Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

Abstract

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology.

Engineering of Human Skeletal Muscle With an Autologous Deposited Extracellular Matrix

“…Cross-linking of fibrin with FXIII significantly impeded force generation (189 ± 64 μN/mm 2 ). The forces we report here are in the same range as what we and others previously reported ( Huang et al, 2005 ; Brady et al, 2008 ; Vandenburgh et al, 2008 ; Fuoco et al, 2015 ; Madden et al, 2015 ; Martin et al, 2017 ; Kasper et al, 2018 ). Forces exerted by excised tibialis anterior muscle were around 1–2 mN ( Cook et al, 2016 ), thus the forces we obtain are only slightly lower.…”

Leucine Supplementation Improves Effort Tolerance of Rats With Hyperthyroidism

“…Pioneering studies by Shah et al (2000) demonstrated that leucine supplementation in rats is able to markedly stimulate protein synthesis in skeletal muscle, activating the mTOR intracellular signaling pathway. Martin et al (2017) observed in a cell culture model of skeletal muscle tissue that leucine improves mTOR signaling, associated with microtubule hypertrophy and increased maximal contractile force by electrical stimulation, providing evidence for the efficacy of leucine as an anabolic nutritional agent which may influence the functional capacity of muscle.…”

Cored in the act: the use of models to understand core myopathies

“…For example, electrical and mechanical stimulation can induce tissue maturation, increasing myosin heavy chain expression and myoblast differentiation in vitro (Niu et al, 2013;Player et al, 2014), as well as other tissue maturation benefits, i.e. improved metabolic flux, increased Ca 2+ transient amplitude, tetanic force and fatigue resistance (Davis et al, 2019;Khodabukus et al, 2019;Martin et al, 2017;Mills et al, 2019;Takahashi et al, 2018). These simulated 'exercise protocols' have greatly increased myotube width and force (Powell et al, 2002;Raman et al, 2016).…”