Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts

Abstract

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

Cessation of cyclic stretch induces atrophy of C2C12 myotubes

“…While the orientati on of the myotube on the membrane would still affect the direction of deformation , the myotubes had a tendency to align in the same plane with maturati on and exposure to stretch as observed previousl y [22]. We have previously reported that cyclic stretching or L-NAME treatment of C2C12 cells does not reduce cell viability as measured by trypan blue exclusion [23]. Further, in the present study, counting of DAPI-stained nuclei indicate no differences in the total number of nuclei between groups (data not shown).…”

Pro-Inflammatory Mediation of Myoblast Proliferation

“…PDTC has been shown to be effective at decreasing NF-κB activation in C2C12 myotubes [68]–[71] and myoblasts [72] at concentrations ranging from 10 μm to 100 μM. In order to observe this effect in proliferating C2C12 myoblasts, NF-κB promoter activity and BrdU incorporation were measured following treatment with IL-1β or TNF-α, with and without 50 μM PDTC.…”

EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading

“…In contrast, a new publication indicates that nNOS is necessary in triggering an anabolic signaling pathway involving Nox4 and mTOR, which responds to overloading and regulates muscle hypertrophy (15). Furthermore, nitric oxide and NF-B were also found to be essential for stretch-induced proliferation of myoblasts (40). Indeed, inhibition of nitric oxide synthase has been shown to attenuate muscle remodeling and fiber-type shift in response to altered mechanical loading, whether the magnitude be increased or decreased (15,38,42).…”