Pyrroloquinoline Quinone Resists Denervation-Induced Skeletal Muscle Atrophy by Activating PGC-1α and Integrating Mitochondrial Electron Transport Chain Complexes

<div><p>Denervation-mediated skeletal muscle atrophy results from the loss of electric stimulation and leads to protein degradation, which is critically regulated by the well-confirmed transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α). No adequate treatments of muscle wasting are available. Pyrroloquinoline quinone (PQQ), a naturally occurring antioxidant component with multiple functions including mitochondrial modulation, demonstrates the ability to protect against muscle dysfunction. However, it remains unclear whether PQQ enhances PGC-1α activation and resists skeletal muscle atrophy in mice subjected to a denervation operation. This work investigates the expression of PGC-1α and mitochondrial function in the skeletal muscle of denervated mice administered PQQ. The C57BL6/J mouse was subjected to a hindlimb sciatic axotomy. A PQQ-containing ALZET® osmotic pump (equivalent to 4.5 mg/day/kg b.w.) was implanted subcutaneously into the right lower abdomen of the mouse. In the time course study, the mouse was sacrificed and the gastrocnemius muscle was prepared for further myopathological staining, energy metabolism analysis, western blotting, and real-time quantitative PCR studies. We observed that PQQ administration abolished the denervation-induced decrease in muscle mass and reduced mitochondrial activities, as evidenced by the reduced fiber size and the decreased expression of cytochrome <i>c</i> oxidase and NADH-tetrazolium reductase. Bioenergetic analysis demonstrated that PQQ reprogrammed the denervation-induced increase in the mitochondrial oxygen consumption rate (OCR) and led to an increase in the extracellular acidification rate (ECAR), a measurement of the glycolytic metabolism. The protein levels of PGC-1α and the electron transport chain (ETC) complexes were also increased by treatment with PQQ. Furthermore, PQQ administration highly enhanced the expression of oxidative fibers and maintained the type II glycolytic fibers. This pre-clinical <i>in vivo</i> study suggests that PQQ may provide a potent therapeutic benefit for the treatment of denervation-induced atrophy by activating PGC-1α and maintaining the mitochondrial ETC complex in skeletal muscles.</p></div

Western blot of OXPHOS mitochondrial complexes.

<p>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 7^th (A) and 21^st days (B) after denervation.</p

Effect of PQQ on energy metabolism in denervated gastrocnemius muscle.

<p>On the 21^st 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. *, <i>P</i><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.</p

mRNA expression of myosin following denervation.

<p>Quantitative real-time RT-PCR for myosin subtypes of the gastrocnemius muscle in the denervated (Den) and PQQ administered groups (Dep) on the 21^st 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 *, <i>P</i><0.05, indicating significant differences compared to the Con (control) and Den (denervation) groups, respectively.</p

Decreased PGC-1α level in the gastrocnemius muscle following denervation and recovery by PQQ treatment.

<p>(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 *, <i>P</i><0.05 indicating a significant difference compared to the Con and Den groups, respectively.</p

Summary of sciatic denervation-stimulated signaling transduction leading to skeletal muscle twitch and atrophy, and the possible actions of PQQ.

<p>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).</p

JARID1B-silencing suppresses tumorsphere formation and increases cisplatin sensitivity.

<p>(A) Tumorspheres generated from wild type and JARID1B-shRNA 1- and 2- infected SK-N-BE(2) cells. (B and C) JARID1B knockdown caused significant reduction in number and sizes of tumorspheres formed. (D) Cell viability assay show that the cytotoxicity effect of cisplatin was significantly enhanced by JARID1B silencing. All assays were repeated at least three times. * and ** indicate p<0.05 and p<0.001.</p

Silencing JARID1B downregulates Jagged/Notch signaling transduction pathway.

<p>The expression of Notch and its ligand in wild type and JARID1B-silenced SK-N-BE(2) cells were analyzed by Western blot assay. (A) JARID1B-silencing disrupted Jagged1, Notch1 and Notch 2 signaling. (B) Comparative bar diagrams demonstrate the downregulation of Notch signaling in the wild type SK-N-BE(2) cells, compared to the JARID1B-silenced SK-N-BE(2) cells. The intensity was measured relative to loading control (β-actin) from three independent experiments. *, p<0.05 (C) Immunofluorescent staining showing that JARID1B-silencing downregulated the expression of Notch1. Notch1 (green) colocalizes with JARID1B (red) in the nucleus (DAPI for nuclear staining, blue).</p

Evaluation of JARID1B expression and stemness functions in <i>MYCN</i> and <i>non-MYCN</i> neuroblastoma (NB) cell lines.

<p>(A) Western blot analysis was performed to investigate the expression of JARID1B and α-MYCN in <i>MYCN</i> amplification (MNA⁺) and <i>non-MYCN</i> amplification (MNA^-) NB cells. (B) The SP percentage was analyzed in MNA⁺ and MNA^- NB cells by Hoechst staining and flow cytometry. SK-N-BE(2) and SK-N-AS cells had 9.05% and 1.45% SP cells (C) Representative Aldeflour assay result of MNA⁺ SK-N-BE(2) and MNA^- SK-N-AS NB cells showed 17.2% and 3.43% ALDH+ subpopulation, respectively. Data was collected from three independent experiments.</p

Silencing JARID1B decreases epithelial to mesenchymal transition (EMT).

<p>(A) Western blot analysis of epithelial and mesenchymal markers’ expression in wild type and JARID1B-silenced SK-N-BE(2) cells show downregulation of N-cadherin and vimentin in response to JARID1B knockdown, while E-cadherin was upregulated. β-actin served as loading control. (B) Bar chart quantification of (A). Expression of EMT markers are normalized against β-actin. Assay was performed three times. *, represent p<0.05.</p