Activation of mTORC1 signalling in rat skeletal muscle is independent of the EC‐coupling sequence but dependent on tension per se in a dose‐response relationship

Abstract

Aim mTORC1 is regarded as an important key regulator of protein synthesis and hypertrophy following mechanical stimuli in skeletal muscle. However, as excitation and tension development is tightly coupled in most experimental models, very little and largely indirect evidence exist for such a mechanosensitive pathway. Here, we sought to examine whether activation of mTORC1 signalling is dependent on tension per se in rat skeletal muscle. Methods To examine the mechanosensitivity of mTORC1, rat EDL muscles were exposed to either excitation‐induced eccentric contractions (ECC), passive stretching (PAS) with identical peak tension (Tpeak) and Tension‐Time‐Integral (TTI), or ECC with addition of inhibitors of the myosin ATPases (IMA). To further explore the relationship between tension and mTORC1 signalling, rat EDL muscles were subjected to PAS of different magnitudes of Tpeak while standardizing TTI and vice versa. Results PAS and ECC with equal Tpeak and TTI produced similar responses in mTORC1 signalling despite different modes of tension development. When active tension during ECC was nearly abolished by addition of IMA, mTORC1 signalling was reduced to a level comparable to non‐stimulated controls. In addition, when muscles were exposed to PAS of varying levels of Tpeak with standardized TTI, activation of mTORC1 signalling displayed a positive relationship with peak tension. Conclusions The current study directly links tension per se to activation of mTORC1 signalling, which is independent of an active EC‐coupling sequence. Moreover, activation of mTORC1 signalling displays a positive dose‐response relationship with peak tension.

Estimation of p70S6K Thr389 and 4E‐BP1 Thr37/46 phosphorylation support dependency of tension per se in a dose‐response relationship for downstream mTORC1 signalling

“…Compared to non‐manipulated controls; ECC produced a 3.2 ± 0.4‐fold increase in phosphorylation of p70S6K Thr 389 and a 1.4 ± 0.2‐fold increase in phosphorylation of 4E‐BP1 Thr 37/46 ; PAS produced a 3.6 ± 0.7‐fold increase in p‐p70S6K Thr 389 and a 1.5 ± 0.1‐fold increase in p‐4E‐BP1 Thr 37/46 and ECC+I MA showed no significant increase in either p‐p70S6K Thr 389 or p‐4E‐BP1 Thr 37/46 (Figure A,B). These results exhibit the same response patterns as for mTOR Ser 2448 and rpS6 Ser 235/236 phosphorylation visualized in figures 2C and 3C of Rindom et al, emphasizing the importance of tension development per se for mTORC1 downstream signalling. Furthermore, the response in the PAS group demonstrates that activation of the EC‐coupling sequence inherent of excitation‐induced force generation is not required for the acute activation of mTORC1 signalling.…”

“…1:1000, cat # 2855), purchased from Cell Signaling Technology, were utilized in the current experiments. For detailed description of immunoblotting procedures and contraction experiments, see Rindom et al.…”

“…In a letter to the editor by Figueiredo et al, entitled ‘On the appropriateness of antibody selection to estimate mTORC1 activity’, the authors question our choice of mTORC1 biomarkers . We much appreciate the input and the possibility to emphasize our conclusions.…”

“…In the study (Rindom et al, 2019), our primary aim was to examine the effect of tension per se on signalling for translation initiation. To this end, we conducted a comprehensive series of experiments, in which the EC‐coupling sequence was manipulated through a combination of standardized electrical activity patterns, chemical inhibitors of the myosin ATPase, as well as variables of the mechanical stimuli through dynamic manipulation of active and passive tension regimens in isolated extensor digitorum longus (EDL) rat muscles . As outcome measures of effect, we employed immunoblotting to quantify the phosphorylation levels of mTOR Ser 2448 and rpS6 Ser 235/236 respectively.…”

On the appropriateness of antibody selection to estimate mTORC1 activity

“…Dear Chief Editor Pontus B. Persson, We read with great interest the recent study from Rindom et al, 1 which undoubtedly will be of great value to the skeletal muscle community, in particular for those studying intramuscular cell signalling and muscle cell adaptations induced by mechanical cues. Although the study was rigorous and well-designed, we were disappointed with the method used to assess mTORC1 activity, the main dependent variable of the study.…”