Fig 1.
Histochemical staining of the gastrocnemius muscle post denervation.
H & E, NADH-TR, and COX stains were performed on 10 μm thick transverse frozen muscle section sections of gastrocnemius muscles from the control (Con), denervated (Den), and PQQ-treated denervated groups (Dep) on the 21st day post denervation. Scale bar: 20 μm; 200 ×. (A-C, arrows point to the hindlimb muscle; D-F, H & E stain; G-I, NADH-TR stain; J-L, COX stain).
Fig 2.
Decreased PGC-1α level in the gastrocnemius muscle following denervation and recovery by PQQ treatment.
(A) The PGC-1α protein level was determined by western blot analysis of the gastrocnemius muscles of the denervated (Den) and PQQ-treated denervated (Dep) groups. β-actin is defined as the housekeeping protein and used as a loading control. (B) Statistical analysis of the data from two independent experiments (n = 4 to 5 per group). # and *, P<0.05 indicating a significant difference compared to the Con and Den groups, respectively.
Fig 3.
Effect of PQQ on energy metabolism in denervated gastrocnemius muscle.
On the 21st day after denervation, the gastrocnemius muscle was excised to determine basal OCR (A), ECAR (B), and the OCR to ECAR ratio (C). (D) The OCR and ECAR values of Den and Dep were plotted to show the difference in the metabolic profile between these groups. Values (n = 3 to 5 per group) represent means ± SEM. *, P<0.05, indicating a significant difference between groups. DenL, DenR, DepL, and DepR represent the left internal control (L) and the right denervated (R) hindlimb muscle from the denervated (Den) or PQQ-treated denervated group (Dep), respectively.
Fig 4.
mRNA expression of myosin following denervation.
Quantitative real-time RT-PCR for myosin subtypes of the gastrocnemius muscle in the denervated (Den) and PQQ administered groups (Dep) on the 21st day after denervation. Data are expressed as the right denervated muscle relative to the contralateral non-denervated left hindlimb muscle of the same mouse, normalized to control group. # and *, P<0.05, indicating significant differences compared to the Con (control) and Den (denervation) groups, respectively.
Fig 5.
Changes in glycolytic and mitochondrial energy metabolism following denervation.
(A) Western blot analysis of GAPDH and TFAM expression in the denervation (Den) and PQQ-administered denervation groups (Dep). (B) Statistical analysis of western blot data (n = 3 to 5 of each). *, P<0.05, indicating a significant difference compared to the Den group.
Fig 6.
Western blot of OXPHOS mitochondrial complexes.
An antibody cocktail against proteins representing the five mitochondrial oxidative phosphorylation complexes was used to examine the expression of mitochondrial proteins in skeletal muscle from the control (Con), sham-operated (Sham), denervated (Den), and PQQ administered groups (Dep) on the 7th (A) and 21st days (B) after denervation.
Fig 7.
Summary of sciatic denervation-stimulated signaling transduction leading to skeletal muscle twitch and atrophy, and the possible actions of PQQ.
In the event of denervation, PGC-1α is attenuated and subsequently UCP-2, TFAM, NRF1 and NRF2 are down-regulated. Thereafter, mitochondrial membrane potential disruption and up-regulation of CPL and PKC occur due to the accumulation of ROS and the increase in [Ca2+], leading to the protein degradation and muscle atrophy (A). Administration of PQQ results in an increase in the PGC-1α protein level, slowing protein degradation and muscle atrophy after denervation. Eventually, the strong antioxidant PQQ has the effect of reprogramming the mitochondrial OXPHOS integrity and metabolic bioenergetics (B).