Two functionally and structurally different mTOR complexes, mTORC1 and mTORC2, exist.⁷ Although each structure has unique components, mTOR is the fundamental functional component of both. The DEP domain-containing mTOR-interacting protein (DEPTOR), the mammalian SEC13 protein 8 (mLST8), and the associated protein of mTOR (raptor) make up mTORC1. The rapamycin-insensitive companion of mTOR and the mammalian stress-activated map kinase-interacting protein 1 are two of the components of the second mTOR complex, mTORC2, which shares mLST8 and DEPTOR with mTORC1 (Figure 1).

By considering its genetic and therapeutical evidence and restricting the present evaluation of the molecular mechanism of mTOR in the upgradation of skeletal muscle mass, this work combines the critical role of mTOR complexes and their signaling in the functioning of skeletal muscle (Figures 2 and 3).

The complex mTORC1 processes multiple signals, such as amino acid availability, energy status, growth factors, and oxygen levels. mTORC1 stimulates protein synthesis by controlling S6 kinase 1 (S6K1) and inhibiting 4EBP1.⁸ In contrast, mTORC2 phosphorylates AGC kinases, glucocorticoid-regulated kinase 1, AKT, and protein kinase C control cell stability and metabolism.⁹

Skeletal muscle myoblasts C2C12 were procured from an approved cell culture bank. The cells were kept in Dulbecco’s Modified Eagle Medium (DMEM), which was enhanced with 90 U/ml of penicillin, 90 μg/ml of streptomycin, and 10% fetal bovine serum (FBS). To provide ideal conditions for cell development, the culture media was changed every 48 hours. To replicate physiological circumstances, cells were incubated at 37°C in a humidified environment with 5% CO₂. The cells underwent two rounds of washing in phosphate-buffered saline (pH 7.4) before being exposed to 0.25% trypsin-EDTA for three minutes at 37°C until they separated. After DMEM supplemented with 10% FBS was added to halt trypsinization, cells were centrifuged at 300 × g for 5 minutes before being resuspended in new culture media.

RNA Extraction and cDNA Synthesis

As directed by the manufacturer, total RNA was isolated from cells using the TRIzol Reagent (Invitrogen, USA). A Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration and integrity of the RNA. DNase I (Promega, USA) was used to treat RNA samples in order to remove genomic DNA contamination. The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) was used to create first-strand cDNA from 1 μg of total RNA using the following protocol: 25°C for 10 minutes, 42°C for 60 minutes, and 70°C for 10 minutes.

Cells were divided into four experimental groups:

Group A: Untreated control cells (no transfection, no stress)

Group B: Cells with mTOR knockdown (CRISPR/Cas9 transfection, no stress)

Group C: Normal cells subjected to mechanical stress

Group D: mTOR knockdown cells under mechanical stress

The Flexcell Tension System FX-5000 (Flexcell International, USA) was used to apply mechanical tension to cells. In order to replicate physiological mechanical loading conditions, cells were seeded on collagen-coated BioFlex plates and exposed to cyclic stretch (10% strain, 1 Hz frequency) for 6 hours every day for 3 days.

Cell Counting Kit-8 (CCK-8) test (Dojindo, Japan) was used to measure cell proliferation. After seeding 5000 cells per well in 96-well plates, the cells were treated for two hours at 37°C with 10 μl of CCK-8 reagent in each well. Utilizing a microplate reader (BioTek, USA), absorbance was determined at 450 nm.

Western blot analysis was used to examine the protein expression of mTOR, 4EBP1, p70S6K, and their phosphorylated variants (p-4EBP1, p-p70S6K). In short: RIPA lysis solution (Thermo Fisher, USA) containing protease and phosphatase inhibitors was used to extract the proteins. After being separated on a 10% SDS-PAGE gel, 30 μg of total protein was put onto PVDF membranes (Millipore, USA). After blocking the membranes with 5% non-fat milk in TBST, primary antibodies (1:1000 dilution) were incubated at 4°C for the whole night. Membranes were cleaned and then incubated for one hour at room temperature with HRP-conjugated secondary antibodies (1:5000 dilution). ECL substrate (Thermo Fisher, USA) was used to create the blots, and a ChemiDoc XRS+ Imaging System (Bio-Rad, USA) was used to visualize them.

GraphPad Prism 9 (GraphPad Software, USA) was used to analyze the data. The mean ± standard deviation is used to display the results. Tukey’s post hoc test was used after a one-way ANOVA for statistical comparisons. P-values less than 0.05 were regarded as statistically significant.

Mechanical stress influenced the proliferation of C2C12 cells, with noticeable differences observed under the microscope. The mTOR knockdown groups exhibited a reduced cell count compared to the control groups. However, when subjected to mechanical stress, this trend was reversed, and the cell numbers in the mTOR knockdown levels increased. Additionally, the mechanical stress caused the cell units to align in the direction of the applied force.⁹

In murine C2C12 myoblasts, we initially observed elevated mTOR expression levels in cells exposed to mechanical stress compared to unstressed controls (Figure 5). Upon mTOR knockdown, cell numbers declined as anticipated. Interestingly, when these knockdown cells were subjected to mechanical stress, their proliferation increased significantly. Microscopic examination revealed that mechanical stress also influenced cell orientation, causing them to align along the force vector (Figure 5). These results proposed that mechanical stress may perform a compensatory character in modulating cellular responses following mTOR knockdown.

Microscopic analysis revealed that mechanical-stress-enhanced cell proliferation in mTOR knockdown groups compared to unstressed conditions. To explore this further, we utilized CRISPR/Cas9 to effectively knock down mTOR in myoblasts. Since p70S6K and 4EBP1 are significant downstream regulators in the PI3K/mTOR pathway, we examined their expression to assess pathway involvement under mechanical stress. RT-PCR analysis revealed that mTOR knockdown significantly reduced 4EBP1 mRNA levels compared to controls. However, when mechanical stress was applied, there was an upregulation of 4EBP1 expression in the mTOR knockdown groups, indicating that mechanical stress can partially restore pathway activity (Figures 4 and 6).

Various studies reveal that IGF1 is a crucial factor for skeletal muscle development and resurgence,^10–15 as well as a well-known upstream activator of mTOR in skeletal muscle. IGF1 gets links to the IGF1 receptor (IGFR) and subsequently activates insulin receptor substrate-1 (IRS-1). The role of the IRS-1 pathway in skeletal muscle is ambiguous.¹⁶ Rather, the Akt/mTOR signaling pathway by IGF1 proves to be crucial in activating muscle hypertrophy.¹ Akt phosphorylates TSC1/2, which blocks the GTPase-activating protein (GAP) activity of TSC1/2 toward small G protein Rheb. Then, GTP-bound Rheb activates mTORC1, resulting in phosphorylation of S6K1and 4EBP1, which stimulate the production of proteins by releasing the translation initiation factor eIF-4E and activating the ribosomal protein S6, respectively. In tissue culture, IGF1 causes skeletal myofiber hypertrophy in accordance with IGF1-Akt-mTORC1 regulation.¹¹ Transgenic mice with muscle-specific IGF1 expression exhibit at least a twofold increase in muscular growth,^12,13 indicating that muscle hypertrophy depends on the IGF1/Akt/mTORC1 pathway. Furthermore, GSK β1 is phosphorylated and deactivated by Akt, which then inhibits the eukaryotic translation initiation factor 2B (eIF2B) in a GSK β1-dependent manner to control muscle mass (Figure 7).^17,18

Despite this fact, a current study revealed that IGF1 and its receptors were not crucial to the activation of mTOR in mechanical signaling.¹⁹ The utterance of dominance negative (DN)-IGFR-I, mainly in skeletal muscles, produced myocyte hypertrophy with the help of an active overload model produced by interactive excision.¹⁹ Interestingly, DN-IGFR-I showing muscle showed an equivalent amount of expression of Akt and p70S6K1. These findings indicate that an unidentified upstream mediator besides IGFR may activate Akt/mTOR signaling in skeletal muscle hypertrophy (Figure 8).

IGFR-independent mTOR’s one of the strongest activators in muscle cells is PA. IGF1-involved involuntary stress enhances PAs, as revealed by Hornberger et al., governed through mTOR regulation.⁴ PA is strictly linked to the FKBP12 unit in relation to rapamycin and regulates signaling in mTOR.²⁰ PA is produced through a number of pathways: by phosphatidylcholine by phospholipase D (PLD) and through diacylglycerol (DAG) by the use of kinases.^21,22 Through the different enzymes used in PA synthesis, PLD function was accelerated by mechanical strain and accompanied by mTOR regulation.²³ Moreover, treating with 1-butanol, a PLD inhibitor, prevented the rise in mTOR functioning and strengthened the character of PLD in mechanical stains.²³ Nevertheless, the PA number used to retain a high number after the regulated PLD activation went back to the primary level 15–16 minutes after stretch using [3H] arachidonic acid, indicating that other enzymes synthesized PA through mechanical stretch. Hornberger et al. revealed that DGK synthesized PA with the help of mechanical activation, which is governed by mTOR activation.²⁴ Mechanical indication does not induce PLD activation under the influence of [3H] myristic acid, indicating that labels PC and a PLD inhibitor did not block mechanical activation. Besides, both DAG and DGK outer layer activity, crucial for mTOR regulation and activation, has been enhanced through mechanical stimulation. However, the past data reported that PLD1-produced Pas displace DEPTOR, governing the activation of mTORC1.²² Therefore, interrogation into whether PA synthesized by DGK during muscle extension combines with the FRB or regulates mTOR with the help of an FRB-independent process is justified.

It is being reported that mTORC1 moves to the lysosome by the activation of the Rag regulator in amino acid signaling.^25,26 Lysosomal migration of mTOR does not regulate mTOR specifically; instead, it gives a close location to Rheb, a crucial activator of mTOR.²⁷ mTORC1 is regulated by straight linkage with the GTP-linked form of Rheb,^25,26 which is activated by the TSC complex TSC1, TSC2, and Tre2-Bub2-Cdc16-1 domain family member 7 (TBC1D7),²⁸ a GAP of Rheb.²⁹ The occurrence of Rheb, shown to be on the lysosomal units, was not altered by either insulin or amino acid. In spite of that, the TSC regulates the GTPase functioning of Rheb on the outer 30 layers of the lysosome and moves to the lysosome, at least half by its linkedness with Rheb-GDP when the growth components are not present. Insulin regulates Akt, which gradually activates the TSC complex by phosphorylation, forming the breakout of the TSC complex from Rheb before the mTORC1 activation.³⁰

mTOR is a significant serine/threonine protein kinase that manages homeostasis in cells and organisms by processing anabolic and catabolic processes in response to nutrient, energy, and oxygen availability and growth factor signaling.

Cells and living creatures encounter various stresses that disrupt mTOR-governed homeostatic systems. Stress can arise from imbalances in upstream signals or genetic disruptions in upstream pathway effectors, influencing mTOR’s role.

mTOR forms two large protein complexes that are crucial in buffering cells from stress and are linked to stress-related conditions such as aging, cancer, and diabetes (Figure 9).