Research ArticleHEALTH AND MEDICINE

Exercise mimetics and JAK inhibition attenuate IFN-γ–induced wasting in engineered human skeletal muscle

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Science Advances  22 Jan 2021:
Vol. 7, no. 4, eabd9502
DOI: 10.1126/sciadv.abd9502
  • Fig. 1 Exercise-mimetic electrical stimulation (E-stim) counteracts IFN-γ–induced deficit in myobundle contractile function.

    (A) Schematic overview of myobundle culture, treatment, and characterization. Human primary myoblasts were expanded in 2D culture and mixed with hydrogel to form 3D myobundles, which were cultured in growth media (GM) for 4 days, then in differentiation media (DM) for 7 days, after which E-stim and/or IFN-γ (20 ng/ml) was applied for an additional 7 days. (B) E-stim protocol consisted of alternating 1-hour stimulation (S) at 10 Hz and 7-hour rest (R). (C to F) Representative twitch (Twi, 1 Hz) and tetanus (Tet, 20 Hz) force traces from myobundles (C) without IFN-γ or E-stim (IFN-γ, E-stim), (D) IFN-γ+, E-stim, (E) IFN-γ, E-stim+, and (F) IFN-γ+, E-stim+. (G) Twitch and tetanic force amplitudes averaged over three independent donors and shown relative to the IFN-γ, E-stim group (n = 5 to 10 myobundles per donor, 20 to 23 myobundles per group). (H and I) Time-to-peak tension (H; TPT) and half-relaxation time (I; 1/2RT) derived from contractile force recordings in myobundles (n = 47 to 75 data points from 20 to 23 myobundles from three donors per group). *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+. NS, nonsignificant. Data are presented as means ± SEM.

  • Fig. 2 E-stim prevents IFN-γ–induced structural deterioration of myobundles.

    E-Stim prevents IFN-γ–induced structural deterioration of myobundles. (A to C) Representative myobundle cross sections immunostained for (A) F-actin (green; scale bars, 500 μm), (B) sarcomeric α-actinin (SAA; red; scale bars, 500 μm), and (C) dystrophin (Dys; red; scale bars, 25 μm). (D to G) Quantification of (D) myobundle cross-sectional area (CSA), (E) number of nuclei per cross section, (F) F-actin+ area, and (G) myotube diameter [n = 19 to 27 images from three donors per group, 3 to 5 myobundles for each donor for (D) to (F), n > 900 myotubes from three donors per group for (G)]. (H) Force amplitude normalized per myobundle CSA (specific force, n = 20 to 23 myobundles from three donors per group). *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+, **P < 0.05. NS, nonsignificant. Data are presented as means ± SEM.

  • Fig. 3 E-stim prevents IFN-γ–induced decrease in myotube length, sarcomere abundance, and contractile protein expression.

    (A and B) Representative longitudinal myobundle sections immunostained for (A) F-actin (green; scale bars, 100 μm) and (B) SAA (red; scale bars, 50 μm). (C and D) Quantification of (C) projected myotube length (n > 350 myotubes from three donors per group) and (D) SAA cross-striation frequency (n ≥ 9 images from each donor, n ≥ 37 images per group). (E) Representative Western blots from a single donor showing expression of the sarcomeric proteins dystrophin, myosin heavy chain (MYH; MF20), SAA, and myosin light chain (MYL; F-5), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a loading control. (F to I) Quantification of Western blots averaged for three donors with protein abundance normalized to GAPDH and shown relative to IFN-γ group. *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+, **P < 0.05. NS, nonsignificant. Data are presented as means ± SEM.

  • Fig. 4 E-stim counteracts IFN-γ–induced deficit in myobundle calcium handling and up-regulation of STAT1 signaling.

    (A to D) Representative peak Fluo-8 AM fluorescence intensity during E-stim (1 and 20 Hz) showing amplitudes of Ca2+ transients; scale bars, 500 μm. (E) Quantified amplitudes (ΔF/F) of electrically stimulated Ca2+ transients based on Fluo-8 AM recording (n = 8 myobundles from one donor per group). (F) Representative Western blots for RYR1, CASQ1, and GAPDH (loading control). (G and H) Quantified (G) RYR1 and (H) CASQ1 protein expressions normalized to that of GAPDH and shown relative to IFN-γ group (n = 6 samples from three donors per group). (I) Representative Western blots for pSTAT1, STAT1, and GAPDH (loading control). (J to L) Quantified (J) STAT1, (K) pSTAT1, and (L) pSTAT1/STAT1 protein expressions normalized to that of GAPDH and shown relative to the IFN-γ+ group (n = 6 samples from three donors per group). *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+. NS, nonsignificant. Data are presented as means ± SEM.

  • Fig. 5 JAK inhibitors prevent IFN-γ–induced deficits in myobundle force generation and structural organization.

    (A) Schematic overview of myobundle culture, treatment, and characterization. Human primary myoblasts were expanded in 2D culture and mixed with hydrogel to form 3D myobundles, which were cultured in GM for 4 days, then in DM for 6 days, after which JAK inhibitors (JAKi) tofacitinib (Tofa, 500 nM) or baricitinib (Bari, 500 nM) was applied for additional 8 days, the last 7 of which in the presence or absence of IFN-γ. (B) Contractile force amplitude per myobundle CSA (specific force, n = 11 to 15 myobundles from three donors per group). (C) Quantified CSA of myobundles. (D) Quantified myotube diameter from myobundle cross sections (n ≥ 720 myotubes from three donors per group). (E and F) Quantified (E) projected myotube length (n > 150 myotubes from two donors per group) and (F) SAA cross-striation frequency (n ≥ 30 images from three donors per group) from longitudinal sections of myobundles. *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+. NS, nonsignificant. Data are presented as means ± SEM.

  • Fig. 6 JAK inhibitors prevent IFN-γ–induced protein down-regulation and STAT1 activation in myobundles.

    (A) Representative Western blots from a single donor showing expression of dystrophin, MYH (MF20), SAA, and MYL (F-5), with GAPDH serving as a protein loading control. Tofa + I: tofacitinib + IFN-γ, Bari + I: baricitinib + IFN-γ. (B and C) Quantifications of Western blots for (B) MYL and (C) MYH averaged for three donors with protein abundance normalized to GAPDH and shown relative to IFN-γ group (n = 6 samples from three donors per group). No difference in dystrophin or SAA expression was observed. (D) Quantified amplitudes of electrically stimulated (1 and 20 Hz) Ca2+ transients (n = 8 myobundles from one donor per group). (E) Representative Western blots for RYR1, CASQ1, and GAPDH. (F) Quantified RYR1 and CASQ1 protein expression normalized to that of GAPDH and shown relative to the IFN-γ group (n = 6 samples from three donors per group). (G) Representative Western blots for pSTAT1, STAT1, and GAPDH. (H and I) Quantified (H) pSTAT1 and (I) pSTAT1/STAT1 protein expression normalized to that of GAPDH and shown relative to the IFN-γ+ group (n = 3 samples from three donors per group). *P < 0.05 versus IFN-γ, #P < 0.05 versus IFN-γ+; **P < 0.05. NS, nonsignificant. Data are presented as means ± SEM.

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