# Advances in identifying functional groups in carnosol analogues to address their efficacy in skeletal muscle function

A. Berger, J. Sarniguet, G. Hugon, N. Cubedo, Sylvie Morel, Béatrice Chabi, Barbara Vernus, Anne Bonnieu, A. Brun, S. Matecki, et al.

► To cite this version:

A. Berger, J. Sarniguet, G. Hugon, N. Cubedo, Sylvie Morel, et al., Advances in identifying functional groups in carnosol analogues to address their efficacy in skeletal muscle function. Biomedicine and Pharmacotherapy, 2025, 190, pp.118397. 10.1016/j.biopha.2025.118397. hal-05258847v2

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# Advances in identifying functional groups in carnosol analogues to address their efficacy in skeletal muscle function

A. Berger<sup>a,b</sup>, J. Sarniguet<sup>b</sup>, G. Hugon<sup>a</sup>, N. Cubedo<sup>b</sup>, S. Morel<sup>c</sup>, B. Chabi<sup>d</sup>, B. Vernus<sup>d</sup>, A. Bonnieu<sup>d</sup>, A. Brun<sup>e</sup>, S. Matecki<sup>a</sup>, M. Rossel<sup>b,1</sup>, G. Carnac<sup>a,* ,1</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

Keywords:

Carnosol
Skeletal muscle
Human
Zebrafish
Diterpenes
Isorosmanol
Rosmanol

## ABSTRACT

Carnosol, a diterpene derived from rosemary, has recently emerged as a potential strategy to counteract skeletal muscle loss associated with aging, during diseases or sedentary lifestyle. It may protect skeletal muscle cells from inflammation, oxidative damage, and atrophy through multiple mechanisms. Here, to investigate the structure-activity relationship of carnosol, both natural and synthetic derivatives were evaluated for their ability to reduce oxidative damages and to enhance skeletal muscle hypertrophy and function. The natural analogues rosmanol and isorosmanol were selected for their conserved hydroxyl groups at positions C-11 and C-12, while differing in the B-ring moiety. In parallel, dimethylcarnosol and dimethylisorosmanol, featuring methoxy substitutions at C-11 and C-12, were used to assess the functional importance of these hydroxyl groups. In two oxidative damage assays, carnosol, rosmanol, and isorosmanol showed similar antioxidant activity, reducing lipid peroxidation accumulation in post-mortem mouse skeletal muscle tissue and phosphorylation of H2AX, a DNA damage marker, in human skeletal muscle cells. Carnosol and isorosmanol, but not rosmanol, promoted myotube hypertrophy and suppressed the E3 ubiquitine ligase MuRF1 in human skeletal muscle cells, while methoxylated derivatives lacked both antioxidant and hypertrophic effects. The functional efficacy of selected compounds was further evaluated in zebrafish larvae. Carnosol improved locomotion, increased slow myosin heavy chain positive fibers, and downregulated E3 ubiquitin ligases. Isorosmanol also enhanced locomotor performance, whereas its methoxylated analogue showed no effect. The obtained results highlighted the role of specific structural elements, conserved hydroxyl groups at C-11/C-12 and a 20,7-lactone moiety, in mediating the muscle-protective effects of carnosol analogues.

## 1. Introduction

In humans, skeletal muscles represent about 40 % of the body mass. Due to its ability to generate strength, skeletal muscles are essential for mobility, breathing, and posture. They also play a key role as a metabolic regulator through the use of glucose and lipids, particularly during exercise. Skeletal muscle possesses a remarkable capacity for regeneration, primarily due to the presence of resident stem cells known as satellite cells. These adult muscle stem cells reside beneath the basal lamina, adjacent to muscle fibers. In response to injury or stress, satellite

cells become activated and proliferate, giving rise to myoblasts. These myoblasts then undergo biochemical and morphological differentiation into myotubes, which subsequently organize into muscle fibers, thereby restoring muscle structure and function. Satellite cells can be isolated from muscle biopsies and cultured *in vitro*, where they recapitulate the various stages of muscle regeneration, including proliferation and both biochemical and morphological differentiation [1]. Many pathological (e.g., myopathies, cancer, diabetes) and non-pathological (e.g., sedentary lifestyle, immobilization, spaceflights) conditions lead to loss of muscle regeneration, muscle mass and consequently to reduction of the

<footnote><sup>a</sup> PhyMedExp, Montpellier University, CNRS, INSERM, Montpellier 34090, France</footnote>

<footnote><sup>b</sup> MMDN, Montpellier University, EPHE, INSERM, Montpellier, France</footnote>

<footnote><sup>c</sup> CEFE, Montpellier University, CNRS, EPHE, IRD, Montpellier, France</footnote>

<footnote><sup>d</sup> DMEM, Montpellier University, INRAE, Montpellier, France</footnote>

<footnote><sup>e</sup> UMR1019 UNH, INRAE, Clermont Auvergne University, Clermont-Ferrand, France</footnote>

<footnote>* Corresponding author.
E-mail address: gilles.carnac@inserm.fr (G. Carnac).</footnote>

<footnote n="1">¹ Both authors equally contributed to this work.</footnote>
functional capacities and life expectancy [2,3]. Skeletal muscle atrophy may also result in sarcopenia (i.e., the loss of muscle mass, size, strength and functionality because of aging). The mechanisms underlying muscle atrophy involved signaling pathways that control the balance between protein synthesis and degradation in skeletal muscle. Two key pathways govern the renewal of contractile proteins: the PI3K/Akt/mTOR pathway (phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin complex), which is the primary mechanism driving muscle hypertrophy, and the FOXO-dependent proteolytic pathways controlling E3 ubiquitin ligases, including MuRF1 (muscle RING-finger protein-1, TRIM63) and MAFbx (muscle atrophy F-box) and finally proteasome activity [4,5].

Healthy muscles strength and functions are inversely associated to all-cause mortality in human [3]. Therefore, preventing muscle loss may help improve quality of life and reduce the risk of chronic diseases and mortality. Since exercise is essential for maintaining skeletal muscles homeostasis [6], integrating physical activity into rehabilitation programs has become a standard component of care for many chronic diseases. However, compliance with exercise programs and their implementation could be difficult. Therefore, pharmacological intervention that could replace or enhance the effect of exercise sessions, facilitating muscle protein synthesis and avoiding muscles atrophy, are being intensively researched (nutrients, targeting potent negative regulator of muscle mass such as myostatin for example), but have not yet led to a breakthrough in clinical practice [7,8].

Consequently, there is a need for pre-clinical efforts to find effective molecules that might be translated into human interventions. Muscle cells are valuable models for identifying molecules since they can respond to both hypertrophic or atrophic stimuli by altering processes such as cell fusion or proteostasis dynamics [9,10]. However, *in vivo* models are crucial to validate the effects of these molecules on skeletal muscle function. Zebrafish offered a number of significant advantages for studying skeletal muscle responses. Skeletal muscles make up a considerable portion of the zebrafish trunk and has an exceptionally high degree of similarity to human muscle both molecularly and histologically. Zebrafish can also readily absorb xenobiotics added to their water and have been successfully utilized for large-scale drug screens to rescue damage to muscles [11,12]. Locomotor behavior, which begins at one day of life, is quantifiable and easily measurable in a non-invasive and reproducible manner at two and three days of life, as the embryo has developed the ability to escape a mechanical stimulus. Subsequently, the speed and frequency of the swimming pattern can be quantified [13,14].

It was shown that a rosemary leaf extract was sufficient to induce *in vitro* hypertrophy of human skeletal muscle cells and that carnosol, a abietane diterpene, was the active component mediating this effect. The hypertrophic effect of carnosol *in vitro* might be mediated by repressing the ubiquitin-proteasome dependent protein degradation pathway through inhibition of the E3 ubiquitin ligase MurF1 [9].

Carnosol, the major oxidation product of carnosic acid, is present in large amounts within rosemary leaves and possess one of the highest antioxidant activities of the abietane diterpenes [15,16]. Highly oxidized abietane diterpenes rosmanol, isorosmanol, and dimethylisorosmanol were also characterized in the leaves. They derived from carnosic acid by enzymatic dehydrogenation and the participation of activated oxygen [17]. All these structural analogues share conserved hydroxyl groups at C-11 and C-12 but differ in their B-ring lactone moiety. Carnosic acid and carnosol display differences in activity despite their tight structural similarities. Carnosol but not carnosic acid decreased both 26S and 20S proteasome activities at the earliest time assayed in HT-29 cells cancer cells [18], induced hypertrophic activity on human muscle [9] and counteracted C2C12 myotube atrophy induced by C26 medium [19].

Such finding suggested that specific functional groups play a crucial role in determining carnosol protective effects on muscle cells. By extension, the structural differences between natural analogues could

provide a valuable source of molecules with subtle chemical modifications, allowing researchers to identify the key structural elements essential for carnosol's antioxidant and hypertrophic activities.

To address this issue, natural carnosol analogues such as rosmanol, isorosmanol, dimethylisorosmanol, as well as a non-natural carnosol analogue, dimethylcarnosol were evaluated for their antioxidant capacity in mouse muscle tissue and human muscle cells, their hypertrophic effects on human muscle cells, and, for some, their impact on muscle function in zebrafish. This exploratory study identified isorosmanol as a promising carnosol analogue with potent muscle hypertrophy-inducing activity and underscored the importance of specific structural features—namely, the C-11 and C-12 hydroxyl groups and the 20,7-lactone moiety—in mediating the muscle-protective effects of carnosol and its derivatives.

## 2. Material and methods

### 2.1. Reagents

Butylated hydroxytoluene (BHT; B1378, SIGMA ALDRICH/MERCK). Carnosol (HY-NO643), rosmanol (HY-N5015), isorosmanol (HY-N41191), dimethylcarnosol (HY-N12492) and dimethylisorosmanol (HY-N4191) were provided by MedChemexpress (MedChemExpress EU, MedChemTronica, Sweden) with a purity of at least 98 %. Due to solubility constraints, carnosol analogues were dissolved in DMSO at a stock concentration of 10 mM and stored at -80 °C, except for post-mortem experiments where they have been diluted at a stock concentration of 3 mM in ethanol since DMSO freeze à 4°C (see below).

### 2.2. Post-mortem mouse muscle sampling and preparation

The experimental protocol was in strict accordance with the European directives (86/609/CEE) and was approved by the Ethical Committee of the Occitanie Region.

Gastrocnemius and Quadriceps muscles from six-month-old C57BL/6 male mice were removed and immediately placed on ice. Skeletal muscles were then minced with sterile scissors for 5 min and divided in 150 mg batches for each molecule and 300 mg for the control batch. Each batch of minced muscle was mixed with 0.01 % (w/w minced muscle) of each carnosol analogues dissolved in ethanol (CTRL) (15 µL/150 mg), based on the dose-response established for the reference antioxidant butylated hydroxytoluene (BHT) (stock solutions 3 mM in ethanol). Butylated hydroxytoluene (BHT) acts as an antioxidant and prevents lipid peroxidation in mice post-mortem muscle [20]. A control batch was mixed only with ethanol (0.01 %; 30 µL/300 mg). Control batch of minced muscle was divided in two portions (150 mg) using a weighing cup. All portions were packaged in a same polypropylene box with damp paper and covered with a film to maintain a humid atmosphere. The portions were stored at 4 ± 1 °C in the dark for 7 days. One portion of control batch (0 day) was immediately homogenized in 50 mM phosphate buffer (pH 7.0) (1:9) with an Ultra-Turrax homogenizer. The fraction of homogenate needed for thiobarbituric acid reactive substances (TBARS) measurement was quickly frozen. The same procedure was adopted for 7 days' time point. The experiment was performed six times. Results are presented as mean ±/– standard error of mean.

### 2.3. TBARS measurement in post-mortem mouse muscle

The lipid peroxidation index was determined in muscle homogenates by measuring TBARS [21]. Briefly, skeletal muscles homogenates were mixed with 154 mM KCl, phosphoric acid (1 % v/v) and 30 mM thiobarbituric acid (TBA). The mixture was boiled at 100 °C for 1 h. After cooling, it was extracted with n-butanol and centrifuged at 1000 g at room temperature for 15 min. The fluorescence intensity of the organic phase was measured with a spectrofluorometer (Ex: 515 nm; Em:
553 nm). A standard was prepared from 1,1,3,3-tetraethoxypropane (TEP), and results were expressed as nanomoles of TBARS per gram of tissue and were the mean ± SD of six experiments. Statistical analysis was done using Prism 6.01, GraphPad, San Diego, CA. Data were analyzed by one-way ANOVA. When a main effect was identified, Dunnett's multiple comparison test was used to localize the differences. Statistical significance was set at P-values< 0.05.

## 2.4. Human skeletal muscle cell cultures

Quadriceps muscle biopsies were obtained from one 19-year-old male (M19; Healthy male), at the Centre Hospitalier Universitaire Lapeyronie (Montpellier, France). Donor signed an informed written consent after the description of the protocol (Authorization N° DC-2008-594). Briefly, myoblasts (muscle progenitor cells) were purified from the muscle biopsies using an immunomagnetic sorting system with magnetic activated cell sorter (MACS) microbeads linked to an antibody against N-CAM (CD56) (Ref: 130-050-401; Miltenyi Biotec). After purification, H19 cells were at least 96 % positive for desmin (a muscle marker) by immunofluorescence. Myoblasts were cultured on collagen-coated dishes in DMEM/F12 medium with 10 % fetal bovine serum (FBS), 0.1 % Ultroser G and 1 ng/mL of human basic fibroblast growth factor (proliferation medium), as previously described [22]. Myoblasts can differentiate by fusing into long multinucleated myotubes that express specific skeletal muscle proteins. For cell differentiation, confluent cells were cultured in DMEM/F12 medium with 5 % FBS (differentiation medium) for 3 days. Then, cells were treated with carnosol (CO), rosinol (ROS), isorosmanol (ISOR), dimethylcarnosol (DCO) or dimethylisorosmanol (DISOR) for 48 h at 10 µM. Such concentration was selected for all carnosol analogues, based on previously established dose-response data for carnosol in repressing MuRF1 mRNA expression [9]. For induced oxidative damage experiments, human myotubes were treated or not (DMSO; solvent control) for 36 h with 10 µM of carnosol analogues prior to incubation with sub-lethal concentration of 100 µM H₂O₂ for one hour. To compare the effects of carnosol analogues, control and H₂O₂ alone received DMSO at 0,1 % at final concentration in culture medium in all experiments since carnosol and carnosol analogues were diluted in DMSO at the stock solution of 10 mM. Human muscle cells were used between passage 2 and passage 5.

## 2.5. Immunofluorescence staining of human muscle cells

Human myotubes were fixed in 2 % paraformaldehyde (ref: EM-15714, Electron Microscopy Sciences) in PBS and permeabilized with PBS/0.25 % Triton X-100. Cells were then incubated with a mouse monoclonal anti-troponin T antibody (1/200; ref: T6277, Sigma-Aldrich Merck), followed by the Alexa 555-conjugated anti-mouse antibody (1/1000; Thermo Fisher Scientific). Nuclei were revealed by DAPI staining. Images (seven fields for each condition) were taken with a Zeiss epifluorescence microscope, with 5X plan air objective and analyzed with the Image J software. Fusion index was calculated from fluorescence images of myotubes and nuclei using a custom macro developed in ImageJ (version 1.53 t). The macro processes paired images of myotubes and nuclei, applying thresholding (Huang method) to segment the myotube area and leveraging the Stardist plugin (Versatile 2D Fluorescent Nuclei model) for nuclei detection. The total number of nuclei and the number of nuclei outside the myotube regions were quantified. The fusion index was defined as the percentage of nuclei located within the myotube regions relative to the total nuclei count. Results were exported for analysis, including myotube area, total nuclei count, and fusion index. The macro supports batch processing of multiple images and requires the Stardist, CSBDeep, and Read&WriteExcel plugins.

## 2.6. Western blots of human muscle cells

Myotubes grown in 35 mm dishes were resuspended in 100 µL of

Laemml blue buffer (Biorad ref: 1610747) with DTT 50 mM. Samples were denatured at 70°C for 5 min. Cool samples on ice for 5 min. Protein extracts were centrifuged for 30 s at 12,000 RPM. Protein extracts (20 µg/well) were separated by SDS-PAGE gel electrophoresis (25 mA for 1 h) using 12 % and 4–15 % Mini-PROTEAN Precast Gels, and then transferred (1.3 A, 25 V for 5 min) using the Trans-Blot Turbo Transfer System to nitrocellulose membranes (Trans-Blot Turbo 0.2 µm Nitrocellulose Transfer Pack) (Bio-Rad, Schiltigheim, France). Membranes were blocked at room temperature with Odyssey blocking buffer (LICOR, France) and probed with the mouse monoclonal anti-MuRF1 antibodies (1/2000; ref: SC-398608; Santa Cruz Biotechnology,) or rabbit polyclonal anti phosphorylated γ-H2AX (ref: 9718S; 1/3000; Cell Signaling, Danvers, MA, USA) antibodies. Primary antibodies were followed by incubation with IRDye®680RD or IRDye®800RD secondary antibodies (Eurobio Scientific, Les Ulis, France). Fluorescence was quantified with the Odyssey software. Data were normalized to the Revert bands (Revert Total Protein Stain Kit, LI-COR USA).

## 2.7. Zebrafish lines and husbandry

The presented study followed the recommendations of the ARRIVE guidelines and the European Union Directive 2010/63. All experiments were approved by the Animal Welfare Structure of the zebrafish facility (registration number # B3417237). Zebrafish (Danio rerio) AB wild type strain was used and was acquired from the ZebraFish International Resource Center (European Zebrafish Center, EZRC, Karlsruhe, Germany). Adult zebrafish were bred and maintained under standard conditions in an automated fish tank system (ZebTEC, Tecniplast) at 28°C, pH 7, conductivity around 500–550 µS and with a 14 h:10 h light:dark cycle. Eggs were obtained by natural spawning and were maintained in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.05 % methylene blue) at 28°C. Each experimental procedure was carried out in triplicate. Each box contained between 70 and 80 larvae between D0 and D5 and is filled with 90 mL of E3 medium.

## 2.8. Carnosol and isorosmanol toxicity in zebrafish larvae

Toxicity was assessed on zebrafish larvae treated with Carnosol (CO) and Isorosmanol (ISO) between 1 dpf and 5 dpf in accordance with OECD 236 recommendations. Mortality was measured using Kaplan-Meier's curves and the rate of functional and morphological toxicity according to 5 factors criteria: cardiac edema, curved tail, delayed pigmentation and lack of swim bladder. Four different concentrations were used, 1.5–3 - 6–10 µM for CO and ISOR and compared with the control, DMSO (Supplementary Fig. s1). DMSO was kept to a final concentration of 0.1 % for experimental incubations and controls. For zebrafish, a concentration of 1.5 µM for carnosol and isorosmanol (and by extension for dimethylisorosmanol) was determined as the optimal dose, offering efficacy with minimal toxicity and mortality (Supplementary fig. s1).

## 2.9. Visual motor response assay

The locomotor activity of zebrafish larvae was quantified with VMR (visual motor response) assay using an infrared (IR) tracking system (Zebralab®, Viewpoint, Lissieu, France) as previously described [23]. In brief, at 5 dpf, larvae were transferred in a 96-well plate (Whatman, ref: 7701–1651) with 300 µl E3 medium and the locomotor behavior was monitored with an automated video tracking device (Zebrabox®, ViewPoint). The response to light changes was recorded by an IR camera under IR light illumination. The light protocol was as follows: 30 min of acclimatization in the dark (0 % light intensity), then two cycles of 10 min duration light ON (100 % light intensity) or light OFF (0 %) periods. The total duration of the experiment was 70 min. Activity during the experiment was measured in mm/s. The value obtained during OFF periods were subtracted for each larva from their values registered during ON periods to remove inter and intra group variability
in basic locomotion. Each experimental procedure was carried out in triplicate.

## 2.10. Mitochondrial activity measurement in zebrafish larvae

The oxygen consumption rate (OCR) of 5 dpf larvae was measured with the Seahorse XFe24 Extracellular Flux Analyzer (Agilent). Larvae were placed singly individually in wells of a Seahorse XFe24 spheroid microplate containing 500 µl of E3 medium. A grid was placed manually on larvae to keep them at the bottom of the wells throughout the experiment. The blanks were two empty wells of the plate. Four basal cycle readings were recorded, then five recording cycles following oligomycin (25 µM) injection, five recording cycles after carbonyl cyanide-p-trif luoromethoxyphenylhydrazone (FCCP) (8 µM) injection and nine recording cycles after rotenone + antimycin A (1.5 µM) injection. Calculations for specific parameters, such as non-mitochondrial respiration, basal respiration, maximal respiration, proton leak and ATP production, were made. The room temperature was controlled at 28±0.5°C. Measurements of total zebrafish OCR were started immediately and performed according to the manufacturer's instructions.

## 2.11. Western blots of zebrafish larvae

50 larvae (5 dpf) per condition in triplicate (Control DMSO vs. CO-treated at 1.5 µM) were resuspended in 500 µL of Laemmli blue buffer (Biorad ref: 1610747) with DTT 50 mM. Larvae were grinding with a manual grinder on ice for 1 min. Samples were denatured at 70°C for 5 min. Cool samples on ice for 5 min. Protein extracts were centrifuged for 30 s at 12,000 RPM. 12µl (30 µg) of total extract from each sample were loaded per well in triplicate (3 DMSO controls versus 3 CO) on 15 wells 4–15 % precast mini protean TGX gels (BIO-RAD ref: 4561086). Samples were run at 25 mAMP/gel for 1.5 h, then the proteins were transferred the proteins to a 0.2 µm nitrocellulose membrane (Turbo transfer pack BIO-RAD ref:1704159) for 10 min at 1.3 Amp and 25 volts on a BIO-RAD transblot turbo transfer system. After 1 h of drying the nitrocellulose membranes, total proteins of the extracts were quantified with Revert staining (LI-Cor Total Protein Stain Kit ref: 926–11010) according to the procedure given by the manufacturer. The membrane is scanned on the Odyssey LI-Cor and total protein in each well are quantified with image studio software. Then the membranes were blocked with Intercept Blocking buffer TBS (Licor ref: 927–60001) for 45 min at room temperature before incubation with the antibodies. Primary antibodies against Myosin Light Chains MLC (1:1000, DSHB ref: F-310), Neonatal Myosin Heavy Chains MHC (1:150, DSHB ref: N2–261), Slow Myosin Heavy Chains MHCs (1:100, DSHB ref: F-59) and sarcomeric alpha actin (1:5000, Sigma-Aldrich Merck, ref: SA2172) were diluted in blocking buffer with 0.2 % Tween 20 and then incubated at 4°C overnight with shaking, then washed with 0.1 % TBS-T buffer. The membranes were incubated according to their isotype in the anti-mouse IgG and IgG1 secondary antibodies IRDye 800CW (li-Cor ref: 926–32212 and 926–32350) diluted 1:30000 in 0.1 % TBS-T buffer for 45 min at room temperature, dried for 1 h away from light, scanned and quantified with studio image software. Each target protein signal was normalized by dividing the intensity of its band by the corresponding REVERT™ total protein stain signal for the same well.

## 2.12. Whole mount immunofluorescence and confocal microscopy of zebrafish larvae

Immunostaining was performed on whole mounted zebrafish larvae as previously described [24]. Briefly, zebrafish larvae were anaesthetized in 0.6 mM Tricaine (pH 7.0), and then fixed in 4 % paraformaldehyde in PBS for 2 h at room temperature. The zebrafish embryos larvae were washed in PBS/Tween 0.1 % for 3 × 10 min and then permeabilized with 1 % triton for 2 h at room temperature. Immunolabeling was performed with the following primary antibodies

and antagonist: anti-MyHC for slow muscles (F59 1/100, DSHB), anti-SV2 (1/50, DSHB) and α-Bungarotoxin (1/500). Secondary antibodies were Alexa Fluor® 488- or Alexa Fluor 555-conjugated anti-IgG secondary antibodies (ThermoFisher Scientific). For confocal imaging, embryos were mounted in Moviol 4–88, and data collected on the myotomes at position 16–17 somites, near the ventral fin on with an inverted Zeiss confocal 880 FastAi. equipped with a 40X-60X oil-immersion objective lens (Carl Zeiss, Oberkochen, Germany) Acquired image and stacks were subsequently processed using either Fiji or ZEN software (Zeiss).

## 2.13. Image analysis and 3D reconstruction of zebrafish larvae

Image acquisitions with the marker for slow MyHC (F59, DSHB) were obtained with the following settings: in a Z-stack thickness of 14–16-micron, Z-stack at a 40X oil-immersion objective. Images, were processed and analyzed using IMARIS 10.1.1 3D reconstruction software. The “Volume” function in IMARIS enabled to reconstruct the overall 3D volume represented by myosin heavy chains in myotomes 16–17. The “Spot” function in IMARIS enabled to quantify the number of neuromuscular junctions thanks to using the colocalization of Alpha-Bungarotoxin and SV2 immunostainings.

## 2.14. RT-qPCR gene expression analysis in zebrafish

Total RNA was extracted from larvae using the NucleoSpin RNA II Kit (Macherey-Nagel, Hoerdt, France). The RNA concentration for each sample was measured with a Nanodrop (Thermo Scientific). cDNA was synthesized using the Transcription Universal cDNA Master (Roche). Gene expression analysis was then performed as previously described [25] by quantitative polymerase chain reaction (qPCR) using a Light-Cycler device (Roche Diagnostics, Meylan, France) with the following sets of primers:

**trim63a (murf1a)**

Forward: 5'-CAGTGAACTCTACGACTCCAA-3'

Reverse: 5'-CGGTCCAGAACCACCTC-3'

**trim63b (murf1b)**

Forward: 5'-CTGCCGTGTCAGCATAAC-3'

Reverse: 5'-ACCACCAGAGAAACGATAAGG-3'

**fbxo32 (mafbx)**

Forward: 5'-GTGTCCTGACCTGAGTATGT-3'

Reverse: 5'-TTCCATTCCAACTGCCCT-3'

**Hmox-1 (Heme Oxygenase 1)**

Forward: 5'-TTGGCCAGACTGGAGGCTCT-3'

Reverse: 5'-CGAGGTAGCGGGTGAAGCG-3'

**ef-1α (reference gene)**

Forward: 5'-TTCTGTTACCTGGCAAAGGG-3'

Reverse: 5'-TTCAGTTTGTCCACACCCA-3'

## 2.15. Statistical analysis

Statistical analysis was performed using PrismTM v8.0 software (GraphPad, San Diego, CA, USA). Data were analyzed by one-way ANOVA. When a main effect was identified, Dunnett's multiple comparison test was used to localise the differences. To compare two experimental groups, the unpaired t test was used. All data were presented as mean ± SD or SEM. Statistical significance was set at P-values < 0.05. The levels of statistical significance were: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

# 3. Results

## 3.1. Carnosol analogues showed similar antioxidant activity in two oxidative damage assays

Carnosol analogues including carnosol (CO), rosmanol (ROS),
isorosmanol (ISOR), dimethylisorosmanol (DISOR) and dimethylcarnosol (DCO) (structures depicted in Fig. 1) were evaluated for their antioxidant capacity in a mouse mince muscle model (post-mortem) of lipid peroxidation and in human muscle cells treated with a pro-oxidant molecule.

Lipid peroxidation is the process of generating lipid peroxide, where oxygen free radicals oxidize membranes. It appears over time in processed meat (post-mortem) but can be delayed by the addition of antioxidants. This process can be reproduced in laboratory using minced muscle of mice refrigerated at 4°C for 7 days [20]. Minced muscle tissue was treated with ethanol (0.01 %, solvent, control), the synthetic phenolic antioxidant butylated hydroxytoluene (BHT, positive control), carnosol (CO), rosmanol (ROS), isorosmanol (ISOR), dimethylisorosmanol (DISOR) and dimethylcarnosol (DCO), all diluted in ethanol. As expected, the oxidation of lipids quantified by TBARS analysis increased in minced non-treated muscle (CTRL) after 7 days at 4°C and decreased with BHT treatment (Fig. 2A). CO, ROS, ISOR but not DCO and DISOR maintained the TBARS values at very low levels in minced muscle compared to the control group after 7th day of storage (Fig. 2A).

Pro-oxidants like hydrogen peroxide ($H_2O_2$; 100 µM treatment, a sublethal concentration in human muscle cells) increase reactive oxygen species (ROS) levels and induce oxidative damages such as DNA double-strand breaks in cells. The protective effects of carnosol analogues were assessed by evaluating their ability to prevent $H_2O_2$-induced damage in human myotubes. At day 3 of differentiation, when myoblasts had fused and myotubes had formed, cells were treated or not (DMSO; solvent control) with carnosol analogues for 36 h before exposure to 100 µM $H_2O_2$ for one hour. DNA damage was then assessed by measuring phospho-γH2AX protein levels via western blotting, as γH2AX phosphorylation is a marker of DNA double-strand breaks (Fig. 2B). As expected, $H_2O_2$ treatment increased phospho-γH2AX levels. Pre-treatment with CO, ROS, and ISOR significantly reduced phospho-γH2AX

accumulation, whereas DISOR and DCO had no effect (Figs. 2B and 2C).

The results obtained in the presented study demonstrated that CO, ROS, and ISOR exhibited comparable antioxidant activity in both biological assays. They also revealed a relationship between structural features of carnosol analogues and their antioxidant activity pointing that the substitution of hydroxyl groups at C11 and C12 with methyl groups, completely abolished the antioxidant activity.

## 3.2. Carnosol and isorosmanol induced muscle hypertrophy and repressed MuRF1 in human muscle cells

Human skeletal muscle cell cultures were used to evaluate the hypertrophic potential of carnosol analogues. At day 3 of differentiation, when myotubes had formed, cells were treated or not (0.1 % DMSO; solvent control) with 10 µM of carnosol analogues. After 48 h of incubation, cells were fixed, and the expression of troponin T-a cytoskeletal protein exclusively expressed in myotubes- was analyzed using immunofluorescence. The quantification of the area of troponin T-positive myotubes was determined with the Fiji software. Compared with controls (DMSO treated), incubation of myotubes with CO, ISOR but not ROS, DCO or DISOR led to the appearance of large, branched myotubes, resulting in increased muscle cell area, as shown by troponin T immunostaining (Fig. 3A, quantified in Fig. 3B), and unchanged fusion index (Fig. 3C) and number of nuclei (Fig. 3D). Protein levels of the musclespecific E3 ligases MuRF1 were then analyzed. CO and ISOR, but not ROS, DCO or DISOR, were found to significantly reduced MuRF1 protein levels (Figs. 3E and 3F).

Data obtained from the presented study suggested that the hypertrophic activity of carnosol analogues could depend on hydroxyl groups at C11 and C12, as well as specific chemical structures absent in rosinol.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Structure of carnosol analogues. (A) Formation of natural carnosol analogues in rosemary plants. Carnosol, rosmanol, isorosmanol, dimethylisorosmanol are formed from carnosic acid by enzymatic dehydrogenation and oxidation (From [17]). (B) Chemical structure of non-natural dimethylcarnosol with substitution of hydroxyl groups at C11 and C12 with methoxy groups.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** High antioxidant activity for carnosol analogues in “minced-muscle” and human skeletal muscle cell assays. (A) Minced gastrocnemius muscles from six-month-old C57BL/6 male mice were mixed with ethanol (0.01%; solvent; CTRL), BHT (positive control), carnosol (CO), rosmanol (ROS), isoromanol (ISOR), dimethylcarnosol (DCO) or dimethylisorosmanol (DISOR), all dissolved in ethanol and mixed at 0.01% w/w minced muscle. At day 0 and day 7 of refrigerated storage (+4 °C), lipid oxidation was evaluated by TBARS quantification; Mean ± SD, N = 6; p < 0.0001 (****) compared with CTRL at day 7. (B, C) Human myotubes treated for 36 h with 10 µM of carnosol analogues or DMSO (Solvent; CTRL) prior to incubation with sub-lethal concentration of 100 µM H₂O₂ for one hour. (B) Western blot analysis of phosphorvlated γH2AX protein level; (C) Quantification of the Western blot data, A.U. for arbitrary unit represents the ratio of p-γH2AX protein level on revert staining. Mean ± SD, N = 3; \*p < 0.05, p < 0.01 (\*\*), p < 0.001 (\*\*\*\*) compared with H₂O₂.</figcaption></figure>

### 3.3. Carnosol treatment enhanced the level of myosin heavy chain proteins in zebrafish

Given the differential effects of carnosol analogues on human muscle cells, the *in vivo* effects of these compounds were tested in zebrafish larvae. A first approach was to validate the zebrafish as a model by testing the effect of carnosol. The initial step involved evaluating the toxicity of CO on zebrafish larvae. An optimal concentration of 1.5 µM was selected for subsequent experiments due to its efficacy and low toxicity (Supplementary fig. s1).

To determine whether carnosol regulates the expression of various proteins associated with components of muscle structure, the levels of myosin heavy chain, myosin light chain and sarcomeric actin were quantified by western blot in larvae treated or not (DMSO 0.1%; solvent control) with 1.5 µM CO (Fig. 4). The results showed no difference in the levels of sarcomeric actin (SA) (Fig. 4 B and 4E) or myosin light chains (MyLC) (Figs. 4B and 4F). In contrast, our results revealed an increase in slow myosin heavy chains (sMyHC) (Fig. 4A and 4C) and neonatal myosin heavy chain (neoMyHC) (Figs. 4B and 4D). To investigate whether CO treatment induced any significant structural changes in slow Myosin Heavy Chain (sMyHC) fibers in 5 dpf zebrafish larvae (Fig. 5), confocal microscopy was used to visualize sMyHC labeling (Fig. 5A). Confocal acquisitions were reconstructed in 3D using IMARIS

10.1.1 analysis software, enabling visualization of the volume of slow muscle fibers in the myotomes (Fig. 5B; Supplementary Video S1). The analysis revealed that the muscle volume represented by slow muscle fibers was increased following CO treatment compared with the DMSO control (Fig. 5C).

These changes in skeletal muscle structure can be accompanied by an increase in mitochondrial respiratory capacity or a change in muscle innervation. Mitochondrial capacities were addressed first. Oxidative mitochondrial respiration rates were measured in whole zebrafish larvae at 5 dpf larvae treated or not with CO. CO-treated larvae showed no increase in basal respiration, ATP production, maximal respiration, proton leakage, or non-mitochondrial respiration (Supplementary Fig. s2). Neuromuscular junction (NMJ) integrity was determined by co-labeling α-Bungarotoxin (post-synaptic) and SV2 (pre-synaptic). A 3D reconstruction, based on confocal microscopy acquisition of immunolabeled larvae, showed that CO-treated larvae stained for α-Bungarotoxin-DsRed conjugated and SV2 antibody exhibited similar labeling rates to the DMSO treated control group. No differences were observed in the colocalization of SV2 and α-Bungarotoxin in myotome 16–17 in CO-treated larvae, compared with the DMSO-treated control group (Supplementary Fig. s3; Supplement video S2).

All together these data suggested that CO treatment increased the quantity and the volume of slow myosin fibers but did not significantly
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Among carnosol analogues, only carnosol and isorosmanol induced myotube hypertrophy and repressed MURF1 in human skeletal muscle cells. Differen- tiated myoblasts were treated or not (CTRL; 0.1 % DMSO) with CO, ROS, ISOR, DCO and DISOR, at a concentration of 10 µM. Two days later, myotubes were processed for indirect immunofluorescence analysis with an antibody against troponin T (green). Nuclei were visualized by DAPI staining (blue). (A) Representative images. (B,C,D) Quantification of troponin T images (myotube area in µM²), fusion index (in %) and nuclei (average per field). (E) In parallel, myotube protein extracts were analyzed by western blotting with anti-Muscle Ring-Finger Protein-1 (MuRF1; TRIM 63) antibody (E; green). Revert® total protein stain was used as internal loading control (E; red). (F) Quantification of the western blotting data. A.U., arbitrary units (i.e. the ratio of the level of the protein of interest to the Revert® stain level. Mean ± SD, n = 5; \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001 compared with DMSO.</figcaption></figure>

affect mitochondrial function and formation or distribution of neuro-muscular junctions.

### 3.4. Carnosol treatment repressed expression of E3 ubiquitin ligase in zebrafish

It was recently shown in human muscle cells that carnosol is able to
<figure><img src="image_6.png" /><figcaption>Fig. 4. Carnosol enhanced protein levels of myosin heavy chain in zebrafish skeletal muscles. Protein extracts of zebrafish larvae at 5 dpf (50 larvae per condition), treated at 1 dpf during 5 days with 1.5 µM CO or 0.1 % DMSO as control, were analysed by western blot with: (A) anti-slow Myosin Heavy chain (sMHC) antibody (F59); (B) anti-neonatal Myosin Heavy Chain (neoMHC) antibody (N2.261), anti-sarcomeric actin antibody (SA) and anti-myosin light chain (MyLC) antibody (F310). Revert® total protein stain was used as internal loading control. (C, D, E, F) Quantification of the western blotting data; A.U., arbitrary units (i.e. the ratio of the level of protein of interest relative to the Revert® stain level). Mean ± SD, n = 3; \*p < 0.05.</figcaption></figure>

inhibit the expression of the E3 ubiquitin ligase MuRF1, which is involved in protein degradation. To determine whether this regulatory effect is maintained *in vivo*, the expression of MuRF1 was subsequently examined in zebrafish larvae. To this end, larvae of the AB line were treated with 1.5 µM CO for 5 days, while a control group received 0.1 % DMSO. At 5 days post-fertilization (dpf), gene expression levels were analyzed by qPCR. The analysis focused on the expression of three major E3 ligases present in zebrafish muscle tissue: two genes *murf1* are present, *murf1a* (*trim 63a*) and *murf1b* (*trim63b*), *murf1b* expression being residual at early stages of development [26], and *fbxo32* (*mafbx*).

*hmox-1*, a target gene of carnosol in human muscle cells with antioxidant properties was also quantified. A reduction in the expression levels of *fbxo32* (*mafbx*) (Fig. 6A) and *trim63a* (*murf1a*) (Fig. 6B) was observed. *trim63b* and *hmox-1* gene expressions did not appear to be affected by CO treatment(Figs. 6C and 6D).

### 3.5. Carnosol and isorosmanol improved skeletal muscles function in zebrafish

The functional impact of CO, ISOR, DISOR treatments were
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Carnosol induced an increase of slow muscle fibers volume in myotome of zebrafish larvae. (A) Confocal acquisition with F59 (sMHC) immunolabelling on 5dpf larvae treated with 1.5 µM CO or 0.1 % DMSO as control; (B) 3D IMARIS reconstruction based on the confocal acquisition (z-stack); (C) Quantification of myotome volume normalized with its total area. Mean ± SD DMSO: n = 10; CO: n = 10; ** P < 0,01.</figcaption></figure>

measured using a locomotor test developed in zebrafish, the visual
motor response (VMR). By alternating light and dark periods, the VMR
induces an escape reflex that reflect the locomotor capacities, quantified
with automated machine vision measurements of zebrafish motor ac-
tivity in 96-well plates at 5-day post fertilization (dpf) (Fig. 7). Larvae
were treated with DMSO (0.1 % as solvent, control) or CO, ISOR, DISOR
(1.5 µM) from 1 to 5 dpf. The VMR were performed at 5 dpf on groups of
larvae. Sequence protocol was given for DMSO and CO treated larvae
(Fig. 7A). VMR analysis revealed increased larvae motricity after five
days of treatment with CO or ISOR (Fig. 7B). Considering CO, this
enhancement was associated with elevated movement lengths, encom-
passing both small and large bursts of swimming (Figs. 7C and 7D), as
well as augmentation in both mean swimming speed and the percentage
of time spent in activity (Fig. 7 E and 7 F). Isorosmanol treatment
increased only the percentage of time spent in activity (Fig. 7F), with the
effect on movement duration approaching statistical significance
(P = 0.053; Fig. 7E). In contrast, DISOR treatment had no effect on the
motor abilities of the larvae (Fig. 7).

4. Discussion

The initial observations indicated that carnosol, rosmanol, and iso-
rosmanol possess strong antioxidant activity, effectively preserving
muscle cells from oxidative damages. Interestingly, this antioxidant
activity was abolished when the hydroxyl groups at carbon positions

C11 and C12 of carnosol and isorosmanol were methylated. In human
muscle cells, only carnosol and isorosmanol demonstrated the ability to
induce myotube hypertrophy and repress the expression of the E3
ubiquitin ligase MURF-1, a key regulator of muscle protein degradation.
To extend these findings in vivo, the functional impact of some com-
pounds was evaluated in zebrafish larvae. Behavioral analysis using the
Visual Motor Response (VMR) assay revealed that both carnosol and
isorosmanol significantly improved locomotor performance, whereas
dimethylisorosmanol had no effect. To further elucidate the mechanisms
underlying carnosol's effects on zebrafish muscle function, several pa-
rameters were examined that can enhance skeletal muscle function such
as increase mitochondria function [27] and neuromuscular junction
integrity [28]. However, no detectable changes in mitochondrial activ-
ity or neuromuscular innervation were found. Then, the studies focus on
structural components of muscle tissue. Skeletal muscle fibers can be
broadly classified into type I (slow-twitch) and type II (fast-twitch) fi-
bers. Type I fibers are characterized by slow contraction, high fatigue
resistance, and elevated mitochondrial content, whereas type II fibers
contract rapidly but are more susceptible to fatigue [29]. While type II
fibers are often considered to have greater hypertrophic potential,
multiple studies in both rodents and humans have shown that type I
fibers are also capable of undergoing hypertrophy under certain physi-
ological conditions [30–32]. In zebrafish, slow muscle fibers expressing
slow myosin heavy chain 1 (Smyhc1) are among the first to form during
embryonic myogenesis. Smyhc1 is essential for proper sarcomere
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Carnosol repressed expression of E3 ubiquitin ligase *mafbx* and *murf1a* in zebrafish larvae. Relative mRNA expression of (A) *fbxo32* (analogue of the human *MAFBX*), (B) *trim63a* (1st analogue of the human *MURF1*), (C) *trim63b* (2nd analogue of the human *MURF1*) and (D) *hmox-1* (analogue of the human Heme Oxygenase 1, *HO-1*) in the 5dpf larvae treated or not (0.1 % DMSO; solvent control) with 1.5 µM CO. In each group: n = 80 (each dot represents the RNA extraction of 10 larvae). Mean ± SD * P < 0.05; **P < 0.01.*</figcaption></figure>

assembly and muscle function, and its loss has been linked to impaired motility and decreased survival [33]. This study revealed that in zebrafish carnosol treatment significantly increased the levels of slow myosin heavy chains (sMyHC) protein, consistent with previous findings in human skeletal muscle cells treated with carnosol [9]. This upregulation was further confirmed through confocal microscopy and 3D reconstruction, which demonstrated a clear increase in slow muscle fiber volume in zebrafish larvae. Interestingly, carnosol also down-regulated the expression of the E3 ubiquitin ligase MuRF1 in zebrafish, consistent with our observations in human muscle cells. This suggests that this carnosol regulatory pathway involving MuRF1 is conserved across species and supports the idea that carnosol promotes muscle hypertrophy by modulating the expression of specific structural proteins, particularly myosin heavy chains (MHCs), which are known targets of MuRF1-mediated degradation [5]. Consequently, it could be hypothesized that carnosol, by enhancing the size of type I muscle fibers, could enhance skeletal muscle endurance and fatigue resistance in zebrafish larvae.

Recent studies have highlighted the potential of novel plant- or fruit-derived molecules for the treatment or prevention of skeletal muscle atrophy. These bioactive compounds play critical roles in maintaining and promoting muscle tissue growth by regulating protein balance,

which depends on the dynamic equilibrium between protein synthesis and proteolysis pathways. These compounds (the list below is not exhaustive) can be classified based on their mode of action such as: 1) Activation of IGF-I/AKT protein synthesis signaling pathway and downregulation of proteasome activity (*ursolic acid*, a triterpenoid enriched in apple skins and medicinal herbs; *tomatidine*, 3β-hydroxy steroid derived from tomatoes; *20-Hydroxyecdysone*, plant-derived phytosteroids, and *urolithin B*, a metabolite derived from ellagitannins, predominantly found in pomegranates.) [10,34–36]; 2) enhancement of muscle energetics and mitochondrial function (*urolithin A* and *resveratrol*, a polyphenol found in grapes and berries) [37,38]; 3) suppression of inflammation-induced muscle atrophy (*Handelin*, a compound derived from *Chrysanthemum indicum* L.) [39].

Notably, carnosol can simultaneously target multiple signaling pathways in muscle cells, including anti-inflammatory, antioxidant, and protein degradation inhibition mechanisms. Carnosol and chemical derivatives generated through targeted structural modifications have been shown to suppress the activation of the NF-κB pathway in C2C12 myotubes exposed to inflammatory cytokines and in C26 tumor-bearing BALB/c mice, thereby protecting muscles from atrophy [19,40]. Additionally, carnosol enhanced the glutathione metabolism and strengthens the antioxidant system in muscle cells [41]. In this study, carnosol and its natural analogues isorosmanol, but not rosmanol, were found to induce muscle fiber hypertrophy in human muscle cell cultures and zebrafish under normal, non-inflammatory conditions, most likely by inhibiting E3 ubiquitin ligases [9]. Therefore, carnosol and its analogues allowed a multifaceted approach to mitigating muscle loss associated with aging, disease, and inactivity.

Moreover, results obtained from the presented study also offered valuable insights into the structure–function relationship of carnosol and its analogues. Carnosol is an ortho-diphenolic diterpene with an abietane carbon skeleton with hydroxyl groups at positions C-11 and C-12 and a lactone moiety across the B ring. These hydroxyls are conserved in the same position on all carnosol analogues. Monomethyl substitution in carnosic acid (12-O-methyl carnosic acid) and carnosol (12-O-methyl carnosol) abolished their antioxidant activity, suggesting that the presence of an ortho-dihydroxy group on the C-ring is essential for antioxidant function [42]. Similarly, simple natural polyphenol compounds with two or more hydroxyl groups in ortho or para position to each other presented the highest antioxidant properties whereas their methoxy derivatives were shown to be less efficient [43]. In this study, carnosol, rosmanol, and isorosmanol were found to be strong antioxidant in muscle tissue but when the hydroxyl groups at carbon positions C11 and C12 of carnosol and isorosmanol were methylated, this activity was lost. More surprisingly, the impact on skeletal muscles function was also lost in human muscle cells and zebrafish. It is thus tempting to suggest that antioxidant activity is related to hypertrophic activity in the muscular system. This appears to contradict our earlier findings, which demonstrated that while carnosol activates the NRF2 antioxidant signaling pathway in human primary muscle cells, this activation is not essential for its hypertrophic effect [9]. However, the obtained data suggested that the antioxidant activity of carnosol analogues is necessary but not sufficient to mediate muscle hypertrophy. Carnosic acid and rosmanol, which have antioxidant activity and conserved hydroxyl groups at carbon positions C11 and C12, did not exhibit muscle hypertrophy activity (this manuscript; [9,19]). This suggested that other chemical structures may play a crucial role in mediating carnosol analogues effects in skeletal muscles. Carnosic acid differs structurally from carnosol, as carnosol contains a lactone moiety in the B ring, whereas carnosic acid has a free carboxylic acid group. Interestingly, carnosol and isorosmanol share the same 20,7-lactone whereas rosmanol has a 20,6-lactone. Therefore, this 20,7-lactone could be important to mediate carnosol analogues effects in muscle in cooperation with hydroxyl structures in C11 and C12.
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Carnosol and isorosmanol increased locomotor capacity of zebrafish larvae. (A) Sequence protocol: the locomotion is measured for 90 min, with 30 min of training in the dark (OFF), then 3 cycles of light/dark (ON/OFF) of 10 min each. Representative profiles of DMSO (0.1 %) and CO (1.5 µM) treated larvae were illustrated. (B) total distance, (C) small distance, (D) large distance traveled per minute by zebrafish larvae treated or not (DMSO; solvent control) with CO, ISOR and DISOR at 1.5 µM during the different VMR sessions. DMSO: n = 316 CO: n = 192; ISOR: n = 254; DISOR: n = 253. Data were normalized to DMSO. (E, F) Quantification of motility of (E) larvae mean speed (mm/s), (F) % of activity (time active) during the OFF phases. Mean +/- SEM * P < 0.05; *** P < 0.001; **** P < 0.0001.</figcaption></figure>

## 5. Conclusion

Taken together, the results of the presented study indicated that the hydroxyl groups at positions C-11 and C-12, along with the 20,7-lactone moiety spanning the B ring, are critical structural features underlying the muscle-protective effects of carnosol and its analogues. Further structural modifications of carnosol are needed to better identify the structural features essential for mediating its effects on muscle. Ultimately, this could lead to the development of new agents with enhanced skeletal muscle protective potentials.

### CRediT authorship contribution statement

**A. Berger:** Investigation, Data curation, Methodology, Investigation, Formal analysis. **J. Sarniguet:** Investigation. **G. Hugon:** Investigation. **N. Cubedo:** Ressources. **S. Morel:** Ressources. **B. Chabi:** Investigation. **B. Vernus:** Investigation. **A. Bonnieu:** Writing – review & editing. **A. Brun:** Software. **S. Matecki:** Writing – review & editing. **M. Rossel:** Project administration, Methodology, Conceptualization, Validation, Writing – original draft. **G. Carnac:** Project administration, Methodology, Conceptualization, Funding acquisition, Validation, Writing – original draft.

### Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used OPEN AI in order to improve the readability and language of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This work was financially supported by CNRS, INSERM, Montpellier University, SATT AxLR Occitanie-Méditerranée and AFM MYOccitanie, France. We thank Elodie Jublanc for her help and training on confocal microscopy and Imaris software and the imaging facility MRI, member of the national infrastructure France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). We thank Philippe Clair for assistance on pPCR platform, and we thank the zebrafish facility ZeNeuro and ZebraSens phenotyping from the ZEFIX platform.

### Supplementary figure s1. In-vivo toxicity assessment of CO and ISOR

(A) Image representation of the different toxicity events used. (B) Number of larvae presenting at least one of the four toxic events considered. (C) Detail of the number and percentage of occurrence of each toxic event according to the treatment used. (D-E) Kaplan-Meier survival curves of the 1.5 µM CO and ISOR treated group compared to the 0.1 % DMSO treated group. Logrank test was used to determine the statistical significance of the survival.

### Supplementary figure s2. Analysis of mitochondrial respiration in zebrafish larvae treated with CO.

(A-F) Oxygen Consumption Rate (OCR) profiles of zebrafish larvae at 5 dpf treated at 1 dpf during 5 days with CO (1.5µM) or DMSO (0.1 %) as control, (B) basal respiration, (C) ATP production, (D) maximal respiration, (E) proton leak, (F) non-mitochondrial respiration. Errors bars
represent ± SD calculated from three replicates. Each group, CO and DMSO, has 20 larvae. DMSO: n = 22; CO: n = 22; ns = non-significant; unpaired *t*-test.

**Supplementary figure s3. Neuromuscular junction quantification in zebrafish larvae treated with CO.**

Zebrafish larvae at 5 dpf, treated at 1 DPF during 5 days with CO (1.5 µM) or DMSO (0.1 %) as control, were stained: (A) with α-bungarotoxin conjugated with Alexa Fluor 594, (B) with SV2 conjugated with Alexa Fluor 488. (C) Confocal z-stacks were processed on IMARIS reconstruction software. Each labelling point for alpha-bungarotoxin and SV2 was reconstructed as a sphere using the “spot” function. The areas of colocalization were then quantified to determine the number of neuromuscular junctions. (D) The percentage of AlphaBungarotoxin-SV2 co-localization per myotube was measured using the IMARIS internal statistical function. Percentage of co-localization in AB larvae treated with CO or DMSO. ns = not significant; unpaired *t*-test.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118397.

**Data Availability**

Data will be made available on request.

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