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Journal of Science in Sport and Exercise https://doi.org/10.1007/s42978-019-00039-z

ORIGINAL ARTICLE

Skeletal Muscle Fiber Type and Morphology in a Middle‑Aged Elite Male Powerlifter Using Anabolic Steroids

Steven B. Machek1,2 · Kent A. Lorenz1 · Marialice Kern1 · Andrew J. Galpin3 · James R. Bagley1

Received: 31 July 2019 / Accepted: 21 September 2019 © Beijing Sport University 2019

AbstractPowerlifting regularly exposes athletes to extreme stimuli such as chronic heavy resistance training (HRT), and many pow-erlifters choose to augment their performance with anabolic–androgenic steroids (AAS). However, little is known about the myocellular adaptations that occur from long-term HRT and AAS use, especially into middle age. We were presented with the unique opportunity to study muscle cells from an elite-level powerlifter (EPL; age 40 years) with ≥ 30 years of HRT experience and ≥ 15 years of AAS use. The purpose of this case study was to identify myocellular characteristics [myosin heavy chain (MHC) fiber type, fiber size, and myonuclear content] in EPL, as well as compare these data to existing litera-ture. The participant underwent a resting vastus lateralis muscle biopsy and single fibers were analyzed for MHC content via SDS-PAGE. A subset of fibers underwent MHC-specific imaging analysis via confocal microscopy to identify cell size (cross-sectional area, CSA) and myonuclear domain (MND) size. MHC fiber type distribution was 9% I, 12% I/IIa, 79% IIa, and 0% other isoforms. This pure MHC IIa (fast-twitch) fiber content was amongst the highest reported in the literature. Imaging analysis of MHC IIa fibers revealed a mean CSA of 4218 ± 933 μm2 and MND of 12,548 ± 3181 μm3. While the fast-twitch fiber CSA was comparable to values in previous literature, mean MND was smaller than has been reported in untrained men, implying greater capacity for growth and repair. These findings showcase the unique muscle cell structure of an elite powerlifter, extending the known physiological limits of human muscle size and strength.

Keywords Resistance training · Myonuclear domain · Myonuclei · Myosin heavy chain · Confocal microscopy · Muscle biopsy

Introduction

Athletes competing in the sport of powerlifting regularly engage in chronic, extraordinarily heavy resistance training (HRT). While the effects of resistance training on muscle cell characteristics have been well documented [11], little

research exists on elite powerlifter muscle cells. Further-more, most research on HRT is acute and may not accurately represent long-term HRT-induced adaptations. Between 33 and 66% of elite-level powerlifters have reported using ana-bolic–androgenic steroids (AAS) to augment the effects of their training and subsequent recovery [6, 43]. These testos-terone-derived compounds exert ergogenic effects through androgen receptor interaction and subsequent DNA binding, potentiating increases in muscle size and strength [1]. Little is known about the myocellular adaptations that occur from the combination of both long-term HRT and AAS use in elite powerlifting athletes, especially into middle age.

Various myocellular characteristics affect muscle perfor-mance, such as muscle fiber type, fiber size, and muscle nuclei (i.e., myonuclei) content. Muscle fiber types are clas-sified on a continuum based on myosin heavy chain (MHC) isoforms. These include (in humans) slow-twitch (MHC I), fast-twitch (MHC IIa), super-fast-twitch (MHC IIx), or co-expressing hybrids (MHC I/IIa, MHC IIa/IIx, and MHC I/

* James R. Bagley jrbagley@sfsu.edu

1 Muscle Physiology Laboratory, Department of Kinesiology, College of Health and Social Sciences, San Francisco State University, 1600 Holloway Avenue, Gym 101, San Francisco, CA 94132, USA

2 Exercise and Biochemical Nutrition Laboratory, Department of Health, Human Performance, and Recreation, Robbins College of Health and Human Sciences, Baylor University, Waco, TX, USA

3 Biochemistry and Molecular Exercise Physiology Laboratory, Center for Sport Performance, California State University, Fullerton, USA

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IIa/IIx) [30, 34]. Using single muscle fiber isolation meth-ods with confocal microscopy, it is possible to identify fiber type-specific myocellular size (e.g., cross-sectional area, CSA) and myonuclear content (e.g., myonuclear domain size, MND) [2]. Myonuclei are critical in growth and repair processes through their role in transcription and subsequent translation, making them prime targets for elucidating adaptation to exercise stimuli [27, 29, 34]. Charles Epstein originally postulated the concept of a myonuclear domain (MND), where a cell’s size is proportional to the transcrip-tional power of its nucleus [9]. A size threshold is suggested in skeletal muscle, whereby myonuclear accretion is required to support hypertrophy and a tight MND [33]. Nevertheless, this hypothesis is controversial. Recent data demonstrates that myonuclear accretion more likely occurs in response to cellular damage in lieu of cell growth [26].

We had the unique opportunity to recruit a competitive elite powerlifter with a background of AAS use. The sub-ject has squatted more than 450 kg in powerlifting competi-tion, whereas the previously highest recorded squat value in the powerlifting research literature was 317.5 kg [13]. One investigation examined elite powerlifter performance and muscle fiber morphology in conjunction with AAS, but singularly examined the barbell bench press [17]. Conse-quently, the effects of AAS on an elite powerlifter’s squat and associated leg musculature are currently unknown [17]. Both HRT and AAS exert adaptations on muscle fiber type, cell size, and MND [7, 44]. Therefore, our investigation aimed to determine the MHC fiber type distribution, fiber size, and fiber-specific MND in the vastus lateralis (VL) muscle of an elite male powerlifter.

Methods

Subject and Recruitment

This study conformed to standards set forth by the Decla-ration of Helsinki and was approved by the Human Sub-jects Institutional Review Board (IRB) at California State University, Fullerton. The subject received oral and written information about experimental procedures and any poten-tial risks prior to providing written consent. An elite-level male powerlifter subject (EPL; aged 40 years) was recruited to participate in the study. Age, height (180.3 cm), and weight (118.0 kg) were recorded to establish anthropomet-ric descriptive data. One muscle biopsy sample was taken and a total of 250 fibers were analyzed for fiber type, and 50 fibers were imaged and analyzed via laser scanning confocal microscopy. Auxiliary information was acquired from the subject regarding AAS use regimen, training and dietary habits, as well as best competition lifts. The subject’s best competition squat (490.0 kg), bench press (387.5 kg), and

deadlift (347.5 kg) were verified using a comprehensive online database for powerlifting data (OpenPowerlifting.org).

At the time of the biopsy, the  EPL reported current AAS use since age 36 (~ 4 years) under the guidance of a physician. He reports unregimented and experimen-tal past AAS use from age 25, but has consistently been using ≥ 500–600 mg testosterone (cypionate) per week since seeking medical supervision. The EPL reported that prior to his current regimen, he haphazardly explored combina-tions of injectable and oral AAS ranging up to ~ 2000 mg/week. Historically, the subject seldom discontinued AAS use, only ceasing administration between ages 30 and 31 for personal reasons. The EPL occasionally paused use for 2–4-week periods during his current regimen, but otherwise remained consistent. EPL has extensive HRT history, having competed in 29 sanctioned powerlifting meets (in a 10-year timeframe) prior to muscle biopsy. Although online records are unavailable before 2005, the EPL reported competing in powerlifting since 1990. In competition, the EPL has per-formed a combination of Raw (limited supportive equip-ment), Classic Raw (limited supportive equipment with knee wraps), Single-ply, and Multi-ply events (“ply” referring to the degree of supportive material layer[s]) [40]. Spanning his career, the EPL achieved a 673.68, 599.51, and 492.45 Wilks coefficient (a validated powerlifting formula normal-izing total lifted to bodyweight), in multi-ply, single-ply, and raw with wraps, respectively [42]. This sets him in the 99th percentile for multi-ply and single-ply, as well as the 98th percentile for classic raw divisions across all registered lifters (OpenPowerlifting.org). From 2015 to 2018, the EPL went on a powerlifting hiatus and adopted bodybuilding-style training. Lastly, the EPL indicates he is currently fol-lowing “The Carnivore Diet”, which is characterized by a high-protein, high-fat diet of almost exclusively red meat and eggs, and strives to exclude carbohydrates.

Muscle Biopsy and Muscle Fiber Processing

The EPL rested supine for ~ 30 min and then received a local anesthetic (1% lidocaine/xylocaine) at the mid-muscle belly (half-way between the greater trochanter and patella) of the vastus lateralis before a biopsy (Bergström technique with suction), as described previously by our team [3, 25, 38, 39]. Samples were divided into ~ 15 mg bundles and placed in cold skinning solution (125 mM K propionate, 2.0 mM EGTA, 4.0 mM ATP, 1.0 mM MgCl2, 20.0 mM imidazole [pH 7.0], and 50% [vol/vol] glycerol) and stored at – 20 °C.

After 5 days in skinning solution, 250 single fibers were isolated and placed in a microscope well with relaxing solu-tion (7.0 M EGTA, 20.0 M Imidazole, 5.42 mM MgCl2, 79.16 mM KCl, 16.33 μM CaCl2, 14.5 mM CrP, 4.75 mM

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ATP, [pH 7.0]). A subsection of 50 isolated muscle fibers was separated into a ~ 1/4 segment and a ~ 3/4 segment using fine tweezers (See Fig. 1). The ~ 1/4 segment was placed into 40 μL of SDS buffer (1% SDS, 23 mM EDTA, 0.008% bromophenol blue, 15% glycerol, and 715 mM b-mercanp-toethanol [pH 6.8]) for subsequent analysis of MHC fiber type via SDS-PAGE. Next, ~ 3/4 segments of each fiber were placed in Phalloidin (with AlexaFluor 568, Life Technolo-gies; Carlsbad, CA, USA) in relaxing solution for 2 h to stain the muscle fiber (actin filaments). The fibers were then rinsed with relaxing solution and stained with 4,6-diamino-2-phenylindole (DAPI) in a mounting medium (ProLong Gold Antifade with DAPI, Molecular Probes; Eugene OR, USA) to label myonuclei. Fibers were covered (2 fibers per slide) with 0.5 mm glass coverslips and sealed with nail polish. Slides were stored at 4 °C or immediately imaged.

Fiber Type Analysis

A total of 250 (200 whole single muscle fibers and 50 ~ 1/4 single fiber segments) were stored in 40 μL of SDS buffer for later MHC identification via SDS-PAGE. Briefly, 1–2 μL aliquots of single fibers were loaded into individual wells in a 3.5% loading and 5% separating gel at 15 °C for 15.5 h (SE 600 Series; Hoefer San Francisco, CA, USA), and sil-ver stained for MHC identification. Single fiber SDS-PAGE allows identification of the MHC isoform (MHC I, I/IIa, IIa, IIa/IIx, and IIx) in each individual fiber. All 250 EPL fib-ers were analyzed for MHC type, where 50 of the total 250 single fibers were ~ 1/4 segments from full-length isolated fibers. The remaining ~ 3/4 of the segmented fibers were ana-lyzed via confocal microscopy.

Muscle Fiber Imaging (3D Confocal Microscopy)

Remaining ~ 3/4 segmented fibers were longitudinally imaged in three-dimensions (3D) via a confocal micro-scope (Zeiss LSM 710; Carl Zeiss AG, Jena, Germany) using methods described previously by our team [2] (see Fig. 2a). Of the 50 imaged muscle fibers, 20 were selected for analysis of size and myonuclear parameters. An undam-aged region of interest was identified (~ 500 μm long) and imaged using a Plan-Apochromat 20×/0.8 objective lens. Nuclei labeled with DAPI were detected at a laser line of 405 nm using a wavelength range of 410–585 nm. Actin filaments labeled with AlexaFluor 568 (Phalloidin) were excited with the laser line at 594 nm and collected with a wavelength range of 599–712 nm. Data were collected in 12-bit depth for both channels. Two images were obtained per fiber, one two-dimensional (2D) image to obtain sar-comere spacing, and one 3D image to obtain muscle cell size and myonuclear content. Z stacks were acquired by col-lecting consecutive images from the top to bottom of each myofibril. Sarcomere spacing was used as a control, and a range of 1.75–2.50 μm was used as a criterion for inclusion. All fibers outside of this range were excluded from analy-ses, as potential indication of fiber damage/stretch during mechanical isolation. These data were then analyzed using open source imaging software to quantify muscle fiber size (500 μm region volume/500 = CSA in μm2) and MND size (nuclei per 500 μm/500 μm region volume = MND in μm3) (ImageJ, FIJI Distribution) [37]. These data were compared with values in the literature [5, 24, 28, 31, 36]. MHC fiber type distribution is expressed as a percentage of the total fiber pool. All values are reported as mean ± SD.

Fig. 1 Skeletal muscle fiber processing methods for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and laser scanning confocal microscopy (LSCM)

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Results

Fiber Type Distribution, Muscle Fiber Cell Size, and Myonuclear Content

MHC fiber type was 9% MHC I, 12% MHC I/IIa, 79% MHC IIa, and 0% other isoforms (MHC IIx, MHC IIa/IIx, or MHC I/IIa/IIx).

Of the 50 fibers imaged via confocal microscopy, 20 were of sufficient quality as determined by sarcomere spacing cri-teria and lacking characteristics that may impede analysis (i.e., mechanical damage to the fiber or bubbles on the slide). All 20 imaged fibers corresponded to MHC IIa isoforms and were analyzed for fiber cell size and myonuclear content. Two of the 20 fibers were excluded due to both a cell volume and myonuclear content that were ± 2 standard deviations from the mean. Of the remaining 18 MHC IIa fibers, the mean CSA was 4218 ± 933 μm2 with a mean cell volume of 2,108,923 ± 466,619 μm3. The mean MND for MHC IIa fibers was 12,548 ± 3181 μm3. Only 1 MHC I fiber was available. The CSA of this fiber was 990 μm2, with a volume of 989,691 μm3, and an MND of 10,997 μm3.

Sarcomere Spacing

In a 100-μm segment, the average sarcomere spacing was 2.23 μm in the 20 analyzed MHC IIa fibers, and the single MHC I fiber had sarcomere spacing of 2.50 μm.

Discussion

This is the first investigation on an elite powerlifter to ana-lyze single muscle fiber MHC isoform distribution, fiber size, and myonuclear content. Our participant demonstrated an extreme preponderance of MHC IIa fibers, and while these fibers ultimately displayed a fairly typical size, their MND was relatively small. Unsurprisingly, the EPL had few hybrids and a substantial expression of the MHC IIa isoforms. The EPL’s MHC IIa content is comparable to the highest known MHC IIa content to date, recently recorded by our research team in world-class weightlifters (79% vs. 71%, respectively) [38]. His extensive training age and almost 80% MHC IIa proportion matches previous work in powerlifters, weightlifters, and anaerobically trained athletes

Fig. 2 a Representative rendering of single muscle fiber from the elite powerlifter (EPL) imaged longitudinally via confocal microscopy. Actin filaments (F-actin) are labeled with Phalloi-din (AlexaFluor568) and myonuclei are labeled with DAPI (gray bar = 100 μm). b The EPL myosin heavy chain (MHC) IIa fiber size

(cross-sectional area, CSA) compared to various other species data presented as percentage ± standard deviation [24]. c The  EPL myo-nuclear domain (MND) size compared to males across various age ranges. Data presented as percentage ± standard deviation [5, 24]

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that describes a fast-twitch shift [10, 12, 13, 16, 22, 32, 38]. The relationship between training exposure and MHC IIa content is further corroborated by Serrano et al. [38], who described a higher MHC IIa percentage in world-class female weightlifters (WCF) of greater training age relative to less experienced national-class female (NCF) weightlifters. It should be noted that four individual weightlifters (3 WCF and 1 national-class male [NCM]) had a greater proportion of MHC IIa fibers and comparable levels of MHC I fibers to EPL. Nevertheless, our participant has a higher MHC IIa content compared to the majority of world-class male and female weightlifters, achieving this distribution at a much older biological age (40 years [EPL] vs. 23.6 ± 3.9 [NCF] and 25.6 ± 3.8 years [NCM]) [38]. The ability to maintain a fiber type profile at the EPL’s age is unprecedented in the current literature, and it can be assumed that is possible through the synergistic effects of HRT and AAS adminis-tration [10]. Along with a higher MHC IIa content, the EPL has a higher percentage of I/IIa hybrid fibers similar to pre-viously investigated bodybuilders [21]. This is likely due to the EPL’s focus over the previous 3 years on bodybuild-ing-style training. Conversely, the current investigation did show a stark contrast to Kesidis et al. [21], where the average percent distribution of MHC IIa fibers was 30% in student controls and 38.8% in bodybuilders, compared to a daunting 79% in EPL. Furthermore, the mean distribution of MHC I expressing fibers was 40.3%, 35.1%, and only 9% in student controls, bodybuilders, and EPL, respectively. Across vari-ous athletic demographics analyzed via single fiber SDS-PAGE, it is extremely uncommon to have an MHC I content below 10% in the human vastus lateralis. Nevertheless, the results support the findings of Kadi et al. [19], who observed

significantly lower MHC I content in powerlifters compared to sedentary controls. It can be hypothesized that perhaps the culmination of training frequency, training volume, as well as factors related to AAS exposure history and dosing patterns are responsible for the EPL’s differential fiber type profile.

The mean CSA of the EPL’s MHC IIa fibers was higher than sedentary populations and older track athletes in previ-ous research, but not noticeably higher than an elite sprinter or younger track athletes [23, 36, 41]. Previous work by Eriksson et al. [10] showed that elite-level powerlifters using AAS had a higher CSA in type II fibers compared to drug-naïve controls. This does not appear to be the case in the EPL, regardless of his extensive AAS use. This dis-crepancy may be due to the small sample size inherent to a case study approach or to exclusion of data outliers. Spe-cifically, one fiber was omitted from data analysis due to a cellular volume that was more than two standard deviations higher (3,833,006 μm3) than the mean. Mention of this out-lier should not be overlooked from the current discussion because it did fit sarcomere length criteria for analysis. Inter-estingly, this fiber is more than 22% greater than the average CSA of Rhinoceros MHC IIa fibers seen in an investigation by Liu et al. [24] (see Fig. 2b).

This is the first determination of MND size in an elite powerlifter, also utilizing single fiber SDS-PAGE method-ology (see Table 1). Due to lacking MHC isoform expres-sion diversity, MND in the EPL could only be determined in MHC IIa fibers. One MHC IIa fiber was excluded from analysis due to a myonuclear number that was > 2 × stand-ard deviations from the mean value (458 myonuclei/mm vs. mean 170 ± 30 myonuclei/mm). Compared to earlier work

Table 1 List of studies that include skeletal muscle fiber type-specific myonuclear domain (MND) size measures via confocal microscopy in human subjects (i.e., similar methods as the current case study) [5, 24, 28, 31, 36]

1RM 1-rep maximum, AAS anabolic androgenic steroid, ATPase adenosine triphosphatase, CSA cross-sectional area, EDL extensor digitorum longus, GM gluteus medius, GN gastrocnemius, HP healthy population, HRT hormone replacement therapy, LD latissimus dorsi, LDE long distal extensor, MN myonuclear number, MND myonuclear domain, MHC myosin heavy chain, NMRI nuclear magnetic resonance imaging, NP normal population, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis, VL vastus lateralis

References Muscle Fiber Type-Specific Experimental Condition MND Size Δ

Ohira [28] VL YesSDS-PAGE

2- or 4-min bed rest ↓ MHC I MND↔ MN/mm

Cristea et al. [5] VL YesSDS-PAGE

Young (21–31 years) vs. old (72–96 years) men and Young (24–31 years) vs. old (65–96 years) women

↑ MHC I MND size variability with age↓ MHC IIa MND size with age

Qaisar et al. [36] VL YesSDS-PAGE

Female homozygous twin pairs (55–59 years) One twin on HRT

↓ in MHC I MND size with HRT

Liu [24] Various (EDL, GN, GM, LDE, VL)

YesSDS-PAGE

Comparative physiology without inter-vention

Across species: MHC I = smallest MNDMND size scales with body size and fiber

typePaoli [31] LD Yes

SDS-PAGENormal protein (NP: 0.85 g/kg/day) vs.

high (HP: 1.8 g/kg/day) protein diet in young, healthy males + 8-week resist-ance training program

↓ MHC IIx &↑ MHC IIa in NP↔ in HP↑ MND after resistance training, irrespec-

tive of MHC type or CSA

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using similar myonuclear determination methods, the EPL had a lower MHC IIa MND relative to healthy men rang-ing from 21 to 96 years old (12,836 ± 4388 μm3 [EPL] vs. 35,100 ± 2500 μm3 [21–31-year-old men] and 23,500 ± 1400 μm3 [65–96-year-old men]) (see Fig. 2c) [5]. Another investigation examined a human male subject with a MND of 32,900 ± 2100 μm3, ultimately greater than our findings in EPL [24]. Our participant’s chronic HRT experience may consequently impose a smaller MHC IIa MND to meet pro-tein turnover demands and subsequent recovery [35].

Unfortunately, the 50 fibers from  the EPL that were imaged did not yield sufficient MHC I fibers for comparison to MHC IIa. As previously mentioned, type I fibers are pref-erentially affected acutely in response to muscular stimuli (or lack thereof) and aging [14, 20]. Multiple investigations have found a smaller slow-twitch MND due to increased demand for cellular turnover [35]. Furthermore, it would have been illustrative to determine how the EPL’s fiber type-specific MND correlated with both his age and training sta-tus. Work by Cristea et al. [5] described an increased MHC I MND and decreased MHC IIa MND with increasing age. Nevertheless, comparing the singular MHC I fiber that was available, the results corroborate a smaller MHC I MND in prior literature [35].

One unique characteristic to our case subject was his extensive AAS use, which has been shown to cause differ-ential effects on MND size. The EPL’s lower MHC IIa MND size compared to male participants in previous investiga-tions is supported by Kadi et al. [19] and Eriksson et al. [10]. Their work determined AAS-using powerlifters had a higher myonuclear number per cell volume. Conversely, Yu et al. [44] saw no difference in myonuclear density when accounting for fiber size, but the difference may be due to a discrepancy in MND determination methodology, in which the latter investigator utilized immunohistochemistry (i.e., cross sections). Moreover, the subjects were not homoge-nously powerlifters, but rather were a mix of bodybuilders, weightlifters, and strongmen [44]. It is also worth noting that the EPL has had a historically inconsistent AAS dose and usage pattern. The current investigation can only con-firm the EPL’s anecdotal dosage and pattern of AAS use from the last 4 years under medical supervision; however, he had professed to haphazard experimentation with vary-ing doses and combinations of performance enhancing sub-stances since around age 25. Nevertheless, rodent models have shown AAS use can cause higher myonuclear retention and a greater sensitivity to training after drug cessation [8]. This gives credence to speculate the augmentative effects of years of varying AAS compounds and their ability to endure and compound across multiple years.

Very few investigations in human performance-centered AAS administration report substance dose or use patterns [4, 10, 17, 18]. The highest reported dose of 938 ± 527 mg/

week testosterone was in a cross-sectional analysis of pow-erlifters [10]. This is comparable to the ≥ 500–600 mg/week regimen used over the last ~ 4 years by EPL. Regardless, both AAS patterns are confounded by undocumented, intermittent experimentations with cocktails of other illicit performance enhancing drugs, including IGF-1 and other testosterone derivatives [10]. The inability to hitherto control for past AAS use in athletic populations is an issue that is not eas-ily resolved; safe, empirically backed use is undermined by social and moral barriers that stigmatize use for performance enhancement [15].

Limitations and Conclusions

The primary limitation to the current study is the small sub-ject number inherent to a case study approach. While data on an elite powerlifter with extensive AAS-use history are novel, future research should utilize age-matched, seden-tary (and trained) controls to tease out the effects of both chronic HRT and AAS usage. Additionally, because acquir-ing a muscle biopsy sample from the case subject was a rare opportunity, it was unfeasible to impose a standardized training protocol, nor a dose and specific vehicle for AAS usage before the sampling.

The unique opportunity to analyze EPL served to illus-trate the dynamic nature of skeletal muscle plasticity. Along with an astoundingly dominant percentage of MHC IIa iso-form expression, it is hard to ignore the two fibers that were excluded from analysis. The largest fiber examined was nearly double (~ 1.7 times) the size and more than two stand-ard deviations away from the mean fiber volume. The other fiber presenting itself as an outlier had an MND that was under half the mean value and contained 2.5 times as many myonuclei per mm of fiber volume. At least with regards to the context of the EPL’s MHC IIa fibers, these outliers repre-sent the extremes of athletic human muscle fiber physiology.

The desire to augment performance with AAS is enduring and prevalent in powerlifters who compete in official fed-erations [6]. It is imperative to discern how these powerful exogenous compounds can affect the wide range of individu-als who may experiment with their probable illicit use, as well as their impacts across the lifespan. The investigation of a middle-aged, elite-level powerlifter with extensive training history, in conjunction with prolonged anabolic androgenic steroid use, provides research that represents the extreme range of athletic strength. The desire to tease out the physi-cal limits of the human body is the impetus driving muscle physiology and general science forward.

Acknowledgements The authors would like to thank the team members involved in both the Biochemistry and Molecular Exercise Physiology Laboratory at California State University, Fullerton, as well as those

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in the Muscle Physiology Laboratory at San Francisco State Univer-sity (SFSU). Special thanks to Nathan Serrano, M.S., Kara Lazauskas, M.S., and Jeremy Siu, B.S. for performing the muscle biopsy proce-dure and fiber type analysis. Further thanks go to Donny Gregg, M.S. for assisting with fiber typing analysis and Annette Chan, Ph.D. (Cell and Molecular Imaging Center, Department of Biology, SFSU) for her technical assistance with the confocal microscope. This article includes data from the OpenPowerlifting project, https ://www.openp owerl iftin g.org. You may download a copy of the data at https ://gitla b.com/openp owerl iftin g/opl-data.

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