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. 2022 Oct 15;14(10):7063-7079.
eCollection 2022.

Fndc5/irisin is regulated by myogenesis stage, irisin, muscle type and training

Gal Lavi  1 , Avital Horwitz  1 , Ofira Einstein  2 , Reut Zipori  1 , Ofri Gross  1 , Ruth Birk  1
Affiliations

Fndc5/irisin is regulated by myogenesis stage, irisin, muscle type and training

Gal Lavi et al. Am J Transl Res. .

Abstract

Objectives: Irisin, a novel myokine that responds to exercise, was initially identified as a regulator of fat tissue metabolism. We aimed to investigate fibronectin type III domain-containing protein 5 (Fndc5)/irisin, auto/para-crine role in different muscle fibers, different activities, and muscle cell differentiation.

Methods: Using in-vitro, ex-vivo, and in-vivo muscle models, Fndc5 was studied at the physiological and molecular levels.

Results: Following training, C57BL/6 mice (n=10) were subject to fast and slow-twitch muscles dissection and molecular analysis. Isolated mice (C57BL/6, n=14) slow and fast-twitch muscles were subject to electrical aerobic and anaerobic pulses stimulation (EPS). L6 muscle cells differentiation was characterized by Fndc5 differentiation-depended expression pattern parallel with significant hypertrophy, Myogenin elevation, and overlapping Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (Pgc-1α) expression pattern. Exogenous irisin significantly altered Fndc5 expression; augmented at early differentiation (3-4-fold, P<0.05) and decreased (2-fold, P<0.05) at late differentiation. Training induced a significant elevation in Fndc5/irisin and Pgc-1α expression levels in all muscle types compared to the sedentary state, where soleus muscle (slow) Fndc5 expression levels were significantly higher compared to levels in all other fast muscles (3-140-fold, P<0.001). Similarly, following EPS, Fndc5 expression levels were significantly augmented in the soleus slow muscle following both aerobic and anaerobic activity (3-3.5-fold, P<0.05) compared to extensor digitorum longus (fast) muscle.

Conclusions: Muscle cell's Fndc5 expression has a differentiation-depended pattern paralleling Pgc-1α expression and hypertrophy. Irisin autocrinally and significantly regulate Fndc5 and Pgc-1α in a differentiation-depended manner. Muscle Fndc5 expression levels are dependent on fiber type and activity type.

Keywords: FNDC5; Irisin; Pgc-1α; muscle; myogenesis.

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Conflict of interest statement

None.

Figures

Scheme 1
Scheme 1
In vivo training protocol.
Figure 1
Figure 1
Phenotype and molecular characterization of muscle L6 cells during differentiation. L6 muscle cells were grown and differentiated for 10 days using standard methods (materials and methods). The L6 cells were photographed under a light microscope (Olympus microscope IX81) at a magnification of 20X throughout differentiation. The analyses were performed using ImageJ software (1.46 version) software. Results were expressed as mean ± SE of 3 independent experiments (n=3). A. Visual differences in muscle fiber phenotype during the L6 differentiation. B. Mean muscle fiber thickness during L6 differentiation. C. Maximum muscle fiber thickness during L6 cell differentiation. L6 cells were differentiated and subject to mRNA extraction and protein isolation at different days of differentiation as indicated (materials and methods). Following cDNA isolation, Myogenin, Fndc5 and Pgc-1α transcript levels were analyzed by quantitative real time PCR (qRT-PCR) and normalized to transcript of housekeeping gene (S18). Proteins were subject to western analysis. D. Myogenin expression during L6 cells differentiation. E. Fndc5 expression levels during L6 cells differentiation. F. Pgc-1α expression levels during L6 cells differentiation. Results are expressed as mean ± SE of 5 independent experiments (n=3). Asterisks represent statistically significant difference *P<0.05, **P<0.01.
Figure 2
Figure 2
Exogenous irisin treatment during L6 muscle cells differentiation - phenotype and Fndc5 and Pgc-1α expression. L6 muscle cells were grown and differentiated for 10 days. Treatment group received exogenous irisin (60 ng/ml) at the beginning of differentiation (day 0) and at the end of differentiation (day 7). Cells were photographed under a light microscope (Olympus microscope IX81) at a magnification of 20X. The analysis was performed in ImageJ software (1.46 version) software. A. Visualization of muscle fiber thickness during differentiation. B. Mean muscle fiber thickness during L6 cell differentiation. Cells were subject to complete mRNA extraction during differentiation days as indicated. Following cDNA isolation, Fndc5 and Pgc-1α transcript levels were analyzed by qRT-PCR and normalized to transcript of housekeeping gene (S18). C. Fndc5 expression following exogenous irisin treatment in treated and un-treated control groups. D. Pgc-1α expression following treatment with exogenous irisin in treated and un-treated control groups. Results are expressed as mean ± SE of 2 independent experiments (n=3). * Asterisks represent statistically significant difference (P<0.05); number signs (#) represent statistically significant differences (P<0.05) from day 0.
Figure 3
Figure 3
Fndc5 expression in different mice muscles following aerobic and anaerobic states. SOL and EDL muscles were surgically isolated from C57BL/6 male mice. The muscles were subject to different stimulations, generating aerobic and anaerobic states using stimulation electrical pulses system. After the different physical training (ex-vivo) the muscles were subject to total RNA extraction, following by reverse transcription to cDNA and qRT-PCR analysis. The results are expressed as mean ± SE of 3 independent experiments (n=14). A. Fndc5 expression levels in EDL muscle - aerobic activity (red circle full), anaerobic activity (red circle hollow), SOL muscle - aerobic activity (light blue circle full), anaerobic activity (light blue circle hollow). B. Levels of Fndc5 expression in the 2 different muscles after exercise, regardless of the type of activity (aerobic or anaerobic). Asterisks represent a statistically significant difference *P<0.05, ***P<0.001.
Figure 4
Figure 4
Expression levels of Fndc5 in five different muscles in trained and untrained mice. Five different muscles were surgically isolated from C57BL/6 trained and untrained male mice. The muscles were subject to total RNA extraction followed by reverse transcription to cDNA and qRT-PCR analysis. The muscle tissues were subject to protein isolation. Protein analysis was done by western blot using standard protocols (material and methods section). A. Tolerance assay of trained and untrained mice. B. Fndc5 mRNA expression levels in 5 different muscles of trained versus untrained mice. C. Fndc5 protein expression levels in 5 different muscles of trained versus untrained mice. The results are expressed as mean ± SE of 2 independent experiments, total mice (n=10), total muscles (n=100). Asterisks represent statistically significant difference between trained and untrained mice *P<0.05, ***P<0.001; number signs (#) represent statistically significant differences P<0.05 between muscles.
Figure 5
Figure 5
Expression levels of Pgc-1α in five different muscles in trained and untrained mice. Five different muscles were surgically isolated from C57BL/6 trained and untrained male mice. The muscles were subject to total RNA extraction followed by reverse transcription to cDNA and qRT-PCR analysis. The muscle tissues were subject to protein isolation. Protein analysis was done by western blot using standard protocols (material and methods section). A. Pgc-1α mRNA expression levels in 5 different muscles of trained versus untrained mice. B. Pgc-1α protein expression levels in 5 different muscles of trained versus untrained mice. The results are expressed as mean ± SE of 2 independent experiments, total mice (n=10), total muscles (n=100). Asterisks represent statistically significant difference between trained and untrained mice *P<0.05, ***P<0.001; number signs (#) represent statistically significant differences P<0.05 from QUADRICEPS muscle.
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