Molecular and phenotypic responses to mechanical loading in tissue-engineered skeletal muscle

The overriding aim of this thesis was to optimise, scale and utilise an in vitro model of skeletal muscle in order to establish a hypertrophic loading regime of engineered muscle, thereafter, characterising the molecular mechanisms following mechanical load and application of this regime. Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size (hypertrophy). However, skeletal muscle loss (atrophy) as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and sarcopenia, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. Studies in humans are limited by the need for repeated muscle biopsy sampling, and studies in animals have numerous methodological and ethical limitations. In this investigation, skeletal muscle was tissue engineered utilising the murine cell line C2C12, which bears both structural and functional resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The preliminary investigation (Chapter 3) aimed to determine if mechanical loading would induce an anabolic hypertrophic response, akin to that described in vivo following mechanical loading in the form of resistance exercise. This data reported that mechanical loading (construct length increase of 15%) significantly increased Insulin-like growth factor-1 (IGF-1) and Matrix metalloproteinase-2 (MMP-2) mRNA expression 21 hours post overload, and levels of the atrophic gene muscle atrophy factor BOX (MAFbx) was significantly downregulated 45 hours post mechanical overload. In addition, downstream mammalian target of rapamycin (mTORC1) targets, ribosomal protein S6 kinase beta-1 (p70S6 kinase) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP-1) phosphorylation, were upregulated immediately following mechanical overload. Furthermore, maximal contractile force was augmented 45 hours post load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Thus, overall, mechanical loading of tissue engineered skeletal muscle induced hypertrophy and improved force production. Chapter 4 aimed to determine the role of IGF-1 in skeletal muscle hypertrophy utilising a further developed model of mechanical load, allowing a higher throughput and more biomimetic model of skeletal muscle and mechanical stimulation. Data revealed that the IGF-1R inhibitor NVPAEW541 stunted IGF-1 mRNA expression and mTORC1 activation, alongside both myotube hypertrophy and increases in maximal force production temporally following mechanical load. This supports the notion that skeletal muscle hypertrophy in response to mechanical loading is primarily driven through the linear IGF-1-Akt-mTORC1 pathway. Furthermore, this study also demonstrates the application of this model system to study distinct molecular pathways underpinning skeletal muscle atrophy and hypertrophy. Moreover, the model offers potential avenues to peruse for future treatments for skeletal muscle atrophy, within an easily accessible and highly controlled system. Chapter 5 aimed to determine if mechanical load would offset dexamethasone (DEX) induced skeletal muscle atrophy in muscle engineered using the C2C12 murine cell line. Data revealed that mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX, regardless of whether the loading occurred before or after 24 hours of DEX treatment. This is likely to be underpinned by the prevention of upregulation of muscle RING-finger protein-1 (MuRF-1) and MAFbx mRNA expression, critical regulators of muscle atrophy. Therefore, I demonstrated the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy. The data presented in this thesis demonstrates the use of a novel mechanical stimulation bioreactor. Firstly, to investigate the beneficial therapeutic effects of mechanical load on atrophied engineered muscle, but also to further investigate the acute complex cellular and molecular responses. Specifically, this model has shed further light on the mechanoresponsive genes and proteins underpinning skeletal muscle hypertrophy following mechanical stimulation, offering potential therapies for skeletal muscle wasting. Future studies will investigate the use of this model to either feed forward or feedback on human exercise science experimentation to better inform what we already know around the area of exercise as a treatment for muscle wasting.