HIGH-INTENSITY INTERVAL TRAINING FOLLOWING SEVEN WEEKS OF RESISTANCE TRAINING ALTERS MUSCLE CALPAIN AND AUTOPHAGY MARKERS

Daniel L. Plotkin1, J. Max Michel1, Joshua S. Godwin1, Paulo H.C. Mesquita1, Bradley A. Ruple1, Mason C. McIntosh1, Cleiton A. Libardi2, Andreas N. Kavazis1, Michael D. Roberts1,3. 1Auburn University, Auburn, AL. 2Federal University of Sao Carlos, Sao Carlos. 3Edward Via College of Osteopathic Medicine-Auburn Campus, Auburn, AL. BACKGROUND: While the dynamics of muscle protein balance have been explored in the context of many exercise modalities, they have not been studied in a resistance training followed by endurance training (ET) paradigm. Our laboratory recently determined that seven weeks of resistance training (RT) increases vastus lateralis (VL) muscle thickness, and seven weeks of treadmill high-intensity interval training (HIIT) following the RT period reduces VL muscle thickness. Thus, the purpose of this study was to examine calpain and autophagy activity markers after the 7-week RT and HIIT periods. METHODS: Vastus lateralis biopsies and muscle thickness (MT) measures were collected from 11 untrained college-aged males at baseline (PRE), after 7 weeks of RT (MID) and after a subsequent 7 weeks of HIIT (POST). Proteins associated with autophagy and calpain activities were assessed via immunoblotting, and calpain activity was assessed via a commercially available plate-based assay. Data were checked for normality using Shapiro-Wilk tests and analyzed via one-way ANOVAs. RESULTS: Tukey’s Multiple comparisons test showed a significant increase in MT from PRE to MID [2.3 mm, CI95% = (1.1 mm, 3.5 mm), p= 0.001], a significant decrease from MID to POST [1.2 mm, CI95% = (0.30 mm, 2.1 mm), p= 0.012],and a non-significant increase from PRE to POST [1.1 mm, CI95% = (2.1 mm, 2.9 mm), p= 0.213]. Microtubule-associated proteins 1A/1B light chain 3B (LC3 II/I), phosphorylated AMP-kinase (p-AMPKα, Thr172) / pan-AMPKα, and calpain-1 protein abundance did not reach overall ANOVA significance with respective p-values of 0.854, 0.330, 0.365. Phosphorylated-Unc-51 like autophagy activating kinase 1 (p-ULK, Ser555) / pan-ULK protein abundance was not significant between time points. Beclin-1 protein abundance increased from PRE to MID (p= 0.012) and PRE to POST (p = 0.005). Calpain-2 increased from PRE to MID (p = 0.001) and PRE to POST (p = 0.003). Finally, calpain-activity normalized to Calpain-1/2 protein content decreased from PRE to MID (p = 0.001), albeit with no significant differences from PRE to POST and MID to POST. CONCLUSION: These data suggest that an elevation in calpain activity markers (rather than autophagy markers) during seven weeks of HIIT may have been partially responsible for mid-thigh muscle size decreases following a seven-week period of RT

THE UBIQUITIN-PROTEASOME SYSTEM: IMPLICATED IN HIIT INDUCED MUSCLE ATROPHY FOLLOWING SEVEN WEEKS OF RESISTANCE TRAINING

J. Max Michel1, Daniel L. Plotkin1, Joshua S. Godwin1, Paulo H.C. Mesquita1, Bradley A. Ruple1, Mason C. McIntosh1, Cleiton A. Libardi2, Andreas N. Kavazis1, Michael D. Roberts3,1. 1Auburn University, Auburn, AL. 2Federal University of Sao Carlos, Sao Carlos, Brazil. 3Edward Via College of Osteopathic Medicine-Auburn Campus, Auburn, AL. BACKGROUND: Skeletal muscle is a highly plastic tissue that uniquely responds to different external stimuli. While resistance training (RT) induces skeletal muscle hypertrophy, the effects of high-intensity interval training (HIIT) are less clear, with certain evidence indicating mild hypertrophy and other evidence suggesting no change in measurable outcomes. Our laboratory recently observed an increase in vastus lateralis thickness after seven weeks of RT (2 d/wk), with values returning to pre-training levels following seven weeks of treadmill HIIT (3 d/wk). Given that proteolytic mechanisms were not previously investigated, the purpose of this study was to examine the effects of the aforementioned exercise paradigm on markers of the ubiquitin-proteasome system (UPS). METHODS: Vastus lateralis biopsies were collected from 11 untrained college-aged males at baseline (PRE), after seven weeks of RT (MID) and after a subsequent seven weeks of HIIT (POST). Tissue was analyzed for atrogene mRNA expression, proteins associated with the UPS, and 20S proteasome activity. Data were checked for normality using Shapiro-Wilk tests and analyzed via one-way ANOVAs. RESULTS: Muscle really interesting new gene (RING) finger 1 (MuRF1), Atrogin-1, and forkhead box O3a (FOXO3a) all achieved significance at the overall ANOVA level (p≤0.0129). These mRNAs were all significantly upregulated at POST as compared to both PRE (p≤0.0315), and MID (p≤0.0315). Poly-ubiquitinated proteins achieved overall ANOVA significance (p=0.0185) and trended toward a significant increase at both MID (p=0.0694) and POST (p=0.0529). Protein abundance of the 20S proteasome core demonstrated overall ANOVA significance (p=0.0096) and showed increases at POST as compared to both PRE (p=0.0311) and MID (p=0.0485). Finally, 20S proteasome activity demonstrated overall ANOVA significance (p=0.0031) and showed increases from PRE at both MID (p=0.0011) and POST (p=0.0040). CONCLUSIONS: Markers of the UPS were upregulated at POST at the mRNA, protein, and activity level suggesting that the UPS played a role in the HIIT-induced atrophy that occurred following a prior seven-week RT program

A COMPARISON OF TECHNIQUES ASSESSING THE MORPHOLOGICAL CHARACTERISTICS OF SKELETAL MUSCLE FIBERS

Breanna J. Mueller, Paulo H. C. Mesquita, Bradley A. Ruple, Joshua S. Godwin, Casey L. Sexton, Shelby C. Osburn, Mason C. McIntosh, Andreas N. Kavazis, FACSM, Cleiton A. Libardi, Kaelin C. Young, Michael D. Roberts. Auburn University, Auburn, AL. Background: Two-dimensional cross-sectional analysis is a technique that is widely used to assess skeletal muscle fiber morphology in response to resistance training. While this technique affords some advantages in assessing fiber cross-sectional area (fCSA), fiber typing and other variables, it may not be representative of the whole fiber. A more dimensional analysis is needed to render greater accuracy and insight into the morphological characteristics of a three-dimensional fiber. The purpose of this study was to evaluate single fiber analysis as a valid and comparable technique in analyzing fiber morphology. Methods: Vastus lateralis muscle biopsies from 11 untrained males were collected at pre- and post- of a 7-week total body resistance training protocol. Using two-dimensional cross-sectional analysis and single fiber analysis techniques, we measured fCSA, myonuclear number (MNN), and myonuclear domain (MND). T-tests and correlations were performed with the data to determine any significant changes and relationships between techniques. Results: Two-dimensional cross-sectional analysis revealed a significant increase in myonuclear number (35.51%, p = 0.031) and fCSA (19.81%, p = 0.010), but not in MND (1.15%, p = 0.413). In comparison, single fiber analysis also demonstrated a significant increase in myonuclear number (13.52%, p = 0.011), fCSA (32.55%, p= 0.009), and MND (17.35%, p = 0.118). There was no significant correlation between two-dimensional analysis and single fiber analysis in fCSA (r = -0.074, p = 0.828), myonuclear number (r = 0.326, p = 0.327), or myonuclear domain (r = -0.264, p = 0.435) percent change. Conclusion: In conclusion, the measurements taken with two-dimensional cross-sectional analysis and single fiber analysis did not agree, and statistical analysis showed no correlations between the two measurements. However, these results may have been impacted by our limited sample size. Additional investigation utilizing a greater sample size may provide more promising results. Therefore, future investigation of the longitudinal view provided by single fiber analysis may yet yield an advantage in the morphological assessment of the skeletal muscle fiber. Funding: Participant compensation as well as select reagents related to analyses presented herein were funded by a grant awarded by National Strength and Conditioning Association Foundation to Paulo H.C. Mesquita

EFFECTS OF RESISTANCE TRAINING ON MITOCHONDRIAL ADAPTATIONS TO SUBSEQUENT ENDURANCE TRAINING

Paulo H.C. Mesquita, Joshua S. Godwin, Bradley A. Ruple, Casey L. Sexton, Mason C. McIntosh, Shelby C. Osburn, Breanna J. Mueller, Nicholas Kontos, Cleiton A. Libardi, Kaelin C. Young, Michael D. Roberts, Andreas N. Kavazis, FACSM. Auburn University, Auburn, AL. BACKGROUND: While resistance training (RT) has long been appreciated for enhancing muscle mass and strength, it is widely underappreciated and under-utilized by endurance athletes and practitioners. However, several studies have shown beneficial effects of RT on endurance performance, which is usually linked to an improvement of running economy through neuromuscular adaptations. Emerging evidence highlights that RT may also promote positive mitochondrial adaptations, which could ultimately enhance endurance performance. Even though there are several studies investigating the effects of concurrent training, to the best of our knowledge, no study to date has investigated the effects of performing a block of RT-only before initiating endurance training (ET)-only. Therefore, the goal of the present study was to investigate the effects of RT on adaptations to subsequent ET. METHODS: 23 young untrained males were recruited and divided into two groups: 1) ET-only (n=12), which performed 7 weeks of high-intensity interval training; 2) RT+ET (n=11), which performed 7 weeks of RT twice weekly before initiating 7 weeks of the same endurance training performed by ET-only. All participants performed a maximal oxygen uptake test on a treadmill for determination of VO2max and speed at onset of blood lactate accumulation (OBLA) before (PRE) and after (POST) ET. Furthermore, muscle biopsies were obtained from participants’ vastus lateralis at PRE and POST and used to determine the levels of proteins involved in mitochondrial remodeling. RESULTS: Both groups significantly increased VO2max and speed at OBLA (p\u3c0.001) similarly. Protein levels of mitochondrial complexes I, II, and III, and Mitofusin 2 (Mfn2) increased, while Parkin decreased similarly in both groups (main effect of time, p\u3c0.05). Significant interactions were found for complex IV (ET-only (PRE \u3c POST), p=0.039) and PGC-1α (ETonly-POST \u3e RT+ET-POST, p=0.001). CONCLUSIONS: Our results suggest that performing RT prior to ET had no additional benefit on mitochondrial and endurance performance adaptations to ET in young untrained males. Participant compensation as well as select reagents related to analyses presented herein were funded by a grant awarded by National Strength and Conditioning Association Foundation to Paulo H.C. Mesquita