Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion

Abstract

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43

“…Together, these findings would suggest that Ng has a negative role in CaM activation. Ng was once thought to be a neuronal specific protein; however, we have recently discovered that Ng is expressed in mammalian skeletal muscle (rodent and human), and is particularly abundant in slow-oxidative muscle fibers [28]. Using C2C12 cells, our results showed that silencing Ng increased CaM-CaN binding-indicative of a role for Ng in sequestering CaM in these muscle cells.…”

“…Though neither CaN or CaMKII activation have been directly examined in muscles from Gap43-null mice, Caprara et al [45] did not observe any effects of Gap43 deletion (heterozygous or homozygous) on myoblast fusion and differentiation in primary myoblasts. Given the role of CaN in regulating myoblast fusion and myogenic differentiation [27,28], these results suggest that GAP43 may have less of a role in regulating CaN and is rather more important in regulating free intracellular Ca 2+ during muscle contraction and relaxation. Future studies that examine whether genetic deletion of Gap43 alters fibre type distribution and fatigue resistance, similar to that conducted with Nrip-null mice [9], will provide more insight towards the role of GAP43 in regulating CaM activation of CaN and CaMKII.…”

“…Indeed, Chen et al [9] demonstrated that silencing Nrip expression in C2C12 cells attenuated myoblast fusion and differentiation, and that Nrip-null mice had delayed muscle regeneration after cardiotoxin injection. This is not surprising since CaN has long been linked with myogenic differentiation and fusion in vitro and muscle regeneration in vivo by stimulating the expression of proteins such as myogenin, interleukin-4, and stabilin-2 [27][28][29][30][31][32][33][34]. The latter is a phosphatidylserine receptor that plays an important role in promoting myoblast fusion and is regulated by CaN/NFAT signalling [29].…”

“…Associated with enhanced CaN signalling, knocking down Ng also led to a significant enhancement of myoblast fusion and myogenic differentiation [28]. Given the role of CaN in muscle regeneration [27][28][29][30][31][32][33][34], it will be important to determine whether reducing Ng expression and increasing CaM-CaN binding in mice may enhance muscle regeneration in vivo. To date, the regulation of Ng on CaMKII signalling has not been investigated.…”

“…Thus, it will be important to determine whether Ng activates or inhibits CaMKII in rodent skeletal muscle. Finally, the enrichment of Ng in slow-oxidative muscle fibres [28] warrants the investigation of Ng and its physiological role in cardiac muscle where it may regulate CaN, CaMKII, and Ca 2+ handling.…”