Netrin-4 synthesized in satellite cell-derived myoblasts stimulates autonomous fusion.

Satellite cells are indispensable for skeletal muscle regeneration and hypertrophy by forming nascent myofibers (myotubes). They synthesize multi-potent modulator netrins (secreted subtypes: netrin-1, -3, and -4), originally found as classical neural axon guidance molecules. While netrin-1 and -3 have key roles in myogenic differentiation, the physiological significance of netrin-4 is still unclear. This study examined whether netrin-4 regulates myofiber type commitment and myotube formation. Initially, the expression profiles indicated that satellite cells isolated from the extensor digitorum longus muscle (EDL muscle: fast-twitch myofiber-abundant) expressed slightly more netrin-4 than the soleus muscle (slow-type abundant) cells. As netrin-4 knockdown inhibited both slow- and fast-type myotube formation, netrin-4 may not directly regulate myofiber type commitment. However, netrin-4 knockdown in satellite cell-derived myoblasts reduced the myotube fusion index, while exogenous netrin-4 promoted myotube formation, even though netrin-4 expression level was maximum during the initiation stage of myogenic differentiation. Furthermore, netrin-4 knockdown also inhibited MyoD (a master transcriptional factor of myogenesis) and Myomixer (a myoblast fusogenic molecule) expression. These data suggest that satellite cells synthesize netrin-4 during myogenic differentiation initiation to promote their own fusion, stimulating the MyoD-Myomixer signaling axis.

Abundant Synthesis of Netrin-1 in Satellite Cell-Derived Myoblasts Isolated from EDL Rather Than Soleus Muscle Regulates Fast-Type Myotube Formation.

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.