skNAC depletion stimulates myoblast migration and perturbs sarcomerogenesis by enhancing calpain 1 and 3 activity

Abstract

skNAC (skeletal and heart muscle specific variant of nascent polypeptide-associated complex α) is a skeletal and heart muscle-specific protein known to be involved in the regulation of sarcomerogenesis. The respective mechanism, however, is largely unknown. In the present paper, we demonstrate that skNAC regulates calpain activity. Specifically, we show that inhibition of skNAC gene expression leads to enhanced, and overexpression of the skNAC gene to repressed, activity of calpain 1 and, to a lesser extent, calpain 3 in myoblasts. In skNAC siRNA-treated cells, enhanced calpain activity is associated with increased migration rates, as well as with perturbed sarcomere architecture. Treatment of skNAC-knockdown cells with the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) reverts both the positive effect on myoblast migration and the negative effect on sarcomere architecture. Taken together, our data suggest that skNAC controls myoblast migration and sarcomere architecture in a calpain-dependent manner.

The E3 SUMO ligase Nse2 regulates sumoylation and nuclear-to-cytoplasmic translocation of skNAC-Smyd1 in myogenesis

“…4C), an effect that has also previously been described by Riquelme and colleagues (Riquelme et al, 2006b). Interestingly, as in cells that had been transfected with an skNAC-specific siRNA (Berkholz et al, 2013), calpain gene expression was strongly induced in the ginkgolic-acid-treated cells and in cells that had been transfected with siRNA against Ubc9 (Fig. 4D).…”

“…This staining pattern was clearly different from that observed in untransfected cells or those transfected with a scrambled siRNA, in which the typical, homogeneously distributed spotty staining of newly forming sarcomeres was detected (Fig. 3D), and was reminiscent of the MyHC and a-sarcomeric actin staining patterns we had observed previously in myoblasts in which skNAC expression had been knocked down (Berger et al, 2012;Berkholz et al, 2013). The perturbed sarcomerogenesis we had seen previously in cells that had been transfected with siRNA against skNAC was dependent on an induction of calpain gene expression and calpain enzymatic activity (Berger et al, 2012).…”

“…This was most probably due to defects in myofibrillogenesis and/or sarcomerogenesis; embryos in which skNAC expression had been knocked down showed irregular distribution of actin and myosin filaments and no formation of well-structured sarcomeres. Correspondingly, we have recently demonstrated defective sarcomerogenesis in murine C2C12 myoblasts after skNAC depletion using a small interfering (si)RNA-mediated approach (Berger et al, 2012;Berkholz et al, 2013). These data implicate skNAC in the regulation of sarcomere formation.…”

“…skNAC and its known binding partner Smyd1 have been demonstrated to regulate myogenesis, specifically skeletal myoblast migration and sarcomerogenesis (Tan et al, 2006;Li et al, 2009, Li et al, 2011Just et al, 2011;Berger et al, 2012, Berkholz et al, 2013Li et al, 2013). Because both proteins undergo nuclear-to-cytoplasmic translocation during myogenesis (Sims et al, 2002), it is probable that they fulfill specific functions in both cellular compartments; in early myogenesis, they might act as transcriptional modulators, presumably through two specific domains within the Smyd1 protein -the so-called SET domain, which is a histone methyltransferase moiety, and the MYND domain, which is a potential recruiter of HDACs.…”

“…3C). Because we had recently demonstrated a role for skNAC in the control of sarcomerogenesis in mammalian cells (Berger et al, 2012;Berkholz et al, 2013), we hypothesized Fig. 3.…”