Nadège Zanou

Introduction: Repeated sprint training in hypoxia (RSH) or with blood flow restriction (RS-BFR) are known to enhance the repeated-sprint ability. However, studies directly comparing their underlying molecular mechanisms are scarce. Methods: Here we used a comprehensive approach combining pre- and post-intervention testing including sprint performance tests, neuromuscular assessments, skeletal muscle biopsies for protein levels quantification, and blood samples for systemic metabolites analyses to characterize the specific adaptations induced by RSH and RS-BFR. Recreationally active men performed 9 sessions of RSH (FiO₂ ~0.136; n=9) or RS-BFR (45% arterial occlusion; n=8). Results: Both RSH and RS-BFR similarly improved exercise performance without significant changes in knee extensor maximal voluntary contraction force. Both training modalities reduced the levels of oxidative phosphorylation proteins while increasing markers of mitochondrial dynamics and mitochondrial translation proteins. Glycolytic remodelling was evident in both groups, with increased levels of the glucose transporter 4 and phosphofructokinase proteins, accompanied by marked upregulation of the S100A13-Akt signalling. Unbiased muscle proteomics and plasma metabolomics also revealed common and more specific signatures. Both RSH and RS-BFR increased plasma levels of intermediates metabolites of lipid metabolism. RSH specifically upregulated cytoplasmic translation pathways in muscle and increased the kynurenate/kynurenine ratio in plasma, whereas RS-BFR was characterized by an upregulation of immune and inflammatory pathways both in muscle and plasma. Conclusions: In summary, 3-week of RS-BFR elicits performance adaptations comparable to RSH, supported by largely shared muscular and systemic metabolic adaptations. The specific characteristics of the adaptations induced by each method, however, are particularly interesting and could influence the outcome of regular training.

Introduction: Physical training stimulates mitochondrial biogenesis and protein synthesis in skeletal muscle. If the training load is too demanding and prolonged, an overtraining syndrome may develop. Reported overtraining symptoms include recurrent respiratory infections, sleep disturbances, with potential alterations at the skeletal muscle level such as soreness, and atrophy. The aim of the present study was to investigate skeletal muscle (mal)adaptations in response to overtraining, using a translational approach combining human data and an in vitro model. Methods: Electrical stimulation was applied to C2C12 myotubes to mimic endurance exercise. For the simulated training (s-T), stimulation was applied once daily for three days. In the simulated overtraining (s-OT), stimulation was applied three times daily for three days. Proteomic analysis, mitochondrial respiration and imaging were performed in the cellular model. In humans, knee extensor neuromuscular function at rest and in response to a standardized exercise was assessed in overtrained vs. well trained athletes. Muscle biopsies were also collected to assess mitochondrial respiration. Results: In s-T, we observed beneficial adaptations, including myotube hypertrophy, increased expression of myosin heavy chain proteins, and enhanced mitochondrial respiration. In contrast, s-OT showed myotube atrophy, reduced myosin heavy chain content, and impaired mitochondrial respiration. Consistent with these respiratory findings, proteomics analysis revealed a decreased abundance of proteins involved in the mitochondrial respiratory chain in s-OT. Interestingly, mitochondria appeared aggregated in s-OT myotubes, suggesting possible impairments in mitochondrial quality control. Live-cell imaging revealed possible autophagy alterations in the s-OT condition but not in s-T. Human data - neuromuscular function and muscle samples - are being processed and results will be presented during the conference. Conclusions: Our in vitro model provides a molecular platform for further investigations of skeletal muscle maladaptations to overtraining. The integrative approach will help to clarify the molecular mechanisms and ameliorate the clinics of muscle maladaptations triggered by excessive exercise in humans.

The MIME-seq2.0 technique utilizes a chimeric HENMT1 methyltransferase engineered to bind to Argonaute proteins (hereafter referred to MIME enzyme) to protect the terminal sugar of small non-coding RNAs (sncRNAs) from oxidation, a reaction that inhibits unmodified RNA ligation and cloning. In this study, we confirm that the MIME-seq technique efficiently distinguishes oxidized and non-oxidized sncRNAs. Indeed, expression of the MIME enzyme in the insulin-secreting cell line MIN6B1 protected miRNAs from oxidation and permitted their detection by RNA sequencing and quantitative PCR. Comparison of the sncRNA profile between wild type and MIME-expressing cells demonstrated protection from oxidation not only of miRNAs, but also of some Y-RNA and tRNA-derived fragments. Immunoprecipitation with Ago2 antibodies confirmed that the protected Y-RNA and tRNA fragments bind to Argonaute proteins. We also used this system to track miRNA transfer between cells via extracellular vesicles (EVs). miRNAs released inside EVs of MIME-expressing Jurkat T cells or C2C12 myotubes and delivered to MIN6B1 cells were protected from oxidation, enabling sensitive and specific detection of the transferred miRNAs in the receiving cells. Overall, the MIME-seq technique provides a powerful tool for analyzing small RNA binding to Argonaute proteins in living cells and offers new possibilities for tracking RNA transfer across different cell types.

In the hypothalamus, detection of energy substrates such as glucose is essential to regulate food intake and peripheral energy homeostasis. Metabolic interactions between astrocytes and neurons via lactate exchange have been proposed as a hypothalamic glucose‐sensing mechanism, but the molecular basis remains uncertain. Mouse hypothalamic astrocytes in vitro were found to exhibit a stronger glycolytic phenotype in basal conditions than cortical astrocytes. It was associated with higher protein expression levels of the Pyruvate Kinase Isoform M2 (Pkm2) and its more prominent nuclear localization. In parallel, hypothalamic astrocytes also expressed higher levels of the monocarboxylate transporter Slc16a3 (Mct4), which were dependent on Pkm2 expression. The stronger Mct4 expression in hypothalamic versus cortical astrocytes is an intrinsic characteristic, as it was also present after their direct isolation from adult mouse tissue. The high lactate release capacity of hypothalamic astrocytes was demonstrated to depend on the expression of Mct4, but not Mct1. Unlike cortical astrocytes, hypothalamic astrocytes in culture do not respond to glutamate with enhanced glycolysis, but instead, they modulate their lactate production according to glucose concentrations in an AMPK‐dependent manner, an effect observed in both mouse and human hypothalamic astrocytes in vitro. Our study shows that hypothalamic and cortical astrocytes are geared to have distinct glycolytic responses to glucose and glutamate, respectively. These results reveal a metabolic specialization of astrocytes in order to fulfill distinct area‐specific functions: glucose‐sensing in the hypothalamus versus activity‐dependent neuronal energetic supply in cortical regions.

The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy.

Summary: The shift in the energetic demands of proliferating cells during tumorigenesis requires intense crosstalk between the cell cycle and metabolism. Beyond their role in cell proliferation, cell cycle regulators also modulate intracellular metabolism in normal tissues. However, in the context of cancer, where CDK4 is upregulated or stabilized, the metabolic role of CDK4 is barely understood. Using both genetic and pharmacological approaches, we aimed to determine the metabolic role of CDK4 in TNBC cells. Unexpectedly, deletion of CDK4 only slightly reduced triple-negative breast cancer (TNBC) cell proliferation and allowed tumor formation in vivo . Furthermore, proapoptotic stimuli failed to induce appropriate cell death in TNBC cells with CDK4 depletion or long-term CDK4/6 inhibitor treatment. Mechanistically, CDK4 enhances mitochondria-ER contact (MERC) formation, thus promoting mitochondrial fission and ER-mitochondrial calcium signaling. Phosphoproteomic analysis also revealed a role for CDK4 in regulating PKA activity at MERCs to sustain ER-mitochondrial calcium signaling. This proper CDK4-mediated mitochondrial calcium signaling is then required for metabolic flexibility of TNBC cells. Taken together, these results demonstrate that CDK4 inhibition leads to cell death resistance, inhibiting mitochondrial apoptosis and functions through attenuated MERCs formation and ER-mitochondrial calcium signaling in TNBC. Overall, this study provides new insights into the mechanisms of TNBC resistance to CDK4/6i therapy and paves the way to explore potential synergistic therapeutic targeting MERCs-associated metabolic shifts. Graphical

Athletes increasingly engage in repeated sprint training consisting in repeated short all‐out efforts interspersed by short recoveries. When performed in hypoxia (RSH), it may lead to greater training effects than in normoxia (RSN); however, the underlying molecular mechanisms remain unclear. This study aimed at elucidating the effects of RSH on skeletal muscle metabolic adaptations as compared to RSN. Sixteen healthy young men performed nine repeated sprint training sessions in either normoxia (F _I O ₂ = 0.209, RSN, n = 7) or normobaric hypoxia (F _I O ₂ = 0.136, RSH, n = 9). Before and after the training period, exercise performance was assessed by using repeated sprint ability (RSA) and Wingate tests. Vastus lateralis muscle biopsies were performed to investigate muscle metabolic adaptations using proteomics combined with western blot analysis. Similar improvements were observed in RSA and Wingate tests in both RSN and RSH groups. At the muscle level, RSN and RSH reduced oxidative phosphorylation protein content but triggered an increase in mitochondrial biogenesis proteins. Proteomics showed an increase in several S100A family proteins in the RSH group, among which S100A13 most strongly. We confirmed a significant increase in S100A13 protein by western blot in RSH, which was associated with increased Akt phosphorylation and its downstream targets regulating protein synthesis. Altogether our data indicate that RSH may activate an S100A/Akt pathway to trigger specific adaptations as compared to RSN.

Introduction This study aimed at examining the cardiorespiratory and muscular adaptations following 12 weeks of detraining and retraining in a 53-year-old endurance master athlete. Methods Data were collected before and after detraining and after retraining. Maximal oxygen uptake (VO_2max) was evaluated during maximal cycling exercise. Proteins involved in muscle contraction, mitochondrial function and glycolysis were investigated using western blot analysis. Results VO_2max 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, suggesting transformation towards a more glycolytic phenotype. 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 defense proteins as well as the glucose transporter 4 (Glut -4, +36%) and hexokinase (+100%) proteins levels compared to baseline. The mitochondrial dynamic proteins were further increased with the retraining. Discussion/Conclusion 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.

Background: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well‐described feature of recessive RYR1‐related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders.Methods: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle‐specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels.Results: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb‐girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (−75%, P < 0.001) and mitochondrial myopathy (−71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER–mitochondria contact length (−26%, P < 0.001), Ca²⁺ transfer to mitochondria (−48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone‐binding protein/glucose regulated protein 78, GRP78‐Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro‐apoptotic protein CCAAT‐enhancer‐binding protein homologous protein, CHOP‐DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population.Conclusions: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion‐induced ER stress in the pathogenesis of myopathies.

The muscle molecular adaptations to different exercise intensities in combination with hypoxia are not well understood. This study investigated the effect of low- and supramaximal-intensity hypoxic training on muscle metabolic gene expression in mice. C57BL/6 mice were divided into two groups: sedentary and training. Training consisted of 4 weeks at low or supramaximal intensity, either in normoxia or hypoxia (FiO₂ = 0.13). The expression levels of genes involved in the hypoxia signaling pathway (Hif1a and Vegfa), the metabolism of glucose (Gys1, Glut4, Hk2, Pfk, and Pkm1), lactate (Ldha, Mct1, Mct4, Pdh, and Pdk4) and lipid (Cd36, Fabp3, Ucp2, Hsl, and Mcad), and mitochondrial energy metabolism and biogenesis (mtNd1, mtNd6, CytC, CytB, Pgc1a, Pgc1β, Nrf1, Tfam, and Cs) were determined in the gastrocnemius muscle. No physical performance improvement was observed between groups. In normoxia, supramaximal intensity training caused upregulation of major genes involved in the transport of glucose and lactate, fatty acid oxidation, and mitochondrial biogenesis, while low intensity training had a minor effect. The exposure to hypoxia changed the expression of some genes in the sedentary mice but had a moderate effect in trained mice compared to respective normoxic mice. In hypoxic groups, low-intensity training increased the mRNA levels of Mcad and Cs, while supramaximal intensity training decreased the mRNA levels of Mct1 and Mct4. The results indicate that hypoxic training, regardless of exercise intensity, has a moderate effect on muscle metabolic gene expression in healthy mice.