Single Muscle Fiber Gene Expression with Run Taper

Citation: Murach K, Raue U, Wilkerson B, Minchev K, Jemiolo B, et al. ( Single Muscle Fiber Gene Expression with Run Taper Kevin Murach 0 1 Ulrika Raue 0 1 Brittany Wilkerson 0 1 Kiril Minchev 0 1 Bozena Jemiolo 0 1 James Bagley 0 1 Nicholas Luden 0 1 Scott Trappe 0 1 Stephen E. Alway, West Virginia University School of Medicine, United States of America 0 Funding: Funding provided by National Institutes of Health (AG 038576). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 Human Performance Laboratory, Ball State University , Muncie, Indiana , United States of America This study evaluated gene expression changes in gastrocnemius slow-twitch myosin heavy chain I (MHC I) and fast-twitch (MHC IIa) muscle fibers of collegiate cross-country runners (n = 6, 2061 y, VO2max = 7061 mlNkg21Nmin21) during two distinct training phases. In a controlled environment, runners performed identical 8 kilometer runs (30:1860:30 min:s, 8961% HRmax) while in heavy training (,72 km/wk) and following a 3 wk taper. Training volume during the taper leading into peak competition was reduced ,50% which resulted in improved race times and greater cross-section and improved function of MHC IIa fibers. Single muscle fibers were isolated from pre and 4 hour post run biopsies in heavily trained and tapered states to examine the dynamic acute exercise response of the growth-related genes Fibroblast growth factorinducible 14 (FN14), Myostatin (MSTN), Heat shock protein 72 (HSP72), Muscle ring-finger protein-1 (MURF1), Myogenic factor 6 (MRF4), and Insulin-like growth factor 1 (IGF1) via qPCR. FN14 increased 4.3-fold in MHC IIa fibers with exercise in the tapered state (P,0.05). MSTN was suppressed with exercise in both fiber types and training states (P,0.05) while MURF1 and HSP72 responded to running in MHC IIa and I fibers, respectively, regardless of training state (P,0.05). Robust induction of FN14 (previously shown to strongly correlate with hypertrophy) and greater overall transcriptional flexibility with exercise in the tapered state provides an initial molecular basis for fast-twitch muscle fiber performance gains previously observed after taper in competitive endurance athletes. - Athletes routinely reduce training volume prior to a major competition to help facilitate peak performance. A 34 week reduced training phase, known as taper, typically results in a 24% improvement in race performance [13]. In the last 30 years, taper research has identified increased power output at the whole muscle and single muscle fiber level as an important physiological basis for improved performance [1,2,4]. More specifically, tapering has been repeatedly shown to target fast-twitch muscle fibers by increasing their size [13] and power [1,2] with improvements in contractile performance largely accounted for by hypertrophy. However, little is known about molecular alterations that are contributing to performance gains in fast-twitch muscle fibers with tapering. Recent methodological advances in our laboratory have established the ability to examine gene expression at the single muscle fiber level [57]. We sought to apply this novel approach with tapering to better understand potential molecular adaptations in fast-twitch muscle fibers. We were guided into these single muscle fiber gene studies by previous work in cross-country runners (for whom muscle biopsy samples were still available) that had an altered transcriptional response in mixed-muscle homogenate samples after identical 8 km running bouts in the heavily trained versus tapered state [2]. This alteration was intriguing since previous research has shown a blunted transcriptional response to exercise in well-conditioned skeletal muscle [6,8]. Thus, it appears that skeletal muscle of highly trained athletes may be more sensitive at the molecular level to various training phases than previously thought. Further support for conducting these single fiber gene studies was that the runners MHC IIa fibers hypertrophied (+15%) and increased power output (+9%) with taper [2]. The combination of single muscle fiber gene measurements at strategically timed muscle biopsies after a heavily trained and tapered state 8 km run provided a unique opportunity to gauge the transcriptional flexibility of the MHC IIa fibers during these two training periods. For the single muscle fiber gene experiments, we selected six genes that have been implicated in muscle size regulation and remodeling and included FN14, MSTN, HSP72, MURF1, MRF4, and IGF1. In developed human skeletal muscle, Fibroblast growth factor-inducible 14 (FN14/TNFRSF12A) was recently shown to strongly correlate with fast-twitch specific growth in response to exercise [6,9], the cytokines Myostatin (MSTN/GDF8) and Insulin-like growth factor 1 (IGF1) are components of major growth pathways [10], Myogenic factor 6 (MRF4/MYF6) is associated with exercise-induced remodeling [7], Muscle ring-finger protein-1 (MURF1/TRIM63) is a marker of ubiquitin-proteasome mediated myofibrillar breakdown [11], and Heat shock protein 72 (HSP72/HSPA1A) combats protein degradation [12]. We hypothesized that advantageous post exercise regulation of HSP72, MURF1, and MRF4 previously observed in mixed-muscle with taper [2] would be more pronounced in MHC IIa fibers and that favorable FN14, MSTN, Materials and Methods Subjects Seven male runners from Taylor Universitys (Upland, Indiana) cross-country team volunteered to participate in a previous study [2]. Of these seven subjects, six were used for this investigation (age [y] 2061, height [cm] 17865, weight [kg] 66.165.1) (Table 1) as insufficient tissue at one time point for one subject negated molecular analysis. Details of the general study design and taper program are outlined in our previous investigation [2] and briefly profiled here (Figures 1 and 2). Subjects were competitive runners with 8 km lifetime best average performances of 26:3260:32 (min:s). Prior to the investigation, subjects competitively participated in running for ,7 years (range: 4.510.0 y). Subjects were provided with written and oral information about experimental procedures and potential risks prior to providing written consent. Ethics Statement Support was granted by the coaching staff and all procedures were approved by the Ball State University and Taylor University Institutional Review Boards. Experimental Design A schematic of the study design is presented in Figure 3. Identical laboratory procedures were performed in the heavily trained (T1 and T2) and tapered (T3 and T4) conditions. Resting (heavily trained - T1, tapered - T3) and 4 h post exercise (heavily trained - T2, tapered - T4) gastrocnemius muscle biopsies were collected around an 8 km standardized indoor track run. MHC I and IIa fibers were individually isolated and pooled in order to conduct targeted fiber type specific gene expression analysis via reve (...truncated)