# Cardiovascular and muscular plasticity in an endurance-master athlete following 12 weeks of detraining and retraining: a case study

Nadège Zanou, Vincent Gremeaux, Nicolas Place, Romuald Lepers

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Nadège Zanou, Vincent Gremeaux, Nicolas Place, Romuald Lepers. Cardiovascular and muscular plasticity in an endurance-master athlete following 12 weeks of detraining and retraining: a case study. JCSM Communications, 2024, 7 (2), pp.82-90. 10.1002/rco2.93. hal-05097423

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# Cardiovascular and muscular plasticity in an endurance-master athlete following 12 weeks of detraining and retraining: a case study

Nadège Zanou<fnref n="1" />,², Vincent Gremeaux<fnref n="1" />,³, Nicolas Place<fnref n="1" />,² & Romuald Lepers⁴*

## Abstract

**Background** This study examined the cardiorespiratory and muscular adaptations of a 53-year-old endurance master athlete following 12 weeks of detraining and retraining.

**Methods** Data were collected before and after detraining, and after retraining. Maximal oxygen uptake (VO₂max) was evaluated during maximal cycling exercise. Proteins involved in muscle contraction, mitochondrial function and glycolysis were investigated using western blot analysis.

**Results** VO₂max decreased by 7% after detraining and was 5% greater than baseline after retraining. Detraining induced an important increase in the ryanodine receptor type 1 protein levels (RyR1, +44%) with a decrease in the protein levels of its stabilizer FKBP12 (-24%). We observed a 138% increase in the sarco-endoplasmic reticulum ATPase 1 protein and a 42% increase in the myosin heavy chain fast-twitch protein in response to detraining. This was associated with depressed levels of the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) proteins, while the expression of the mitochondrial dynamic proteins appeared stimulated. Twelve weeks of retraining reversed almost all the alterations observed in muscle proteins, but specifically increased mitochondrial biogenesis, OXPHOS and antioxidant defence proteins as well as the glucose transporter 4 (Glut-4, +36%) and hexokinase (+100%) proteins levels above the baseline. The mitochondrial dynamic proteins were further increased with the retraining.

**Conclusions** These data provide novel information on cardiorespiratory and muscular plasticity, suggesting that highly endurance-trained athletes might show substantial muscular adaptations while retrained after a detraining period and call for more extensive clinical trials.

**Keywords** Cycling peak power output; Mitochondrial dynamics; Myosin heavy chain; OXPHOS; RyR1; VO₂max

Received: 16 January 2024; Revised: 24 April 2024; Accepted: 8 May 2024

*Correspondence to: Romuald Lepers, Inserm U1093, Cognition Action et Plasticité Sensorimotrice (CAPS), Université de Bourgogne, Faculté des Sciences du Sport, BP 27877 UFR STAPS, 21000 Dijon, France. Email:romuald.lepers@u-bourgogne.fr

## Introduction

Skeletal muscle has excellent plasticity and can adapt to varying functional demands by reversibly modifying its functional characteristics and structural composition. While physical training-induced adaptations of skeletal muscle increase the muscle's tolerance to exercise, training cessation results in a

partial or complete reversal of these adaptations, compromising physical performance.<fnref n="1" />,²

Detraining in endurance-trained athletes induces many changes in muscular characteristics.³,⁴ It is characterized by a decreased muscle capillary density, which may occur within 2 weeks of training cessation,⁴ but is not systematic.⁵ The arterial-venous oxygen difference, unchanged after a

<footnote n="1">¹Institute of Sport Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland; ²Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland; ³Department of Sports Medicine, Swiss Olympic Medical Center, Lausanne University Hospital, Lausanne, Switzerland; ⁴Inserm U1093, Cognition Action et Plasticité Sensorimotrice (CAPS), Université de Bourgogne, Faculté des Sciences du Sport, Dijon, France</footnote>
short-term training break, decreases if inactivity continues for up to 3 months.⁵ While myoglobin concentration does not appear to be affected by training cessation,⁵ a rapid and progressive reduction in oxidative enzyme activities reduces mitochondrial ATP production. The decrease in arterial–venous oxygen difference and oxidative enzyme activities is accompanied by a reduction in VO₂max in individuals undergoing long-term training cessation.⁵,⁶ Regarding muscle typology, fibre distribution remains unchanged during the initial weeks of inactivity, but there may be a decrease in the proportion of slow-twitch fibres and a large shift towards fast-twitch fibres in endurance athletes after several months of detraining.⁶,⁷

However, to our knowledge, how detraining, followed by retraining, alters key proteins involved in muscle contraction and metabolic fate in master endurance athletes remains unknown.

In this case report, we analysed the maximal aerobic capacity of a highly endurance-trained master athlete (i.e., >40 years old),⁸ a 53-year-old male, in response to 12 weeks of detraining and 12 weeks of retraining. In the meantime, we quantified the levels of proteins involved in skeletal muscle contraction, mitochondrial biogenesis, dynamics, oxidative phosphorylation, and antioxidant defence, as well as muscle glycolytic fate.

# Methods

## Ethical committee

This study was approved by the Committee on the Protection of Human Subjects (CER-VD 2022–01614) of Lausanne, and written informed consent was obtained from the participant.

## General procedures

At the first evaluation date, the subject was a 53-year-old French Caucasian living in France. He had been practicing triathlon since he was 20 years old. At the peak of his amateur career, he placed 5th overall at the French Long-Distance

Championships (age 35) and twice in the top 150 at the Hawaii Ironman triathlon championship. As he entered the master category, he has regularly placed in the top 5 of his categories in the Ironman 70.3 events. For the past 20 years, he has maintained a regular training schedule with no complete break for more than 2 weeks in a row each year. His annual training volume has remained relatively constant at about 500 h per year (120-h swimming, 270-h cycling, and 110-h running). During the summer of 2022, he voluntarily decided to take a 3-month break after his last competition scheduled for 5 September 2022 (ÖTILLÖ Swimrun race in Sweden consisting of 10 km open-water swimming and 65 km trail running where he placed 42th in men's team category in 10 hrs 01 min). As a university researcher, he planned the whole experiment with his colleagues (Figure 1).

Baseline data were collected in the 2 weeks before the start of training cessation. As the day after his last competition, the athlete engaged in no structured exercise and minimal daily physical activity for 12 weeks. He eventually walked twice for 30 min on the weekend at a slow pace and performed twice 15 min of core training (e.g., forearm plank, side bend, and hip dip) per week. Retesting was conducted on week 13 after training cessation. The athlete then began a structured retraining programme for 12 weeks, gradually progressing from approximately 50% of his usual training volume to 100% over the next 6 weeks. By the 7th week of retraining, he had reached his regular training regimen (10–12 h per week).

In the 3 months prior to his last competition, he trained an average of 10.8 h per week, divided as follows: 32% swimming, 38% cycling, and 30% running. During this period, he was mainly trained for a long-distance Swimrun event lasting around 10 h, so he concentrated more on long-duration, slow-, and medium-intensity training than on short-duration, high-intensity training. During the retraining period, his training volume was distributed as follows: 28% swimming, 47% cycling, and 25% running. Compared with his training programme at baseline, the participant incorporated more high-intensity training sessions, especially during the third month, but the total overall volume before the detraining period and during the retraining period was about the same.

<figure><img src="image_2.png" /><figcaption>**Figure 1** Graphical representation of the experimental procedures. Testing consisted of VO₂max measurement and muscle biopsy performed three times: before (Pre), after 12 weeks of detraining, and after 12 weeks of retraining.</figcaption></figure>
## Exercise performance testing

### Maximal aerobic capacity (VO₂max)

VO₂max was determined on an electrically braked cycle ergometer (Lode Excalibur, Groningen, The Netherlands) equipped with automatic pedals. The test began with a warm-up at 100 W for 2 min, after which the power output was increased by 25 W every 2 min until volitional exhaustion. During this incremental exercise, oxygen consumption was assessed with a metabolic analyser K5 (COSMED, Rome, Italy), respectively. The gas analyser was calibrated and used according to company guidelines in a closed room with constant temperature and humidity. The peak power output (PPO) was the highest power completed for 2 min. If the athlete stopped before the end of the 2 min stage, PPO was calculated in proportion to the time spent on the last stage.

### Muscle biopsies

Needle biopsies were taken from the left (non-dominant leg) *vastus lateralis* using previously described and validated procedures.⁹ Briefly, after skin sterilization and local anaesthesia, a 1 to 2 mm long skin cut was made with the tip of a scalpel. Biopsies were collected using an automatic biopsy device (Bard Biopsy Instrument, Bard Radiology, Covington, GA, USA). A 14-gauge disposable trocar mounted in the device was inserted through the cut, perpendicular to the muscle fibres, until the fascia was pierced. Three samples (~15 mg each) were collected from one puncture site at each time point. Muscle samples were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

### Western blot analysis

Western blot analysis was performed as previously reported¹⁰ with slight modifications. Muscle samples were resuspended in a lysis buffer (100 µl/5 mg of tissue) containing the following: 20 mM Tris/HCl (pH 6.8), 2 mM EDTA (pH 8), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM beta-glycerophosphate, 1 mM KH2PO4, 1 mM PMSF, 1 mM NaVO3, 50 mM NaF, and a protease inhibitor mixture (Roche, Complete Mini, Basel, Switzerland). The preparation was homogenized with potter, incubated for 1 h at 4°C on a wheel and then sonicated. The nuclei and debris were removed by centrifugation at 9300 x g at 4°C for 10 min and protein quantification performed using the BCA Kit (Thermo Fisher Scientific, Ecublens, Switzerland). 2x Laemmli sample buffer containing SDS and 2-mercapto-ethanol (Bio-Rad, Hercules, CA, USA) was added to 10 or 20 µg of protein. For PGC-1-α, OPA1, Mitofusine2, DRP1, Tomm20, MCU, and OXPHOS detection, samples were not heated while for all other proteins a 3 min incubation at 95°C was performed. Then, samples

were electrophoresed 40 min on 4–15% SDS-precast gradient gels (Bio-Rad, Hercules, CA, USA) and wet transferred 1 h onto pre-activated PVDF membranes. Total protein staining was detected on membranes with Red Ponceau staining, and bands were quantified using the Image Studio software v.5.2.5 (LI-COR, Lincoln, NE, USA). After red Ponceau was washed out membranes were incubated for 1 h at room temperature with v/v PBS-LI-COR blocking buffer (LI-COR, Lincoln, NE, USA). The membranes were then incubated overnight at 4°C with the appropriate primary antibody prepared in the blocking buffer: rabbit anti-RyR1 antibody kindly provided by Professor Andrew Marks lab (Columbia University, USA, 1:2500), rabbit anti-FKBP12, mouse anti-rabbit anti-SERCA1, mouse anti-SERCA2, rabbit anti-fast MHC, mouse anti-slow MHC, mouse anti-OPA1, mouse anti-mitofusin2, rabbit anti-DRP1, mouse anti-total OXPHOS (Abcam, Cambridge, UK, 1:1000), rabbit anti-SOD1, mouse anti-SOD2, rabbit anti-catalase (Abcam, Cambridge, UK, 1:1000), mouse anti-PGC1-α and rabbit anti-MCU (Sigma-Aldrich, St-Louis, MO, 1:1000), rabbit anti-hexokinase II and mouse anti-Glut-4 (Cell Signalling, Leiden, Netherlands, 1:500). Membranes were washed in PBS-Tween-20 0.1% and incubated for 1 h at room temperature with secondary antibody IRDye 680-conjugated donkey anti-mouse and IRDye 800-conjugated donkey anti-rabbit IgG (LI-COR, Lincoln, NE, USA, 1:10 000 and 1:5000, respectively) or IRDye 680-conjugated goat anti-mouse and IRDye 800-conjugated goat anti-rabbit IgG (LI-COR, Lincoln, NE, USA, 1:100 000 and 1:10 000, respectively). Immunoreactive bands were visualized using infrared fluorescence (IR-Odyssey scanner, LI-COR, Lincoln, NE, USA), and band densities were quantified using Image Studio v 5.2.5 (LI-COR, Lincoln, NE, USA). Protein intensity signal was normalized to that of total protein staining, and protein quantifications were expressed as a percentage of the Pre values. Two independent blots were performed for each protein detection, and the average of the quantifications was represented in the graphs.

## Results

### Retraining improves maximal exercise capacity

To investigate how detraining and retraining altered the athlete's performance, we measured the changes in absolute VO₂max and peak power output. Absolute VO₂max decreased from 4.58 to 4.27 L min⁻¹ (−7%) over the 12-week detraining period and then increased to 4.81 L min⁻¹ during the retraining period, reaching a value 5.0% greater than baseline. As the athlete's body mass was slightly altered during the protocol, from 74.0 to 76.1 kg and to 73.1 kg at Pre, Detraining, and Retraining, respectively, we also expressed the VO₂max relatively to the body mass. We observed almost
similar alterations from 62.0 to 56.2 mL kg⁻¹ min⁻¹ (−9%) with detraining and to 65.8 mL kg⁻¹ min⁻¹ (+6% compared with baseline) with retraining (Figure 2A). Peak power output was equal to 375 W, 338 W (−10%), and 388 W (+3% compared with baseline) before, after detraining, and after retraining, respectively (Figure 2B).

## Retraining after detraining alters proteins involved in muscle Ca²⁺ handling and phenotype

We investigated the levels of key muscle contractile proteins in response to detraining and retraining. The detraining induced a 44% increase in the levels of the main skeletal muscle Ca²⁺ release channel protein, the RyR1, and this was recovered after the retraining (only 3% higher than baseline) (Figure 3A,B). The small molecule FKBP12 (also called calstabin1) that stabilizes the RyR1 showed a decrease in its protein levels with the detraining (−24%) but did not recover with the retraining (Figure 3A,B). The Sarco/Endoplasmic reticulum Ca²⁺ ATPase type 1 (SERCA) protein 1 levels were upregulated in response to detraining (+138%) and returned almost to the baseline in response to retraining (−5% compared with baseline) (Figure 3C,B). SERCA2 protein levels were by opposite slightly depressed (−7%) with detraining and recovered after retraining (+14% compared with baseline) (Figure 3C,D). As SERCA1 is known to be mostly expressed in fast-twitch muscle fibres,¹¹ due to the huge increase of SERCA1 protein levels with detraining, we then measured the protein levels of the fast and slow myosin heavy chain (MHC). Fast MHC protein levels were increased by 42% with detraining and stayed slightly upregulated with retraining (+14% compared with baseline) (Figure 3C,D). No major changes were observed in the slow MHC protein levels: +16% and +14% compared with baseline with detraining and retraining, respectively (Figure 3C,D). Together, these data

suggest that detraining enhanced the expression of fast-twitch muscle proteins, which is reversed after a retraining programme.

## Aerobic exercise retraining increases proteins involved in muscle mitochondrial dynamics

As skeletal muscle constitutes an important system contributing to human oxygen consumption, we next investigated whether alterations in muscle mitochondrial proteins might support the observed alterations in VO₂max. We first measured the protein levels of the well-known regulator of mitochondrial biogenesis, the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which showed 19% decrease in response to detraining, followed by 16% increase compared with baseline in response to retraining (Figure 4A,B). We then measured the levels of proteins related to mitochondrial dynamics. The levels of the mitochondrial inner membrane fusion protein, the optical atrophy 1 (OPA1), and of the mitochondrial outer membrane fusion protein, mitofusin 2 (Mfn2) as well as of the mitochondrial fission protein, the dynamin related protein 1 (DRP1), were all upregulated in response to detraining (+17, +31%, and +42%, respectively) and further increased with retraining (+64%, +81%, and +96%, respectively, compared with baseline) (Figure 4A,B). This set of data suggests that aerobic exercise detraining in the endurance-trained master athlete initiates mitochondrial dynamics, which are enhanced with retraining.

## Retraining also improves muscle oxidative phosphorylation and glycolytic proteins associated with improved mitochondrial antioxidant defence

We first quantified the levels of Tom20, a mitochondrial outer membrane protein, and did not observe large changes

<figure><img src="image_3.png" /><figcaption>**Figure 2** Values of relative VO₂max (A) and peak power output (B) before (Pre), after 12 weeks of detraining, and 12 weeks of retraining.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 4** Endurance exercise detraining-retraining alters mitochondrial biogenesis and dynamics proteins. (A) Representative immunoblots of the per-oxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein, the optical protein atrophy 1 (OPA1), mitofusine2 (Mfn2) protein and the dynamin related protein 1 (DRP1); and (B) their quantifications. All gels were run twice, and the mean of their quantifications is represented.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

(-9% and +7% in response to detraining and retraining, respectively, compared with baseline) (Figure 5A,B). The mitochondrial Ca²⁺ uniporter (MCU) protein levels were almost

not changed in response to detraining (-3%) but were increased by 29% after retraining, compared with baseline (Figure 5A,B). We then measured the mitochondrial oxidative
<figure><img src="image_6.png" /><figcaption>**Figure 5** Endurance exercise retraining after detraining increases mitochondrial OXPHOS and mitochondrial antioxidant defence proteins. (A) Representative immunoblots of the mitochondrial outer membrane protein Tom20, the mitochondrial calcium uniporter (MCU) protein and the mitochondrial oxidative phosphorylation (OXPHOS) proteins complex I, CI, complex II, CII, complex III, CIII, complex IV, CIV and CV, CV ATPase; and (B) their quantifications. (C) Representative immunoblots of superoxide dismutase 1 (SOD1), 2 (SOD2) and catalase, and (D) their quantifications. All gels were run twice, and the mean of their quantifications is represented.</figcaption></figure>

phosphorylation (OXPHOS) proteins. The levels of OXPHOS complex protein I, CI, were decreased (–10%) with detraining and increased by 10% compared with baseline with retraining (Figure 5A,B). CII protein levels did not show many changes (–2% and +5% with detraining and retraining, respectively compared with baseline) (Figure 5A,B), while the levels of CIII, IV and V ATPase proteins showed a decrease of –8%, –13%, and –3%, respectively with detraining, they were increased by 24%, 20%, and 28%, respectively, after retraining, compared with baseline (Figure 5A,B). This was accompanied by important modifications of the levels of the antioxidant defence proteins. Indeed, the superoxide dismutase 1, SOD1, protein levels were increased by 16% with detraining and returned almost to the baseline with the retraining (+6% compared with baseline) (Figure 5C,D). SOD2 protein levels were by opposite depressed by 30% with detraining, while showing an important increase (+66% compared with baseline) in response to retraining (Figure 5C,D). Catalase proteins levels were decreased by 48% with detraining and

did not recover with retraining (–29% compared with baseline) (Figure 5C,D). We also observed important modifications of the muscle glycolytic proteins. The protein levels of the glucose transporter 4, Glut-4, the main glucose transporter of skeletal muscle, and of hexokinase II, HK II, the first limiting enzyme of the glycolytic pathway were slightly modified with detraining (+10% and –11%, respectively) but were clearly upregulated by 36% and 100% respectively after retraining, compared with baseline (Figure 6A,B).

Together, aerobic exercise detraining slightly alters mitochondrial OXPHOS and glycolytic proteins, which are boosted in response to retraining, accompanied by improved mitochondrial antioxidant defence.

## Discussion

Although these data originate from a case study, our report provides new information on cardiorespiratory and muscular
<figure><img src="image_7.png" /><figcaption>**Figure 6** Endurance exercise retraining after detraining boots the expression of muscle glycolytic proteins. (A) Representative immunoblots of glucose transporter 4 (Glut-4) and hexokinase II (HK II) proteins; and (B) their quantifications. All gels were run twice, and the mean of their quantifications is represented.</figcaption></figure>

plasticity in an experienced endurance master athlete after 12-week periods of detraining and retraining. The results suggest that highly endurance-trained master individuals may exhibit substantial muscular adaptations, superior to baseline when retrained, even after a prolonged detraining period.

## Detraining period

### Detraining and performance

Following the 12-week detraining period, the maximal aerobic capacity decreased by ~7% when expressed in absolute values and by ~10% when expressed relatively to body mass. Data on changes in maximal aerobic capacity after prolonged detraining (12 weeks) are scarce in the literature.<sup>12-14</sup> In young endurance-trained athletes (<30 years old), the decrease in relative VO<sub>2max</sub> after a 12-week detraining period was around 15–20%.<sup>5,15,16</sup> The two combined studies that looked at changes in VO<sub>2max</sub> after a 12-week detraining period in older endurance athletes (mean age: 59 ± 8 years, mean VO<sub>2max</sub> before detraining: 50 ± 5 mL kg<sup>-1</sup> min<sup>-1</sup>) showed a reduction ranging from 11 to 20%.<sup>12,17</sup> The present 53-year-old master athlete falls in the lower range of decline values observed in the literature.<sup>13</sup> However, it is unknown if the number of years of endurance exercise training (over 30 years in our study) could influence the rate of VO<sub>2max</sub> decline when exercise training stops.

### Detraining and muscle alterations

There is very little data in the literature on the alterations of muscle proteins in response to detraining, especially in healthy endurance master athletes submitted to a detraining protocol. We here showed that a 12-week of aerobic exercise detraining leads to major changes in the RyR1 protein levels, a result that has not been reported before. A study on young, recreationally-active adults submitted to 56 days of bed rest

showed a 27% decrease in RyR1 protein levels, which was prevented when the participants were submitted to resistance exercise during the bed-rest period.<sup>18</sup> These results might suggest that (1) human training level and/or age potentially determines the RyR1 alterations upon exercise cessation and (2) complete inactivity, such as bed rest, might be hugely detrimental for muscle cells. Of note, our experienced master athlete was performing his regular and daily duties, a situation which completely differs from bed rest, while mimicking a sedentary lifestyle.

Another study on rats submitted to hindlimb unloading for 14 days in contrast showed a huge increase in sarcoplasmic reticulum proteins, including RyR1 (+94%), a mechanism that the authors have proposed to lead to nucleocytoplasmic Ca<sup>2+</sup> overload, initiating apoptosis.<sup>19</sup> A potent cytosolic Ca<sup>2+</sup> overload linked to RyR1 protein increase might be enhanced in our study by the decrease in the RyR1 stabilizer protein FKBP12 (−24%).

Other major changes observed in our study with the detraining are the expression of fast-twitch muscle proteins: an increase in SERCA1 and fast MHC protein levels, associated with decreased levels of proteins involved in mitochondrial biogenesis and OXPHOS. However, we did not investigate muscle fibre composition in this study. A 15-day detraining of well-trained runners has also led to similar alterations as ours.<sup>4</sup> These results indicate that proteins involved in muscle aerobic capacity can quickly be altered in response to endurance exercise cessation. A previous study has reported an increase in mitochondrial oxidative enzymes in young and sedentary people in response to 15 weeks of high-intensity interval training protocol, which was lost after 7 weeks of detraining.<sup>20</sup> In our study, aerobic exercise detraining of the endurance master athlete also triggered mitochondrial dynamics, possibly to prepare the muscle cells for better aerobic adaptations in case of retraining, likely to enhance the efficiency of the system (improved physiological effects of training during retraining).
Together, our results suggest that a long-term aerobic training lifestyle does not protect muscle cells against the alterations occurring in response to 12 weeks of detraining. However, the extent of muscle protein alterations in response to the detraining seems larger than that of the change in performance. Discrepancy between alterations in muscle changes and performance has also been reported previously in a shorter time of a detraining/retraining protocol.⁴ Our data, however, point to a contribution of muscle alterations in the observed changes in the athlete performance.

## Retraining period

### Retraining and performance

Surprisingly, at the end of the retraining period, the athlete's VO₂max was 5% higher than the baseline, even though he was already in good shape at baseline. Although we do not expect it, we cannot exclude that the small changes in training modalities at retraining compared with before baseline may have played a role in this improvement. Nor can we rule out the possibility that the 12 weeks of detraining boosted the physiological effects of training during the retraining. The description of changes in cardiorespiratory parameters following prolonged periods of detraining and retraining in the same individuals is rare in the literature. A 26% decrease in VO₂max has been observed in a female cyclist master athlete (aged 49) following a 4-week detraining period due to traumatic injury.²¹ However, her VO₂max was only 2% below the pre-injury value after 4 weeks of retraining.²¹ These data suggest that maximal aerobic capacity remains highly plastic, even in master endurance athletes.

### Retraining and muscle adaptations

Our results showed different alterations of muscle proteins in response to retraining. The important increases in the levels of RyR1, SERCA1, and fast MHC proteins observed with detraining were all reversed with retraining. However, FKBP12 protein levels were not recovered, a situation that could prolong any cytosolic Ca²⁺ overload linked to a potent RyR1 instability, and that could also explain the increase in MCU protein levels, possibly as an adaptive way to buffer cytosolic Ca²⁺ towards more active mitochondria. Indeed, all mitochondria proteins, either involved in biogenesis, dynamic or OXPHOS, as well as the glycolytic proteins Glut-4 and HK II were clearly increased with retraining. This suggests muscle cell adaptation towards more powerful energy production. Accordingly, the mitochondrial antioxidant protein SOD2 was highly upregulated with retraining, when catalase protein levels stayed depressed and SOD1 protein levels returned to baseline levels. Together, these data support specific mitochondrial adaptations with the retraining programme. They also indicate that 12-week periods of detraining/retraining seem much more beneficial for muscle

metabolic proteins dynamics than short-time protocols. Indeed, 15 days of detraining/retraining on well-trained endurance participants were shown to depress maximal oxygen uptake and muscle oxidative and glycolytic enzymes at the detraining stage, then restored the VO₂max to nearly baseline values with the retraining but with no restoration of muscle enzymatic activity.⁴ Our data on 3-month detraining/retraining protocols clearly show an increase in muscle mitochondrial and glycolytic proteins after retraining, which might potentially correlate with increased enzymatic activity, even though not measured in this study. Our results suggest that restoration of cardiorespiratory function might be quicker than those of muscle proteins in well-trained athletes in case of retraining after a period of detraining.

In conclusion, this case report indicates that a 12-week detraining programme was beneficial for our well-trained master athlete to boost muscle metabolic phenotype and aerobic capacity after the completion of a retraining protocol. In terms of performance, 12 weeks of detraining reduced the VO₂max of the master endurance athlete by 7%, while 12 weeks of retraining increased it by 5% compared with baseline. At the muscle level, detraining induced a decrease in mitochondrial OXPHOS proteins associated with an increase in RyR1, fast MHC, and SERCA1 proteins, whereas retraining increased not only mitochondrial proteins but also glycolytic proteins all above baseline, likely improving skeletal muscle metabolism. This study may be used as a reference to determine the basis for periodization of training sessions or the management of injury periods in athletes.

## Acknowledgements

We thank Professor Bengt Kayser for his contribution to the muscle biopsies for this study.

## Conflict of interest statements

There are no competing interests.

## Funding

This work was supported by University of Lausanne financial support to NZ, VG, and NP.

## Ethics Statement

All human and animal studies have been approved by the appropriate ethics committee and have been performed in
accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All the patients gave their informed consent prior to their inclusion in the study.

Data availability statement

All data generated for this study are included. All authors have read and approved the manuscript.

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