AMP Deaminase 3 Overexpression Reduces Mitochondrial Content in C2C12 Myotubes by Decreasing PGC‐1α Promotor Activation
Authors: Miller Spencer , Brault Jeffrey 
Published: June 2021

Skeletal muscles undergoing atrophy have decreased intracellular [ATP], PGC‐1α expression, and mitochondrial content. This combination of findings is unexpected considering that decreased [ATP] is typically associated with increases in [ADP] and [AMP], activation of AMPK and PGC‐1α, and subsequent mitochondrial biogenesis in order to increase energy production. Therefore, it is unclear why atrophying muscles display deficits in PGC‐1α and mitochondrial content despite having decreased [ATP]. A possible explanation is increased activity of the enzyme AMP Deaminase 3 (AMP→IMP+NH3), which is highly upregulated during muscle atrophy. We tested the hypothesis that increased expression of AMPD3 can reduce mitochondrial content in skeletal muscle cells by decreasing the AMPK→ PGC‐1α→ mitochondrial biogenesis intracellular signaling cascade by decreasing [AMP].

Methods

C2C12 myotubes were transduced with adenovirus encoding AMPD3 or GFP (control) and allowed to differentiate further for 5 days. Myotubes were then collected and analyzed for nucleotide concentrations (UPLC), protein expression of AMPK, phosphorylated AMPK(Thr172), AMPKα substrates, mitochondrial OXPHOS proteins (Western Blot), 2 Kb PGC‐1α promotor activity (PGC‐1α‐Luciferase assay), and citrate synthase enzyme activity (marker of mitochondrial content).

Results

Increased AMPD activity was confirmed by the presence of IMP (0.20 μmol/g) in AMPD3 groups compared to undetectable in GFP controls. Overexpression of AMPD3 decreased [ATP] by 32%, [ADP] by 19%, [AMP] by 28%, and the total adenine nucleotide pool + IMP by 31% (p<0.001 for all). Notably, despite the pronounced decrease in [ATP], the [AMP]/[ATP] did not differ between groups. This was reflected by no differences in protein expression of AMPK, pAMPK(Thr172), and AMPKα substrate. However, PGC‐1α‐luciferase activity was significantly lower after 1 and 5 days of AMPD3 overexpression compared to GFP (p<0.0001, p<0.05), suggesting decreased PGC‐1α promotor region activation despite no measurable difference in pAMPK(Thr172). Furthermore, citrate synthase activity was 17% lower in myotubes overexpressing AMPD3 compared to GFP (p=0.01), yet no differences in mitochondrial OXPHOS proteins were detected.

Summary/Conclusions

Our results show that increased expression of AMPD3 can decrease adenine nucleotide concentrations without disrupting the relative ATP:ADP:AMP ratios. Consequently, we do not detect measurable differences in AMPK activation. However, we do find significantly lower PGC‐1α promotor activity and citrate synthase activity. These findings indicate that increased expression of AMPD3, as occurs during muscle atrophy, can reduce PGC‐1α activation and mitochondrial biogenesis, ultimately resulting in reduced mitochondrial content. Interestingly, our data suggests that the reduction in PGC‐1α activity occurs independent of AMPK activation. Future studies should be conducted to determine the specific mechanism of PGC‐1α activation along with direct measures of mitochondrial biogenesis, such as, mitochondrial protein synthesis rate.

Support or Funding Information

Funded by NIH R01 AR070200

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.