ECSS Paris 2023: CP-PN29
INTRODUCTION: Black chokeberry extract (BCE) is rich in polyphenols such as anthocyanins, which are known for a variety of bioactive properties. However, potential anabolic effects of BCE on skeletal muscle cells and the underlying molecular mechanisms have not yet been systematically investigated. Therefore, the aim of the present study was to examine the anabolic effects of BCE and its bioactive fractions in an in vitro model using differentiated C2C12 myotubes. METHODS: BCE as well as its anthocyanin (ANTH) and copigment (COPIG) fractions were prepared at the University of Hannover. Dose-dependent effects on myotube hypertrophy and cell viability were investigated in differentiated C2C12 myotubes. Cell viability was assessed using an MTT assay. Myotube diameter was quantified by microscopic image analysis. The mRNA expression levels of insulin-like growth factor 1 (IGF-1) and mechanistic target of rapamycin (mTOR) were determined by quantitative RT-qPCR, while protein expression of myosin heavy chain was analyzed by Western blotting. Binding affinities to androgen and estrogen receptors were analyzed using a yeast-based transactivation assay. RESULTS: Treatment with BCE did not affect C2C12 cell viability across the investigated concentration range. BCE induced a significant dose-dependent increase in myotube diameter at concentrations between 10 ng/mL and 1 µg/mL. The mRNA expression of IGF-1 and mTOR was significantly stimulated by BCE treatment in C2C12 myotubes. In parallel, BCE treatment resulted in a significant increase of myosin heavy chain protein expression. Co-treatment with dexamethasone was associated with reduced catabolic responses in BCE-treated myotubes. Only at very high concentrations were antiandrogenic (100 µg/mL) and antiestrogenic (10 µg/mL) activities observed. Fractionation experiments indicated that anabolic activity was mediated by the ANTH fraction, whereas the COPIG fraction was associated with catabolic changes in C2C12 myotube diameter. CONCLUSION: BCE induced significant anabolic and anticatabolic effects in differentiated C2C12 myotubes. These effects were associated with enhanced myosin heavy chain protein synthesis and activation of molecular signalling pathways, particularly the IGF-1/mTOR signalling. The results further indicate that the ANTH fraction represents the primary bioactive component responsible for the observed anabolic effects. Future studies should investigate the anabolic and anticatabolic potential of BCE and its ANTH fraction in additional in vitro models and human intervention studies.
Read CV Tihomir KostovECSS Paris 2023: CP-PN29
INTRODUCTION: Sarcopenia is an age-related decline of skeletal muscle mass and function that contributes to frailty, loss of independence, metabolic dysfunction, and increased mortality. A key feature is anabolic resistance, defined as blunted stimulation of muscle protein synthesis (MPS) and reduced hypertrophic adaptation to anabolic stimuli such as exercise and nutrient intake. Although the extent varies across conditions, anabolic resistance is frequently observed in aged muscle. However, the molecular basis of these impaired anabolic adaptations remains poorly defined. METHODS: Male young (10-week) and aged (24-month) C57BL/6J mice completed 12 weeks of resistance training (RT), with age-matched sedentary controls. To quantify anabolic resistance to RT, cumulative muscle proteome-wide synthesis rates of mixed, myofibrillar, and mitochondrial proteins were measured during the final 2 weeks using deuterium oxide labeling and tandem mass spectrometry. Whole-body functional muscle mass was assessed using the D3-creatine dilution method. To identify candidate genes associated with anabolic resistance, RNA sequencing was performed followed by protein-protein interaction network analysis. To test the role of the candidate gene caveolin-1 (Cav-1), implicated in glucose and fatty acid uptake and metabolism, C2C12 myotubes were transfected with shCav-1 plasmids and stimulated to contract using electrical pulse stimulation (EPS) to induce hypertrophy, while carbon energy flux were quantified using 13C-metabolic flux analysis. RESULTS: RT significantly increased harvested muscle mass (+12%), strength (+98%), MPS of mixed (+33%), myofibrillar (+46%), and mitochondrial proteins (+15%) in young mice, but these increases were not observed in aged mice. RNA-sequencing showed that broad RT-induced gene expression changes in young muscle, whereas responses in aged muscle were minimal. Protein-protein interaction analysis identified Cav-1 among candidate hub genes linked to anabolic resistance. Proteome kinetic analysis revealed that synthesis rates of 146 of 159 quantified proteins were lower in aged than young muscle, with the largest reductions in proteins involved in muscle contraction, glycolysis, and oxidative phosphorylation. Cav-1 knockdown abolished EPS-induced hypertrophy and stimulation of MPS, accompanied by reduced glucose uptake (-33.3%) and lactate production (-69.1%), with lower glycolytic flux and ATP production (approximately -51.8% and -41.3%), as determined by 13C-metabolic flux analysis. CONCLUSION: Aged mice exhibit anabolic resistance to RT, characterized by failure to muscle hypertrophy and ATP-dependent MPS responses. These data indicate that the absence of hypertrophy and an ATP-dependent MPS response to RT is partly due to reduced glucose uptake, glycolytic carbon flux, and associated ATP production. Cav-1 may therefore serve as a mechanistic link between energy metabolic flux and anabolic remodeling in skeletal muscle.
Read CV Yeongmin KimECSS Paris 2023: CP-PN29
INTRODUCTION: Mechanical loading potently stimulates skeletal muscle adaptation, however excessive mechanical strain can lead to muscle damage, activating inflammatory, regenerative and remodeling processes. Even though mechanical loading has been widely studied in the context of skeletal muscle adaptation, data directly quantifying mechanically induced cellular damage in differentiated myotubes remain sparse. The purpose of this study was to investigate the effects of a prolonged mechanical stretching protocol on the plasma membrane integrity of differentiated C2C12 myotubes, as an indication of mechanical stress-induced cell injury, necrosis or apoptosis, and hence to establish an in vitro model of mechanically induced muscle cell damage. METHODS: C2C12 myotubes (at day 6 of differentiation) underwent a passive cyclic stretching protocol using the Flexcell FX-5000 system. The protocol consisted of sustained 6% elongation with additional cyclic stretching up to 12% elongation at a frequency of 2 Hz for 3 hours. Non-stretched myotubes were used as controls. Potential cell membrane damage was assessed via LDH release into the culture medium at baseline, 1 hour, 24 hours and 72 hours post mechanical loading. RESULTS: Mechanically loaded myotubes presented increased LDH release at 1-hour post-loading (71.42±9.68%; p<0.05), which further increased at 24 (146.40±2.01%; p<0.001) and 72 hours (171.31±16.66%; p=0.01) compared to baseline (12.70±1.09%). Control (non-stretched) cells exhibited lower levels of LDH release at 24 (104.07±0.08; p<0.001) and 72 hours (128.96±11.69; p=0.01) compared to baseline, possibly due to its spontaneous release during the myotubes’ differentiation. These LDH release levels remained significantly lower in comparison to the loaded cells (24 hours: 104.07±0.08 vs 146.40±2.01; p<0.01; 72 hours: 128.96±11.69 vs 171.31±16.66; p=0.08). CONCLUSION: Sustained mechanical loading induces increased and time-dependent cytotoxicity in differentiated C2C12 myotubes, confirming the effectiveness of the protocol utilized in eliciting mechanically induced muscle cell damage. This in vitro model might be utilized as a useful source for future investigation of various cellular and molecular responses following mechanical loading-associated damage of muscle cells.
Read CV EIRINI CHATZINIKITAECSS Paris 2023: CP-PN29