AMP Deaminase 3 knockout mice and loss of mitochondrial proteins and enzyme activity during denervation atrophy

Abstract

Muscle atrophy leads to decrements in muscle function, partly attributable to decreased mitochondrial content. One controller of mitochondrial content is AMP-activated protein kinase (AMPK), which when bound by AMP activates PGC-1[alpha]leading to mitochondrial biogenesis and possibly greater mitochondrial content. AMP Deaminase (AMPD: AMP [right arrow]IMP + NH3) isoform 1 is the dominant isoform in skeletal muscle, yet AMPD3 is robustly upregulated during atrophy of skeletal muscle. This study will determine if loss of AMPD3 gene will reduce mitochondrial content loss typically seen with atrophy. Methods: Whole body AMPD3 knock out, heterozygous (HET), and wildtype (WT) littermate mice (male & female, 10wk old, C57BL6 background) were unilaterally, surgically denervated by sectioning of the sciatic nerve. Contralateral limbs were sham treated. Two weeks later, body composition was measured by Echo MRI. Muscles were collected, weighed, and analyzed for citrate synthase enzyme activity, oxidative phosphorylation proteins, and COX IV protein (measures of mitochondrial content) and expression of AMPD proteins. ANOVA was used to detect significant differences (P[less than]0.05) . Results: AMPD3 genotype had no effect on total body weight, lean mass, or fat mass in either males or females. Likewise, non-denervated EDL and soleus muscle weights were not significantly different among genotypes. As expected, surgical denervation led to substantial muscle atrophy versus innervated muscle in female EDL (~22%) and soleus (~21%), which were not different among genotypes. In males, similar atrophy occurred in EDL (~23%) and soleus (~22%) with no difference among genotypes. Citrate synthase activity in male innervated muscles was not significantly different among genotypes in EDL, but was significantly different in soleus (16.6±;1.8 WT, 22.3±;2.3 HET, 24.7±;1.4 KO)(p [less than]0.05 vs WT). Citrate synthase activity in female innervated muscles was not significantly different among genotypes in EDL or soleus. Predictably, denervation resulted in loss of CS activity in male EDL (~11%) and soleus (~14%), as well as female EDL (~14%) and soleus (~14%), but the loss was not significantly different between genotypes. Complexes I, III, and V proteins, in male EDL, significantly decreased with denervation (p[less than];0.05), while complexes II and IV were not significantly decreased. All male soleus oxidative phosphorylation complexes decreased with denervation (p[less than];0.05). In female EDL, complexes I and III significantly decreased (p[less than];0.05), while complex II (p[less than];0.051) and complex V (p[less than];0.09) trended toward significance, and complex IV increased with denervation (p[less than];0.05). Female soleus complexes all significantly decreased (p[less than];0.05). Male EDL and soleus COX IV protein significantly decreased with denervation, as did female EDL and soleus, yet there were no differences between genotypes (p[less than];0.05). AMPD3 protein was solely expressed in wild type denervated conditions and undetectable in knockout, in both muscles. Male and female EDL AMPD1 protein was significantly decreased (~90%) with denervation (p[less than];0.05). Male and female soleus AMPD1 protein was significantly decreased (42%) with denervation (p[less than];0.05). Female soleus AMPD1 was significantly increased versus wild type (p[less than];0.05). Conclusion: These data suggest AMPD3 does not affect mitochondrial content loss in denervation induced skeletal muscle atrophy. However, since AMPD3 is not present during development in our AMPD3 knockout mice, we cannot exclude compensation from other AMPD isoforms. Future research should investigate either inducible muscle specific AMPD3 knockouts or loss of both AMPD1 and AMPD3, as well the control of AMPD activity.