Cyclic stretch reduces myofibrillar protein synthesis despite increases in FAK and anabolic signalling in L6 cells

Abstract

Muscle protein synthesis is increased after exercise, but evidence is now accruing that during muscular activity it is suppressed. In life, muscles are subjected to shortening forces due to contraction, but may also be subject to stretching forces during lengthening. It would be biologically inefficient if contraction and stretch have different effects on muscle protein turnover, but little is known about the metabolic effects of stretch. To investigate this, we assessed myofibrillar and sarcoplasmic protein synthesis (MPS, SPS, respectively) by incorporation of [1-13 C]proline (using gas chromatography-mass spectrometry) and anabolic signalling (by phospho-immunoblotting and kinase assays) in cultured L6 skeletal muscle cells during 30 min of cyclic stretch and over 30 min intervals for up to 120 min afterwards. SPS was unaffected, whereas MPS was suppressed by 40 ± 0.03% during stretch, before returning to basal rates by 90-20 min afterwards. Paradoxically, stretch stimulated anabolic signalling with peak values after 2-30 min: e.g. focal adhesion kinase (FAK Tyr576/577; +28 ± 6%), protein kinase B activity (Akt; +113 ± 31%), p70S6K1 (ribosomal S6 kinase Thr389; 25 ± 5%), 4E binding protein 1 (4EBP1 Thr37/46; 14 ± 3%), eukaryotic elongation factor 2 (eEF2 Thr56; −47 ± 4%), extracellular regulated protein kinase 1/2 (ERK1/2 Tyr202/204; +65% ± 9%), eukaryotic initiation factor 2α (eIF2α Ser51; −20 ± 5%, P < 0.05) and eukaryotic initiation factor 4E (eIF4E Ser209; +33 ± 10%, P < 0.05). After stretch, except for Akt activity, stimulatory phosphorylations were sustained: e.g. FAK (+26 ± 11%) for ≥30 min, eEF2 for ≥60 min (peak −45 ± 4%), 4EBP1 for ≥90 min (+33 ± 5%), and p70S6K1 remained elevated throughout (peak +64 ± 7%). Adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was unchanged throughout. We report for the first time that acute cyclic stretch specifically suppresses MPS, despite increases in activity/phosphorylation of elements thought to increase anabolism.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain

“…Thus, scientists still seek feasible strategies to obtain mature, long, functional muscle fibers in volumetric constructs, with proper vascularization and innervation of the tissue. Proposed approaches to engineer muscle (-like) tissue range from scaffold anchoring [10,40,58] or micropatterning of biomaterials for guided myotube formation [39,60,61] to electrical [36][37][38][39]62] or mechanical stimulation in bioreactors in 2D [29,31,[63][64][65] and 3D settings [6,9,11,14,27,[66][67][68]. However, while there is a clear consensus in the field concerning the necessity for mechanical stimulation in 3D skeletal muscle tissue engineering, not all available systems offer programmable and individually adjustable control over strain parameters.…”

“…With the notion that mechanotransduction plays a central role in myogenic differentiation [26], mechanical stimulation has been demonstrated to induce cellular alignment along the axis of strain [6,9] and to stimulate muscle growth in vitro and in vivo [27,28]. While static mechanical stimulation is largely considered a trait related to induction of myogenic differentiation, inconsistent findings have been reported regarding the role of cyclic mechanical stimulation on signaling and myogenic marker expression, respectively [29][30][31][32].…”

Body weight-dependent troponin T alternative splicing is evolutionarily conserved from insects to mammals and is partially impaired in skeletal muscle of obese rats

“…Atherton et al, 2009;Hornberger et al, 2005;Spangenburg, 2009). It is becoming apparent that many alternative splicing events require the activity of the same signaling pathways (Lynch, 2007).…”

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

“…The influence of osmotic stress on activity-related phosphorylation of ERK1/2 and p38 MAPKs in chondrocytes was investigated at the protein level, as previously described (1,39). Specific antibodies for the phosphorylated form of ERK1/2 and p38 MAPKs were used in Western blot experiments to investigate the phosphorylation of ERK1/2 and p38 MAPKs following exposure of EACs to osmotic stress for 90 min and 3 h. ERK phosphorylation was significantly (Ͼ2-fold) increased in response to hypo-osmotic stress at 90 min (P Ͻ 0.001) and was reduced at 3 h (Fig.…”