Skeletal muscle mitochondrial volume and myozenin-1 protein differences exist between high versus low anabolic responders to resistance training Michael D. Roberts1, Matthew A. Romero1, Christopher B. Mobley1, Petey W. Mumford1, Paul A. Roberson1, Cody T. Haun1, Christopher G. Vann1, Shelby C. Osburn1, Hudson H. Holmes1, Rory A. Greer1, Christopher M. Lockwood2, Hailey A. Parry1 and Andreas N. Kavazis1 1 School of Kinesiology, Auburn University, Auburn, AL, USA 2 Lockwood, LLC, Draper, UT, USA ABSTRACT Background: We sought to examine how 12 weeks of resistance exercise training (RET) affected skeletal muscle myofibrillar and sarcoplasmic protein levels along with markers of mitochondrial physiology in high versus low anabolic responders. Methods: Untrained college-aged males were classified as anabolic responders in the top 25th percentile (high-response cluster (HI); n = 13, dual x-ray absorptiometry total body muscle mass change (D) = +3.1 ± 0.3 kg, D vastus lateralis (VL) thickness = +0.59 ± 0.05 cm, D muscle fiber cross sectional area = +1,426 ± 253 mm2) and bottom 25th percentile (low-response cluster (LO); n = 12, +1.1 ± 0.2 kg, +0.24 ± 0.07 cm, +5 ± 209 mm2; p < 0.001 for all D scores compared to HI). VL muscle prior to (PRE) and following RET (POST) was assayed for myofibrillar and sarcoplasmic protein concentrations, myosin and actin protein content, and markers of mitochondrial volume. Proteins related to myofibril formation, as well as whole lysate PGC1-a protein levels were assessed. Submitted 14 May 2018 Results: Main effects of cluster (HI > LO, p = 0.018, Cohen’s d =0.737)and Accepted 9 July 2018 time (PRE > POST, p =0.037,Cohen’s d = -0.589) were observed for citrate 27 July 2018 Published synthase activity, although no significant interaction existed (LO PRE = 1.35 ± Corresponding author 0.07 mM/min/mg protein, LO POST = 1.12 ± 0.06, HI PRE = 1.53 ± 0.11, HI POST = Michael D. Roberts, 1.39 ± 0.10). POST myofibrillar myozenin-1 protein levels were up-regulated in the [email protected] LO cluster (LO PRE = 0.96 ± 0.13 relative expression units, LO POST = 1.25 ± 0.16, Academic editor Justin Keogh HI PRE = 1.00 ± 0.11, HI POST = 0.85 ± 0.12; within-group LO increase p =0.025, Cohen’s d = 0.691). No interactions or main effects existed for other assayed Additional Information and Declarations can be found on markers. page 20 Discussion: Our data suggest myofibrillar or sarcoplasmic protein concentrations DOI 10.7717/peerj.5338 do not differ between HI versus LO anabolic responders prior to or following a Copyright 12-week RET program. Greater mitochondrial volume in HI responders may have 2018 Roberts et al. facilitated greater anabolism, and myofibril myozenin-1 protein levels may represent Distributed under a biomarker that differentiates anabolic responses to RET. However, mechanistic Creative Commons CC-BY 4.0 research validating these hypotheses is needed. How to cite this article Roberts et al. (2018), Skeletal muscle mitochondrial volume and myozenin-1 protein differences exist between high versus low anabolic responders to resistance training. PeerJ 6:e5338; DOI 10.7717/peerj.5338 Subjects Cell Biology, Kinesiology Keywords MYOZ1, Muscle hypertrophy, Citrate synthase INTRODUCTION Numerous studies have reported resistance exercise increases both whole/mixed fraction muscle protein synthesis (MPS) and myofibrillar protein synthesis (MyoPS) rates several days following a single exercise bout (Damas et al., 2016; Mitchell et al., 2012; Phillips et al., 1997, 1999; Wilkinson et al., 2008). Several studies have also reported weeks to months of resistance exercise training (RET) increases muscle fiber cross sectional area (fCSA) (Mitchell et al., 2013; Mobley et al., 2017; Petrella et al., 2008; Reidy et al., 2016; Staron et al., 1994). These parallel findings have led to a general consensus that RET-induced increases in fCSA likely coincide with increased myofibrillar protein content. The addition of myofibrils to pre-existing myofibrillar structures involves coordinated actions from proteins such as alpha-actinin 2 (ACTN2), myozenin 1 (MYOZ1), myotilin (MYOT), and Sorbin and SH3 Domain Containing 2 (SORBS2) (Sanger et al., 2002). This process has been observed in rapidly growing cardiomyocytes (LoRusso et al., 1997), skeletal muscle myotubes (White et al., 2014), developing zebrafish (Sanger et al., 2009), and embryonic chicken heart rudiments (Ehler et al., 1999). Given that resistance exercise acutely upregulates MyoPS, it seems logical RET would upregulate these genes in order to increase myofibril protein content in hypertrophying muscle fibers. From a bioenergetics perspective RET-induced increases in MPS and MyoPS are energetically costly given upwards of four ATP molecules are required per peptide bond synthesized (Stouthamer, 1973). Thus, increases in mitochondrial function or volume are likely needed to sustain muscle growth during RET due to the increased energy demand required for intracellular protein accretion. Contrary to this hypothesis, a recent review by Groennebaek & Vissing (2017), which included 16 studies examining how chronic “high load” RET affected markers of mitochondrial volume in whole-tissue lysates, cited 14 of these studies observed no change or a decrease in these biomarkers. While this report suggests RET likely does not increase markers of mitochondrial volume, it remains possible that high anabolic responders to RET may experience greater increases in biomarkers related to mitochondrial volume in order to better support anabolism. We recently published a study examining skeletal muscle biomarkers related to ribosome biogenesis, inflammation, and androgen signaling that were differentially expressed between high versus modest and low anabolic responders following a 12-week full body RET program (Mobley et al., 2018); notably, vastus lateralis (VL) thickness changes was the clustering variable. Herein, we adopted a refined approach similar to Davidsen et al. (2011) in order to define high versus low anabolic responders in these subjects based upon three hypertrophic indices including total muscle fCSA, VL thickness, and total body skeletal muscle mass (TBMM) assessed using dual x-ray absorptiometry (DEXA). Next, we sought to examine if total myofibrillar and sarcoplasmic protein concentrations, myosin, and actin protein content, myofibrillar proteins involved with myofibril formation, or markers of mitochondrial physiology differed between clusters (high-response cluster (HI) = anabolic responders in the top 25th percentile and Roberts et al. (2018), PeerJ, DOI 10.7717/peerj.5338 2/25 low-response cluster (LO) = anabolic responders in the bottom 25th percentile). We hypothesized HI responders would exhibit greater changes in myofibrillar and sarcoplasmic protein concentrations relative to LO responders following RET. Additionally, we hypothesized HI responders would exhibit greater indices of mitochondrial volume or biogenesis relative to LO responders prior to and/or following RET. MATERIALS AND METHODS Ethical approval and study design This study was approved by the Institutional Review Board at Auburn University (approved protocol #: 15-320 MR 1508) and conformed to the standards set by the latest revision of the Declaration of Helsinki. In the current study we analyzed muscle specimens from select participants that participated in a study we previously published (Mobley et al., 2017) and registered on ClinicalTrials.gov (Identifier: NCT03501628, date registered: April 18, 2018). However, the current study is not a clinical trial per the definition of the World Health Organization or National Institutes of Health given that health-related biomedical outcomes were not assessed. Apparently healthy, untrained college-aged male subjects provided written consent to participate in this study and performed a testing battery prior to (PRE) and 72 h after the last training bout (POST) following a 12-week full body RET program. The testing battery consisted of a VL muscle biopsy, full-body dual DEXA scan, and VL thickness assessment using ultrasound. More in-depth descriptions regarding the RET protocol as well as assessments of body composition, VL thickness, and fCSA can be found in past publications by our group (Mobley et al., 2017, 2018). Muscle tissue processing Muscle biopsies from PRE and POST testing sessions were collected using a five gauge needle under local anesthesia as previously described (Mobley et al., 2017). Immediately following tissue procurement, ∼20–40 mg of tissue was embedded in cryomolds containing optimal cutting temperature media (Tissue-TekÒ; Sakura Finetek Inc, Torrence, CA, USA) for fCSA assessment. The remaining tissue was teased of blood and connective tissue, fl wrapped in pre-labelled foils, ash frozen in liquid nitrogen (LN2), and subsequently stored at -80 C until protein and citrate synthase activity analyses described below. Western blotting of tissue lysates ∼ For whole tissue lysate protein analysis, 30 mg tissue was powdered on a LN2-cooled ceramic mortar and placed in 1.7 mL microcentrifuge tubes on ice containing 500 mLof – general cell lysis buffer (20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton; Cell Signaling, Danvers, MA, USA) pre-stocked with protease and Tyr/Ser/Thr phosphatase inhibitors (2.5 mM sodium pyrophosphate, 1 mM b m -glycerophosphate, 1 mM Na3VO4,1 g/mL leupeptin). Samples were then homogenized on ice by hand using tight micropestles, insoluble proteins were removed with centrifugation at 500g for 5 min, and obtained sample