphorylated and total p70S6k (antibodies #9206 and #9202, respectively; Cell Signaling Technology, Inc, Danvers, MA). The membranes were subsequently washed and incubated at room temperature for 1 hour in secondary antibodies in 1 : 10 000 dilutions of phosphorylated p70^S6k (antibody #A32729; Goat Anti-Mouse IgG [H + L], Superclonal Recombinant Secondary Antibody, Alexa Fluor 680; Thermo Fisher Scientific) and total p70^S6k (antibody #A32735; Goat Anti-Rabbit IgG [H + L] Highly Cross-Absorbed Secondary Antibody, Alexa Fluor Plus 800; Thermo Fisher Scientific).

The levels of phosphorylated and total p70^S6k were quantified using an Odyssey infrared imaging system (LI-COR Inc) and Image Studio software (LI-COR Inc). After the background was subtracted, the captured intensities of the phosphorylated and total p70^S6k bands were individually quantified. To minimize the effects arising from multiple blots and improve data reliability, we calculated an in-lane total p70^S6k normalization factor for each blot. We accomplished this by identifying the highest total p70^S6k signal on each blot and using this signal intensity to normalize all other total p70^S6k signal intensities. The phosphorylated p70^S6k signal was divided by the total p70^S6k in-lane normalization factor to produce the normalized experimental signal for each lane. To determine the percentage increase or decrease in protein abundance relative to that of unexercised control animals, the percentage response equation was applied to each lane (Equation):

Fiber damage was measured using positive myofibrillar staining. This technique assesses damage to the cellular membrane by quantifying muscle fibers that are lysed and permit the influx of the large extracellular immunoglobulin G (IgG) complex into the fiber. The IgG infiltration can be visualized using fluorescent dye.²⁹  In preparation for IgG staining, we sectioned frozen midbelly cross-sections of the VL (8 μm) and mounted them on glass slides. Slides were then fixed in ice-cold acetone (100%) for 10 minutes, washed 3 times with phosphate-buffered saline (PBS), and incubated in 5% bovine serum albumin solution in PBS for 20 minutes at room temperature. The primary antibody (Anti-Mouse IgG [whole molecule] FITC antibody, F0257; Sigma-Aldrich, Inc) was applied, and the slides were incubated in this solution overnight at 4°C. Next, we washed the slides twice in the 5% bovine serum albumin in PBS solution and once in PBS. Slides were then stained with 4′,6-diamidino-2-phenylindole (product D9542; Sigma-Aldrich, Inc) and coverslipped with VECTASHIELD Antifade Mounting Medium (product H-1000; Vector Laboratories, Burlingame, CA). Images were captured using a Zeiss Axio Imager M1 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Five random fields of each muscle were photographed at a magnification of ×100 and subsequently analyzed using ZEN blue software (Carl Zeiss Microscopy GmbH). Fluorescent intensity was quantified by the densiometric mean of each fiber (in arbitrary units). A trained, blinded assessor (M.S.W.) counted the IgG infiltration, a marker of overt fiber damage.

For the comparisons of exercise groups, 1-way analysis of variance was performed using GraphPad Prism statistical software (version 8.2.0; GraphPad Software, San Diego, CA). When we observed a difference, we applied a post hoc Tukey test to identify the differences between groups. Outliers that were 2 standard deviations from the group mean were removed from the analysis. All values are reported as mean ± standard error of the mean. We set the α level a priori at .05.

Control group rats were used to provide normalized signaling of the phosphorylated p70^S6k signal and total p70^S6k relative to exercise groups (Equation). Therefore, this group was not included in the analysis of variance, as it was mathematically accounted for in the percentage response equation. A difference in VL muscle protein response was detected between exercise groups (F_3,54 = 4.50, P = .006; Figure 1). The post hoc test revealed that rats in the eccentric exercise group euthanized at 24 hours postexercise had a higher VL muscle percentage response than rats in the eccentric exercise group euthanized at 6 hours postexercise (P = .02) and rats in the concentric exercise group euthanized at 6 (P = .03) or 24 (P = .03) hours postexercise. No other between-groups differences were observed.

We observationally quantified the fluorescent intensity of 9446 fibers via the densiometric mean and inspected them for damage using positive myofibrillar staining (Figure 2). Notably, only 1 fiber was infiltrated. This fiber was found in the concentric exercise group euthanized at 6 hours postexercise (Figure 2B). No between-groups differences in fluorescent intensity were detected (F_4,30 = 0.184, P = .94).

The acute ability of eccentric exercise to promote muscle growth and limit muscle damage is not well understood. This misunderstanding is, in part, due to classic animal experiments in which investigators^{6–8,15–17,22,30,31}  isolated muscles in situ and single fibers in vitro and applied lengthening stretches of a very high magnitude to induce fiber injury, far exceeding the physiological strains experienced during in vivo muscle contractions. Other researchers have counteracted the concerns with these experiments by studying in vivo exercise protocols (eg, the muscle is kept intact and the system is not physiologically altered). Specifically, Armstrong et al²²  examined the acute effect of eccentric exercise on an untrained muscle by using downhill and uphill walking protocols in rats to eccentrically and concentrically strain the quadriceps muscle, respectively. However, the dose of exercise used in these experiments (90 minutes of activity) was specifically chosen to cause injury, limiting the clinical translation.²²  A similar experiment has been conducted in humans, with researchers³¹  exposing novice men to eccentric exercise. However, again, the authors selected such high doses that muscle injury was unavoidable (30 minutes of continuous eccentric contractions to the untrained quadriceps muscle).³¹  Remarkably, if the dose of eccentric exercise is tempered, little is known about the acute response of the untrained muscle to eccentric exercise.

Our data demonstrated that a single 15-minute dose of eccentric exercise to an untrained muscle promoted a protein synthesis response and was not associated with muscle-fiber damage. We believe these data are critically important because knowing if an acute bout of eccentric exercise causes severe muscle damage that precedes long-term adaptations^3,13,14  is essential to encouraging patient compliance during the initial phases of exercise. These findings have great potential for musculoskeletal rehabilitation, as strength is not commonly restored after joint injury^1,2,10  and incorporating eccentric exercise to promote an anabolic response is a simple, universally available therapeutic intervention to promote muscle growth.

Distinctly different from the actions of muscle during concentric contractions, eccentric contractions mechanically tension the muscle in a unique way that triggers key molecular events that promote muscle growth.^12,32  Specifically, titin, the giant elastic protein that spans the entire length of the sarcomere, is involved in a mechanotransduction-signaling complex that regulates tissue growth.¹²  Briefly, when the sarcomere is elongated, titin unfolds at the I-band. This unfolding allows for the phosphorylation of titin kinase.¹²  Phosphorylation of titin kinase begins a cascade of events that ultimately promote muscle growth.¹²  Importantly, only a positive strain of the sarcomere, achieved via eccentric contractions, triggers this pathway, and more force per active fiber increases the mechanical stimulus that, in turn, increases protein synthesis rates.^12,32  Over time, this unique mechanism will likely lead to the accumulation of new protein,^21,25,26  suggesting that the immediate incorporation of eccentric exercise in an exercise regime is likely an effective intervention to promote muscle growth.

The mTOR pathway is arguably the most well-recognized pathway that leads to protein synthesis. Total p70^S6k is a downstream target of the mTOR pathway and is associated with greater muscle growth in both animals^21,25,26  and humans.^27,28  Enhanced signaling can be detected by probing the muscle for evidence of phosphorylated relative to total p70^S6k (eg, newly created protein relative to housekeeping levels of protein). Most data^21,27  have shown that a greater increase in muscle protein synthesis rates generally leads to long-term adaptations in muscle size and strength. However, some investigators^33,34  have shown that elevated p70^S6k signaling does not always lead to muscle growth. In the case of overtraining or exercising to exhaustion, phosphorylated p70^S6k levels can be increased, but oxidative stress is thought to inhibit growth,³³  ultimately leading to muscle loss.³⁴  Other researchers³⁵  have also reported that mTOR may be involved in the repair of muscle damage associated with eccentric exercise. Emerging contemporary data³⁶  have demonstrated that protein translation through eukaryotic translation initiation factor 4E–binding protein 1 activation, which is believed to be triggered by p70^S6k, is not always sufficient to promote translation. Given the limited exposure to continuous exercise in our experiments (three 5-minute bouts of exercise separated by 2 minutes of rest) and the sparse fiber damage observed, we contend that the acute activation of the mTOR pathway in our study will likely lead to muscle growth. Future experiments with protracted time-series data are needed to confirm or refute this hypothesis.

The time course of protein synthesis rates can vary. An animal experiment²¹  showed that, after a single high-frequency electrical-stimulation protocol, acute activation of p70^S6k peaks at 6 hours postexercise. Other researchers³⁷  observed that human muscle protein synthesis rates peaked at 24 hours after a single bout of biceps exercises. Given the variance in the muscle-protein synthesis rates in the literature, we probed 2 key time frames postexercise to detect a response. Our data demonstrated that protein synthesis peaked after 24 hours of a single bout of in vivo eccentric exercise. These results vary from those previously reported²¹  in animal experiments. We believe this difference may be due to the experimental environment (in vivo versus in situ) and method of exercise (eccentric versus electrical stimulation).

Compared with many classic animal experiments in which researchers^{6–8,22,30,31}  studied eccentric exercise using in situ and in vitro exercise protocols, very limited fiber damage was observed in our experiment. We believe this was primarily due to the elastic properties of the intact muscle-tendon unit. Tendons act as a mechanical buffer that reduces impact to the muscle. Specific to eccentric contractions, tendons absorb energy during a brief rapid event (eg, landing from a jump) and then release this energy to the active muscle during lengthening contractions. This system is inherently built to protect muscles from damage. Another consideration is that the quadriceps muscles are highly pennate. During contraction, a change in muscle length is the net result of cross-bridging and the rotation of muscle fibers along the muscle's line of action, which leads to a change in muscle-pennation angle. An increased pennation angle (due to more fiber rotation) is a protective mechanism of the muscle and an indicator that the muscle-tendon unit has become decoupled from the muscle fascicle.³⁸  This decoupling leads to relatively minimal stretching of the fascicle, as the muscle-tendon unit absorbs most of the lengthening contractions.³⁸  In this way, pennate muscles are uniquely protected from fascicle strain and, thus, muscle-fiber damage. These data provide further evidence that muscles such as the quadriceps would uniquely benefit from eccentric exercise. However, a limitation of our work was that the time frames were selected to optimize capture of muscle protein synthesis rates. Evidence of muscle damage can occur up to 72 hours postexercise. Given the limited fiber damage observed at 24 hours, we anticipate that muscle damage present at later times would not be statistically different from that at earlier times. However, these data need to be confirmed with future experiments.

A single 15-minute dose of eccentric exercise to a novice muscle was better at exerting an anabolic effect on muscle than a comparable dose of concentric exercise. Furthermore, no muscle-fiber damage was observed. The possibilities for the clinical application of eccentric exercise are promising for patients seeking to build muscle tissue.

Funding for this study was provided by Research & Education Foundation New Investigator award (1516NIGP002 [Dr Lepley]).