Resistance Training Diminishes Mitochondrial Adaptations to Subsequent Endurance Training

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into RT+ET (n=13), which underwent seven weeks of RT followed by seven weeks of ET, and ET-only (n=12), which performed seven weeks of ET. Body composition, endurance performance, and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, post RT for RT+ET and baseline for ET), and after ET (T3). Immunohistochemistry was performed to determine fiber cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number, and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodeling. Citrate synthase activity and markers of ribosome content were also investigated. Resistance training improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (p<0.050). In response to ET, both groups similarly decreased body fat percentage and improved endurance performance (e.g., VO2max, and speed at which the onset of blood lactate accumulation occurred during the VO2max test). Levels of mitochondrial complexes I-IV in the ET-only group increased 32–66%, while the RT+ET group increased 1–11%. Additionally, mixed fiber relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group. In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET.


KEY POINTS SUMMARY 27 28
• Resistance training is largely underappreciated as a method to improve endurance 29 performance, despite reports showing it may improve mitochondrial function. 30 • Although several concurrent training studies are available, in this study we investigated 31 the effects of performing a period resistance training on the performance and molecular 32 adaptations to subsequent endurance training. 33 INTRODUCTION 5 91

MATERIALS AND METHODS 92
Ethical approval 93 The current study was reviewed and approved by the Institutional Review Board at 94 Auburn University  and conformed to the standards of the Declarations of 95 Helsinki, except that it was not registered as a clinical trial. 96 97 Participants 98 Twenty-five healthy young male participants (baseline characteristics in Table 1) were 99 recruited to participate in this study. Participants should not have participated in structured (more 100 than once weekly for at least two months) RT over the last three years or ET over the last six 101 months prior to joining the current study. All participants were informed of the procedures and 102 risks of the current study before providing written consent. 103 104 Abbreviations: RT+ET, group that performed 7 weeks of resistance training followed by 7 weeks 106 of endurance training; ET-only, group that performed 7 weeks of endurance training only; BMI, 107 body mass index; VO2peak, peak aerobic capacity 108 109

Familiarization session 110
Participants visited the laboratory to become familiarized with the exercises and tests 111 used in the study. First, participants performed a maximal cardiorespiratory test on a motorized 112 treadmill. The incremental treadmill test was composed of several two-minute stages and started 113 with a fast-paced walk (6.4 km/h, 0% inclination) as a warm-up for three minutes. After that, the 114 speed of the treadmill increased by 1 km/h and inclination by 1% after every stage until the 115 participant reached volitional exhaustion. There was a 30-second break after each stage, during 116 which participants stopped running and reported their ratings of perceived exertion (RPE) 117 according to Borg's CR10 scale (17). Peak oxygen consumption (VO2peak) was determined by the 118 highest 30-second average value using a metabolic cart (True Max 2400, ParvoMedics, Salt Lake 119 City, UT, USA). After the maximal cardiorespiratory test, participants were taught how to 120 properly perform the leg press, bench press, leg extension, cable-bar pull-down, and leg curl 121 exercises. Participants were allowed to perform a few sets and repetitions until they 122 demonstrated proper lifting technique. 123 124

Experimental design 125
VO2peak values obtained during the familiarization visit were used to assign participants to 126 each group in a balanced manner, RT+ET (n=13, VO2peak = 40.4 ± 8.9 ml/kg/min) and ET-only 127 (n=12, VO2peak = 38.9 ± 6.2 ml/kg/min). Importantly, there was no significant difference 128 between groups (p=0.634). Testing sessions (T1, T2, and T3) 145 exercises (i.e., bench press, cable pull-down, leg curls). Volume and load for the quadriceps were 194 progressively increased throughout the seven weeks and can be seen in Table 2. Although the 195 load increment was pre-planned as shown, participant feedback was taken into consideration for 196 load adjustments. After each set for each exercise, participants reported their repetitions in 197 reserve (RIR) by answering how many more repetitions they think they could have done (20). If 198 RIR > 2, the load was increased by approximately 5-10 lbs for upper-body exercises and 10-20 199 lbs for lower-body exercises. If participants failed to perform the programmed number of 200 repetitions, load was decreased in a similar fashion. Participants rested for two to three minutes 201 between sets of exercises. Appropriate range of motion was ensured using the range of motion 202 recorded at T1-testing. 203 204 Abbreviation: 1RM, one-repetition maximum. Symbol: *Total number of sets and reps per 206 session for exercises targeting quadriceps (e.g., 6 x 6 = 3 sets of 6 repetitions for the leg press 207 and 3 sets of 6 repetitions for the leg extension; 9 x 6 = 5 sets of 6 repetitions for the leg press 208 and 4 sets of 6 repetitions for the leg extension). 209 210

Endurance training 211
All participants performed seven weeks of a high-intensity interval training (HIIT)-based 212 ET on a motorized treadmill, and in RT+ET participants, this training occurred the week 213 immediately following their seven week-RT period. A HIIT-based training protocol was chosen 214 because HIIT induces mitochondrial and cardiovascular adaptations in a time-efficient manner 215 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023. For each training session, participants started with a 3-minute warm-up, followed by 5-216 10 sets (5 sets in the 1st week; 8 sets in the 2nd week; 9 sets in the 3rd week; 10 sets for the 217 remaining weeks) of 1 minute running at a high intensity interspersed by 1.5 to 3 minutes 218 running at a low intensity. The intensity of the "sprints" and the recovery was determined using 219 the speed and inclination values achieved in the VO2max test (Table 3). Similar to the RT 220 program, participant feedback was taken into consideration to adjust the intensity of training. At 221 the end of each "sprint" bout, participants rated their perceived exertion (RPE) using the CR-10 222 Borg Scale. In the first week, if participant's final RPE was lower than 5 ("strong"), the intensity 223 of the "sprint" bout was increased in the next training session by 5%. From the second week 224 onward, if participant's final RPE was lower than 7 ("very strong"), intensity (i.e., treadmill 225 speed) was also increased by 5%. If participants were not able to complete the programmed 226 number of "sprints", intensity was decreased by 5%. The ET program can be seen in Table 3:  227 228 Approximately 30 mg of muscle tissue that was flash-frozen in foil was retrieved from -233 80 o C, weighed using an analytical scale, and homogenized in a sucrose homogenization buffer 234 using a glass Dounce homogenizer according to Spinazzi et al. (22). Samples were centrifuged at 235 600 × g for 10 minutes at 4 o C. Protein concentrations from the resulting supernatants were 236 determined using a commercially available BCA kit (Thermo Fisher Scientific, Waltham, MA, 237 USA). Supernatants were then used for citrate synthase (CS) activity and western blotting. 238 239 240 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint (i.e., µg/mg wet tissue). In an attempt to account for muscle size changes, "absolute" RNA 272 content was estimated by multiplying relative total RNA by mixed fiber cross-sectional area 273 (fCSA) determined by immunohistochemistry (described later) and by vastus lateralis thickness. 274 For gene expression analyses, 2 µg of cDNA was synthesized using a commercial qScript 275 cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA). RT-qPCR was performed in an 276 RT-PCR thermal cycler (Bio-Rad) using SYBR green-based methods with gene-specific primers 277 designed with primer designer software (Primer3Plus, Cambridge, MA, USA). For all primer 278 sets, pilot qPCR reactions and melt data indicated that only one amplicon was present. The 279 forward and reverse primer sequences of all genes are listed in Table 4 Citrate synthase activity 290 Citrate synthase activity was determined by monitoring the increase in absorbance at 412 291 nm from the reduction of 5,5′-dithiobis (2-nitrobenzoic acid) coupled to the reduction of acetyl-292 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A portion of skeletal muscle samples preserved in OCT were sectioned at 7 µm thickness 298 using a cryotome (Leica Biosystems; Buffalo Grove, IL, United States) and adhered to positively 299 charged histology slides. Slides were then stored at -80°C until batch processing. Slides were 300 mounted in a manner that all time-points for each participant were analyzed concomitantly to 301 avoid batch-to-batch variation. 302 For fiber type-specific fCSA and myonuclei number quantification, slides were air-dried 303 for 90-120 minutes prior to a 5-minute acetone incubation at -20 o C. Slides were washed 3x5 304 minutes in 1x phosphate buffered saline (PBS) and incubated with 3% H2O2 for 10 minutes at 305 room temperature. After washing, slides were incubated with autofluorescence quenching 306 reagent for 1 min (TrueBlack; Biotium, Fremont, CA, USA). Slides were washed again in PBS 307 and blocked with a 5% goat serum and 2.5% horse serum solution for 1 hour at room 308 temperature. After blocking, slides were incubated overnight at 4 o C with a primary antibody Scientific; catalog #: D3571) for 15 minutes at room temperature before coverslips were applied 317 using PBS + glycerol as mounting medium. 318 For fiber type-specific satellite cell content quantification, a similar protocol was used. 319 However, additional steps were performed to amplify satellite cells. After blocking slides with 320 5% goat serum and 2.5% horse serum, slides were blocked with streptavidin and biotin solutions 321 at room temperature for 15 minutes each. Thereafter, slides were incubated overnight at 4 o C with 322 primary antibody cocktail containing 1:20 Mandra (dystrophin) (Developmental Studies 323 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. For fiber type-specific mitochondrial content, the translocase of outer mitochondrial 331 membrane 20 (TOMM20) protein was stained as previously described and validated (14)  Single fiber analyses were also performed to quantify myonuclei content. As stated 342 above, muscle tissue (~10 mg) was fixed in 4% paraformaldehyde for 48 hours at room 343 temperature following biopsies. Tissue was then washed in PBS and stored at 4 o C until batch 344 processing. Tissue was subsequently incubated in 40% NaOH in slow rotation for approximately 345 2 hours to facilitate extracellular matrix digestion and myofiber disaggregation. Tissue was then 346 washed in PBS through a 40 µm cell strainer and transferred to PBS. Small myofiber bundles 347 were mechanically teased apart under a light microscope, placed in PBS, and centrifuged at 348 13,000 rpm. PBS was removed and myofibers were stained with DAPI for 15 minutes. Following mounting, digital images for each analysis were captured with a fluorescence 353 microscope (Nikon Instruments) using the 20x objective. Fiber type-specific fCSA and 354 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Melville, NY, USA) and are reported as PAX7 positive per 100 fibers. Mitochondrial content 357 was determined in serial sections using ImageJ (NIH) as previously described (14) and reported 358 as percentage change from baseline. In short, the red channel of TOMM20 images was converted 359 to grayscale and a threshold function was applied. Fibers were then manually traced and 360 mitochondrial area was determined as a percentage of the fiber area. Absolute mitochondrial 361 content via TOMM20 was estimated by multiplying the percentage of TOMM20 by mixed, type 362 I, and type II fiber cross-sectional areas. For single fiber nuclei content, a brightfield image was 363 taken to determine fiber border. Thereafter, three images of the DAPI filter were taken at 364 different depths to capture the maximum number of nuclei. Single fiber and nuclei measurements 365 were made by a blinded investigator using ImageJ (NIH In response to ET in both groups, there were no significant effects of group (G), time (T), 391 or interaction (GxT) for body mass (G, p=0.400; T, p=0.111; GxT, p=0.870; Fig. 2A), lean body 392 mass (G, p=0.797; T, p=0.242; GxT, p=0.324; Fig. 2B), or fat mass (G, p=0.118; T, p=0.199; 393 GxT, p=0.818; Fig. 2C). A significant effect of G (p=0.048) and T (p<0.001), but no GxT 394 (p=0.087), was evident for body fat percentage (Fig. 2D)   p<0.001), with no significant main effect of G (p=0.354) or GxT (p=0.315) (Fig. 3D). 422 423 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023.  p=0.041). There were no significant main effects of G, T, or GxT for NRF1 (G, p=0.192; T, 457 p=0.912; GxT, p=0.099) or TFAM (G, p=0.062; T, p=0.078; GxT, p=0.458). Regarding 458 mitochondrial dynamics markers (Fig. 4D), no significant main effects of G, T, or GxT were 459 evident for MFN1 (G, p=0.555; T, p=0.079; GxT, p=0.717) or DRP1 (G, p=0.857; T, p=0.122; 460 GxT, p=0.096). MFN2 significantly increased from T2 to T3 (0.03 ± 95% CI [0.02], p=0.030), 461 but there was no significant main effect of G (p=0.066) or GxT (p=0.161). 462 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint Notes: t-test p-values are for the RT period in the RT+ET group, and the two-way ANOVA main 470 effect and interaction p-values are for the ET period in both groups. 471 472

Ribosome content 473
In response to RT, there were no significant increases in relative total RNA levels in the 474 RT+ET group (p=0.443). However, when accounting for muscle size, there was a significant 475 increase in absolute RNA estimated by fCSA (944,387 a.u. ± 95% CI [866,900], p=0.035), 476 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made In response to ET in both groups, there were no significant effects of G or T for relative 480 total RNA (G, p=0.690; T, p=0.779), absolute RNA by fCSA (G, p=0.740; T, p=0.451), or 481 absolute RNA by VL thickness (G, p=0.815; T, p=0.896) (Fig. 5A-C)  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made fiber analysis, however, did not reach statistical significance (p=0.081). In response to ET in both 513 groups, there were no significant main effects of G, T, or GxT for mixed (G, p=0.329; T, 514 p=0.244; GxT, p=0.748), type I (G, p=0.392; T, p=0.257; GxT, p=0.753), or type II (G, p=0.345; 515 T, 0.476; GxT, p=0.886) myonuclear number in cross-section (Fig. 6B). Single fiber myonuclei 516 number increased from T2 to T3 (2.0 ± 95% CI [1.2], p=0.004), with no main effect of G 517 (p=0.087) or GxT (p=0.996) (Fig. 6C). 518 Myonuclear domain (MND). In response to RT, there was a decrease in cross-sectional 519 MND in type I fibers (934 µm 2 /myonucleus ± 95% CI [900], p=0.043) in the RT+ET group, but 520 not in mixed (p=0.483) or type II fibers (p=0.487). However, MND values assessed through 521 single fiber analysis exhibited no significant change (p=0.176). In response to ET in both groups, 522 ET-only exhibited greater cross-sectional MND values in type I fibers compared to RT+ET 523 group (G effect: 941 µm 2 /myonucleus ± 95% CI [741], p=0.028). There was no main effect of T 524 (p=0.589) or GxT (p=0.571) for type I MND (Fig. 6D). In addition, there were no significant 525 main effects of G, or T, or GxT in mixed (G,p=0.055;T,p=0.956;GxT,p=0.946) or type II (G, 526 p=0.431; T, p=0.751; GxT, p=0.391) fibers. For single fiber MND, there were no significant 527 effects of G (p=0.090), T (p=0.894), or GxT (p=0.608) (Fig. 6E). 528 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 Satellite Cells. In response to RT, there was a significant increase in satellite cell content 529 in mixed (5.2 ± 95% CI [2.7], p=0.002), type I (5.4 ± 95% CI [4.9], p=0.014), and type II (5.5 ± 530 95% CI [3.1], p=0.004) fibers in the RT+ET group. In response to ET in both groups, there were 531 no significant main effects of G or T, or GxT for mixed (G,p=0.378;T,p=0.114;GxT,532 p=0.109), type I (G, p=0.179; T, p=0.169; GxT, p=0.606), or type II (G, p=0.747; T, p=0.144; 533 GxT, p=0.079) fibers (Fig. 6F). 534 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint 535 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint Data are expressed as mean ± SD, and individual respondent values are also depicted. 542 Abbreviations: RT+ET, group that performed 7 weeks of resistance training followed by 7 weeks 543 of endurance training; ET-only, group that performed 7 weeks of endurance training only; GxT, 544 group x time interaction. Notes: t-test p-values are for the RT period in the RT+ET group, and 545 the two-way ANOVA main effect and interaction p-values are for the ET period in both groups. 546 547 548 Mitochondrial content assessed using CS activity and TOMM20 IHC 549 In response to RT, relative CS activity remained unaltered (p=0.152), but absolute CS 550 activity significantly estimated by fCSA (481,561 nmol/min/mg.µm 2 ± 95% CI [335,161], 551 p=0.009) and by VL thickness (152 nmol/min/mg.cm ± 95% CI [117]) increased in response to 552 RT. There were no significant changes in relative mitochondrial content as assessed through 553 TOMM20 IHC in mixed (p=0.296), type I (p=0.982), or type II (p=0.070) fibers in the RT+ET 554 group. Regarding the estimation of total mitochondrial content, which accounts for fCSA 555 changes, there was a significant increase in type II fiber mitochondrial content (45% ± 95% CI 556 [38], p=0.026) but not in mixed (p=0.113) or type I (p=0.476) fibers. 557 In response to ET in both groups, mitochondrial content assessed through relative CS 558 activity was higher in the ET-only group compared to RT+ET group (main effect of G: 92.7 559 mmol/min/mg ± 95% CI [81.3], p=0.036) (Fig. 7A). However, there was no significant main 560 effect of T (p=0.461) or GxT (p=0.162). Additionally, there were no significant effects of G, T, 561 or GxT in total mitochondrial content estimated by mixed fCSA (G, p=0.170; T, p=0.117; GxT, 562 p=0.132) or by VL thickness (G, p=0.074; T, p=0.257; GxT, p=0.080) in response to ET (Fig.  563 7B-C). 564 There were significant GxT for mitochondrial content as assessed through TOMM20 IHC 565 in mixed (p=0.043) and type II fibers (p=0.012). Post-hoc tests for mixed fibers returned p-566 values > 0.050 for all comparisons. Mitochondrial content in type II fibers was greater in the 567 RT+ET group at T2 compared to ET-only group at T2 (33.8% ± 95% CI [20.2], p=0.036), but 568 there were no significant main effects (for mixed: G, p=0.853 and T, p=0.935; for type II G 569 p=0.463 and T, p=0.801) (Fig. 7D). Type I fibers exhibited no significant main effects of G 570 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint (p=0.609) or T (p=0.474), or GxT (p=0.158). Similar responses were observed for total 571 mitochondrial content estimations (Fig. 7E). A significant GxT was observed in type II fibers 572 (p=0.018), where mitochondrial content was higher in the RT+ET group at T2 compared to  only group at T2 (52.2% ± 95% CI [30.6], p=0.032). No significant main effects of G (p=0.162) 574 or T (p=0.519) were detected for type II fibers. In addition, no significant effects of G, T, or GxT 575 were found in mixed (G,p=0.348;T,p=0.385;GxT,p=0.054)  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint sectional staining. (F) Dystrophin (red), MHCI (green), DAPI (blue). (G) Dystrophin (green), 585 TOMM20 (red). T1 = Pre-RT; T2 = Pre-ET; T3 = Post-ET. Data are expressed as mean ± SD, 586 and individual respondent values are also depicted. Abbreviations: RT+ET, group that performed 587 7 weeks of resistance training followed by 7 weeks of endurance training; ET-only, group that 588 performed 7 weeks of endurance training only; GxT, group x time interaction. Notes: t-test p-589 values are for the RT period in the RT+ET group, and the two-way ANOVA main effect and 590 interaction p-values are for the ET period in both groups. 591 592 Correlations 593 Correlation between the values at T2 from select variables (e.g., relative total RNA, 594 fCSA, nuclei, SCs) and the percent change of mitochondrial content variables (i.e., relative CS 595 activity, mixed fibers, type I and type II mitochondrial content (TOMM20)) in response to ET 596 were analyzed in a group-specific manner due to the distinctive responses of each group to ET. 597 The only significant correlation found was between relative CS activity and mixed fCSA in the 598 Resistance training has long been appreciated for increasing muscle mass and strength, 603 and emerging evidence highlights that RT may also promote positive mitochondrial adaptations. 604 Most studies investigating the differences and interplay between RT and ET adaptations have 605 compared concurrent training to single-mode training, using various experimental designs. To 606 the best of our knowledge, this is the first study to investigate the effects of performing a period 607 of RT-only on the adaptations to a subsequent period of ET-only. Our main findings demonstrate 608 that RT performed prior to ET had no additional benefits to ET adaptations. Moreover, even 609 though both groups improved endurance performance similarly, prior RT seemed to impair most 610 mitochondrial adaptations to subsequent ET. 611 In the current study, seven weeks of RT elicited adaptations commonly reported in the 612 literature, which demonstrates the effectiveness of the RT protocol adopted herein. Participants 613 in the RT+ET group improved body composition and strength, and increased VL thickness, 614 mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell 615 number. Various methods can be implemented to increase endurance performance, with the most 616 common being MICT and HIIT. High-intensity interval training protocols have been shown to 617 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 improve endurance performance in as little as two weeks (26). VO2max and the speed at lactate 618 threshold are considered key determinants of endurance performance (1, 2). In the current study, 619 relative VO2max improved 13.4% in the RT+ET group and 10.6% in the ET-only, which is 620 within the range reported in the literature (6,27). In addition, the speed at OBLA increased 7% 621 in the RT+ET group and 12% in ET-only group. However, performing a block of RT before 622 initiating ET did not significantly enhance these adaptations to ET. Additionally, much of our 623 molecular data suggest that seven weeks of RT performed prior to seven weeks of ET may 624 interfere with mitochondrial adaptations, and this will be the crux of the remainder of the 625 discussion. 626 The mitochondrial adaptations to RT are not well defined based on prior literature. For 627 instance, while it is commonly believed that RT is not an effective method to achieve positive 628 mitochondrial adaptations, different researchers have reported increases in markers of 629 mitochondrial content and function in younger (11, 12, 28) and older (13, 29-31) individuals. In 630 the current study, there were no significant changes in mitochondrial protein complex 631 concentrations or markers of mitochondrial content (TOMM20 and CS activity) with RT. 632 However, type II fiber total mitochondrial content (as estimated by considering changes in 633 fCSA) increased. This increase in total, but not relative mitochondrial content, suggests that the 634 expansion of the mitochondrial network occurred in line with type II myofiber size increases. 635 Alternatively stated, we speculate that the metabolic demands of RT did not facilitate 636 mitochondrial expansion per se, but that the expansion of the mitochondrial network occurred in 637 proportion to myofiber size to optimize a mitochondrial-to-myofiber volume ratio. 638 Satellite cells and myonuclear accretion have been extensively studied in the context of 639 RT and skeletal muscle hypertrophy. Whether or not these events are required for hypertrophy is 640 still a topic of debate (32, 33), albeit satellite cells and myonuclei content are commonly reported 641 to increase with RT (34-36). However, the effects of ET on satellite cells and myonuclear counts 642 have received less attention in the literature. In line with our expectations, seven weeks of RT in 643 the RT+ET group increased mixed, type I and type II myofiber myonuclear number and satellite 644 cell number. However, in both RT+ET and ET-only groups, HIIT training did not elicit 645 significant changes in fCSA, cross-sectional myonuclei or satellite cells number. Our results 646 agree with previous studies that showed no changes in type I and II satellite cell or myonuclear 647 number after different forms of ET (37,38), and continue to support that RT (but not ET) acts as 648 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint a stimulus to affect these variables. As with the sparse research examining how ET affects 649 satellite cell number, studies that have examined the effects of ET on ribosome biogenesis 650 markers are also limited. There is a common dichotomous viewpoint whereby RT promotes 651 ribosome biogenesis and ET promotes mitochondrial biogenesis, with an interference effect 652 between the two processes if RT and ET are performed concurrently (39, 40). However, it is 653 possible that untrained individuals can present a generic response to exercise training whereby 654 ribosome and mitochondrial biogenesis can occur in response to both RT and ET (39). In 655 response to the HIIT period in the current study, the RT+ET group presented decreases in 656 ribosome content while the ET-only group presented paradoxical increases in these variables. 657 While these events are difficult to reconcile, the RT+ET response may be related to the cessation 658 of RT and not a response to ET per se, as ribosome content has been previously shown to 659 decrease rapidly upon RT cessation. Hammarström et al. (41), for example, showed a similar 660 decrease in total RNA concentrations (19.3% ) after eight days of detraining in humans. 661 Furthermore,Figueiredo et al. (42) found that the decrease in ribosome content during muscle 662 disuse was correlated with the decrease in muscle CSA. Therefore, it is possible that ET did not 663 provide sufficient stimulus for ribosome maintenance, as has been shown by Romero et al. (43) 664 when providing treadmill ET in rats over a 12-week period. Indeed, this hypothesis is speculative 665 given that we do not have time coursed biopsies to examine markers of ribosome degradation in 666 the RT+ET group, and more research is needed in this regard. The increase in ribosome content 667 in the ET-only group is novel and equally as intriguing. Prior rodent work from our laboratory 668 suggests that 12 weeks of HIIT-style treadmill ET increases ribosome biogenesis markers in lieu 669 of decreasing skeletal muscle ribosome content (43). Subsequent work from Figueiredo and 670 collaborators (42) indicated that a bout of resistance exercise upregulates several markers in 671 skeletal muscle indicative of increased ribosome biogenesis, whereas this does not occur in 672 response to a steady-state bout of cycling. Others have also shown that weeks of concurrent 673 training enhances ribosome biogenesis relative to resistance training alone (44). Hence, these 674 prior and our current data suggest that the mode of exercise (e.g., HIIT versus steady state) and 675 (perhaps) species differences may affect the ribosome biogenesis response to ET. 676 The majority of studies investigating molecular adaptations in response to ET have 677 focused on mitochondrial variables due to their importance in oxidative metabolism. Several 678 studies have shown increased mitochondrial content and function in response to various forms of 679 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint ET (5,45,46). Considering that approximately 98% of the proteins that make up mitochondria 680 are encoded by the nuclear genome (47), we hypothesized that RT-mediated increases in 681 myonuclei and ribosomes would increase both the transcriptional and translational capacity of 682 myofibers, allowing for enhanced mitochondrial adaptations. In fact, Lee and collaborators (15)  683 reported that prior RT facilitated mitochondrial adaptations to a subsequent block of RT in rats. 684 Using both rodent and cell models, these authors also demonstrated that higher myonuclear 685 number was related to a greater expression of mitochondrial genes and proteins in response to 686 exercise. However, even though RT led to increased myonuclei and ribosome content in the 687 current study, most mitochondrial adaptations to subsequent ET were blunted. For example, the 688 protein levels of mitochondrial complexes I-IV in the ET-only group showed increases from 689 32% to 66%, while the RT+ET group only increased from 1% to 11%. Moreover, mixed fiber 690 relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the 691 RT+ET group. Once more, the reasons for such distinctive responses to ET are difficult to 692 reconcile. However, given that the RT+ET group also exhibited decreases in several other 693 variables (e.g., VL thickness, fCSA, and RNA levels), we speculate that these participants 694 existed in an enhanced catabolic/proteolytic state during the duration of the seven week ET 695 period. In support of this hypothesis are certain lines of evidence that have used stable isotopes 696 to ascertain mixed and fractional synthetic protein turnover rates. It is well-known that resistance 697 exercise acutely stimulates increases in both muscle protein synthesis and breakdown, albeit with 698 chronic training, increases in muscle protein synthesis generally exceed increases in muscle 699 protein breakdown (48-50). These events promote a longer-term net positive in protein balance 700 and result in myofiber and whole-tissue skeletal muscle hypertrophy. On the other hand, while 701 ET increases muscle protein synthesis and breakdown (49, 51), the increases in muscle protein 702 synthesis may be specific to mitochondrial (rather than myofibrillar) protein synthesis (52). In 703 addition, ET has been shown to increase several proteolytic markers in skeletal muscle (53,54). 704 When considering these prior data and our current observations, it remains possible that the 705 transition from RT to ET in the RT+ET group promoted a sustained elevation in muscle protein 706 breakdown mechanisms while diminishing the protein synthetic response. An ultimate 707 consequence of this shift may have included myofiber atrophy accompanied by a decrease in 708 cellular mitochondrial and ribosome content. While this is an attractive hypothesis to explain 709 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint several of the RT+ET observations herein, it is speculative and further investigation is needed to 710 confirm this hypothesis. 711 712

Experimental considerations 713
There are limitations to the current study. First, the n-sizes and biopsy time points were 714 limited in scope. Moreover, only younger adult men were examined herein. Hence, these data 715 should be viewed with these limitations in mind. Additionally, it is important to note that we did 716 not ascertain muscle protein synthesis or breakdown rates, and proteolytic markers were not 717 assayed. As such, much of our speculations regarding the RT+ET adaptations require further 718 inquiry. Markers of mitochondrial function were also not measured in the current study, and it is 719 possible that mitochondrial function improved in response to RT and/or ET. Finally, the 720 inclusion of a control group with a detraining period after RT is lacking, and the inclusion of 721 such a group would have helped distinguish the effects of RT cessation from ET adaptations. 722

723
Conclusions 724 In conclusion, the results of the present study showed that prior RT had no additional 725 benefits on performance adaptations to ET. Additionally, several mitochondrial adaptations to 726 ET (as well as other molecular outcomes) were blunted in the RT+ET group following the ET 727 period. Whether these maladaptive responses at the molecular level have longer-term functional 728 consequences remains to be determined. 729 730 critical assistance in manuscript preparation. All co-authors edited the manuscript, and all authors 744 approved the final submitted version. 745 746 DATA AVAILABILITY STATEMENT 747 Several raw data files can be obtained upon reasonable request by emailing the latter co-748 corresponding/senior author (ank0012@auburn.edu). 749 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 6, 2023. ; https://doi.org/10.1101/2023.04.06.535919 doi: bioRxiv preprint