Characterizing the effects of heterozygous neurogranin deletion on myosin heavy chain and calcineurin expression in the murine unloaded soleus

Abstract

Background Neurogranin (Ng) is a small IQ motif protein that binds to the C-terminal on CaM limiting its cellular availability and sequestering it away from its downstream targets such as calcineurin. Calcineurin is a Ca2+/calmodulin (CaM)-dependent protein that promotes the slow oxidative fibre phenotype in skeletal muscle as well as myoblast fusion. Recent research from our lab shows that Ng is expressed in skeletal muscle, particularly in slow-oxidative muscles such as the soleus. The soleus is a postural muscle that acts to withstand the downward pull of gravity and unloading the soleus muscle leads to muscle atrophy and a slow-to-fast fibre type shift. However, activating calcineurin can attenuate the muscle atrophy and fibre type transition that occurs with soleus unloading. In the present study, we determined the effects of heterozygous Ng deletion on muscle mass and myosin heavy chain (MHC) isoform expression in the unloaded soleus. We hypothesized that lowered Ng expression would enhance calcineurin signaling thereby minimizing muscle atrophy and the slow-to-fast fibre type shift. Methods To this end, 3-4 month-old male wild-type (WT) (n=6) and Ng+/- (n=8) mice were subjected to tenotomy surgery, where the soleus and gastrocnemius tendons were severed. Soleus muscles were collected two weeks post-surgery for Western blotting to examine differences in MHC I, IIa, and calcineurin content. Results Soleus to bodyweight ratio was significantly reduced in the unloaded/tenotomy group for both WT (-55%) and Ng+/- mice (-51%) compared with their respective sham controls (p<0.001). Western blot analyses revealed that tenotomy-induced unloading significantly lowered MHC I (-60%, p=0.03) and MHC IIa (-63%, p=0.0001) content, while increasing calcineurin levels (1.4-fold, p=0.003). However, these effects were blunted in the Ng+/- mice with only a 43% reduction in MHC I (p=0.01), 40% reduction in MHC IIa (p=0.01), and a non-significant change in calcineurin (1.0-fold, p=0.98). Two-way ANOVA analyses revealed a significant interaction (genotype x tenotomy) for MHC IIa (p=0.01) and calcineurin (p=0.007) inidicating that WT mice were more impacted by tenotomy-induced soleus unloading. Conclusions Our results show that heterozygous Ng deletion did not reduce the muscle atrophy observed with soleus unloading. However, with respect to MHC isoform expression, Ng+/- mice were less impacted by unloading with only 40-43% reductions in MHC I and IIa. This could suggest a blunted slow-to-fast fibre type shift. Interestingly, calcineurin was significantly upregulated in the unloaded WT soleus, which we believe is a compensatory response elicited to combat the effects of tenotomy. However, this response was completely absent in the Ng+/- mice, perhaps due to sufficient activation of calcineurin with lowered Ng content. In conclusion, this study shows that Ng+/- mice respond differently to soleus unloading compared with WT mice.