Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation

Abstract

Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0-100 ng/ml) and cocultures ± α-IL-6 (0-200 μg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6-21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of myoblast differentiation. Specific biophysical strain patterns modify these outcomes, and suggest that myofascial release after repetitive strain increases myoblast differentiation and thus may improve muscle repair in vivo. Neutralization of IL-6 in coculture significantly reduced differentiation, suggesting fibroblast-IL-6 is necessary but not sufficient in this process.

Fig. 1.

Uniculture and coculture systems in Bioflex wells. Myoblasts (A, C) were seeded on nondeformable coverslips and situated 2 mm above Bioflex membranes. The orientation of the coverslips is such that the cell side faces the fibroblasts. Fibroblasts (B, C) were seeded on Bioflex membranes and subjected to mechanical strain paradigms as described.

Fig. 2.

Images of C2C12 skeletal muscle in uniculture (A) and coculture (B) 96 h after establishing the coculture. Quantification of myotubes per square millimeter (1.7, 6.9), nuclei per myotube (3.0, 7.2), and fusion efficiency (1.1%, 6.7%) demonstrate fibroblast's ability to induce myoblast differentiation. Examples of multinucleated myotubes are shown by arrows.

Fig. 3.

Myotube densities in fibroblast-conditioned media after 48, 72, and 96 h. Three experiments with 3 cell cultures per strain group (n = 9) were combined. CSDS, cyclic short-duration stretches; ALDS, acyclic long-duration stretch; CSDS + ALDS, CSDS combined with ALDS. Symbols show: * > 48 h, ψ > nonstrain (P < 0.05).

Fig. 4.

Differentiation for myotubes per square millimeter (A), nuclei per myotube (B), and fusion efficiency (C) were combined from 7 experiments with 3 cell cultures per strain group (n = 21). The black bars designate coculture and white bars uniculture. Symbols show: * > uniculture, δ > CSDS (P < 0.05).

Fig. 5.

Human IL-6 secretion from fibroblasts at 24 h (A) and 96 h (B) poststrain. The black bars designate coculture and white bars uniculture fibroblasts. Immunoreactive IL-6 was measured from 3 experiments with 3 cell cultures each (n = 9). Symbols show: * > uniculture, δ > CSDS, ω > ALDS + CSDS (P < 0.05).

Fig. 6.

Differentiation measures in the presence and absence of exogenous IL-6 doses (0–100 ng/ml) after 96 h in uniculture. The vehicle was untreated media (2% FBS in DMEM). Data for myotubes per square millimeter (A), nuclei per myotube (B), fusion efficiency (C), and myoblasts per square millimeter (D) were combined from 4 experiments with 3 cell cultures per IL-6 dose (n = 12). hrIL-6, human recombinant IL-6.

Fig. 7.

Differentiation measures in coculture supplemented with 20 μg/ml IL-6 neutralizing antibody 96 h poststrain. Data for myotubes per square millimeter (A), nuclei per myotube (B), and fusion efficiency (C) were combined from 2 experiments with 3 cell cultures per treatment (n = 6). Results were normalized to respective untreated co-culture strain group and shown as a percent change from untreated (%CFU). Symbols show: * > untreated coculture (P < 0.05).