Myricanol prevents aging-related sarcopenia by rescuing mitochondrial dysfunction via targeting peroxiredoxin 5

Abstract

Aging is a process that represents the accumulation of changes in organism overtime. In biological level, accumulations of molecular and cellular damage in aging lead to an increasing risk of diseases like sarcopenia. Sarcopenia reduces mobility, leads to fall-related injuries, and diminishes life quality. Thus, it is meaningful to find out novel therapeutic strategies for sarcopenia intervention that may help the elderly maintain their functional ability. Oxidative damage-induced dysfunctional mitochondria are considered as a culprit of muscle wasting during aging. Herein, we aimed to demonstrate whether myricanol (MY) protects aged mice against muscle wasting through alleviating oxidative damage in mitochondria and identify the direct protein target and its underlying mechanism. We discovered that MY protects aged mice against the loss of muscle mass and strength through scavenging reactive oxygen species accumulation to rebuild the redox homeostasis. Taking advantage of biophysical assays, peroxiredoxin 5 was discovered and validated as the direct target of MY. Through activating peroxiredoxin 5, MY reduced reactive oxygen species accumulation and damaged mitochondrial DNA in C2C12 myotubes. Our findings provide an insight for therapy against sarcopenia through alleviating oxidative damage-induced dysfunctional mitochondria by targeting peroxiredoxin 5, which may contribute an insight for healthy aging.

Keywords: healthy aging; mitochondria; myricanol; peroxiredoxin 5; redox homeostasis; sarcopenia.

FIGURE 1

MY protects against aging‐related loss of muscle mass and strength in mice. (A) body weight. (B) Grip strength. (C) Forced swimming time. (D) The weight ratios of Quad, Gast, and TA muscle to the body weight. (E) Representative H&E staining of Gast. Top: cross section; bottom: longitudinal section. Scale bar = 100 µm. A microscope with 20× objective was used to capture the images. (F) Electron microscope analyses in TA muscle. Scale bar = 1 µm (top); Scale bar = 500 nm (bottom). Young: 3‐month‐old mice, PEG 400 solution; young+MY.H: 3‐month‐old mice, PEG 400 buffer with 50 mg/kg MY; aged: 18‐month‐old mice, PEG 400 solution; aged+MY.L: 18‐month‐old mice, PEG 400 buffer with 10 mg/kg MY; aged+MY.H: PEG 400 solution with 50 mg/kg MY. Data are displayed as mean ± SD, n = 6. ^# p < 0.05, ^## p < 0.01, young versus aged. *p < 0.05, **p < 0.01, aged versus aged+MY.L. ^& p < 0.05, ^&& p < 0.01, aged versus aged+MY.H.

FIGURE 2

MY alleviates oxidative damage and mitochondrial dysfunction in skeletal muscle from aged mice. (A) Cellular H₂O₂ content. (B) 8‐OH‐dG level in mtDNA was measured by ELISA kit. (C) The expression of key proteins in muscle atrophy including MuRF1, myogenic differentiation including MyoD and myogenin, antioxidative capacity including UNG1 and Nrf2, and mitochondrial homeostasis including PGC‐1α, Drp1and Mfn1 in Gast muscle. β‐Actin was used as a loading control. (D) The expression of nuclear Nrf2. Histone H3 was used as the loading control. Young: 3‐month‐old mice, PEG 400 buffer; young+MY.H: 3‐month‐old mice, PEG 400 solution with 50 mg/kg MY; aged: 18‐month‐old mice, PEG 400 buffer; aged+ MY.L: 18‐month‐old mice, PEG 400 buffer with 10 mg/kg MY; aged + MY.H: PEG 400 buffer with 50 mg/kg MY. Data are shown as mean ± SD, n = 6. ^# p < 0.05, ^## p < 0.01, young versus aged. *p < 0.05, **p < 0.01, aged versus aged+MY.L. ^& p < 0.05, ^&& p < 0.01, aged versus aged+MY.H.

FIGURE 3

MY rescues C2C12 myotubes against TBHP‐induced oxidative damage through improving mitochondrial biogenesis and function. (A) LDH level in TBHP‐treated C2C12 myotubes. (B) ROS production. (C) Cellular H₂O₂ content. (D) Immunostaining of MyHC in C2C12 myotubes. (E) The expression levels of MuRF1, MyoD, and myogenin, β‐actin was used as control. (F) Mitochondrial content assessed by MitoTracker Green staining. (G) ATP production. (H) Mitochondrial ROS content determined by MitoSOX Red. (I) Cellular levels of 8‐OH‐dG in mtDNA from TBHP‐treated C2C12 myotubes was measured by ELISA. (J) The expression of key proteins in antioxidative capacity including UNG1, Nrf2 and NQO1, and mitochondrial homeostasis including PGC‐1α, Drp1 and Mfn1 in TBHP‐treated C2C12 myotubes. β‐actin was used as a loading control. (K) The expression of nuclear Nrf‐2. Histone H3 was used as a control. Data are displayed as mean ± SD, n = 6. #p < 0.05, ##p < 0.01, vehicle versus TBHP, *p < 0.05, **p < 0.01, MY versus TBHP.

FIGURE 4

Target identification of MY in protecting oxidative damage in TBHP‐treated C2C12 myotubes. (A) The structure of the probe MY‐P. (B) Pull‐down labeling with MY‐P in C2C12 myotubes. CBB: Coomassie Brilliant Blue. (C) Volcano plot of MY‐P binding proteins compared with control group (10 µM MY‐P). (D) Pull‐down/Western blotting identified the target protein PRDX5. (E) The PRDX5 expression in Gast muscle from young or aged mice. ##p < 0.01, young versus aged. **p < 0.01, aged versus aged+MY.L. &&p < 0.01, aged versus aged+MY.H. (F) The PRDX5 expression in TBHP‐treated C2C12 myotubes. ##p < 0.01, vehicle versus TBHP, *p < 0.05, **p < 0.01, MY versus TBHP. (G) ITC titration of MY (100 µM) into recombinant PRDX5 protein (5 µM). (H) Western blotting‐based CETSA validation of thermal stabilization of PRDX5 in response MY treatment at the concentration from 0 to 100 µM. β‐Actin was used as a control. Data are displayed as mean ± SD, n = 6.

FIGURE 5

MY protects C2C12 myotubes against oxidative damage through targeting PRDX5. (A) Protein expression levels of PRDX5, MuRF1, UNG1, and Nrf2 in si‐Prdx5 and scrambled cells. β‐Actin was used as a loading control. (B) Protein expression level of nuclear Nrf‐2 in si‐Prdx5 and scrambled cells. Histone H3 was used as the loading control. LDH level (C) and ATP concentration (D) in TBHP‐treated scrambled and si‐Prdx5 cells. (E) Mitochondrial content in TBHP‐treated scrambled and si‐Prdx5 cells, assessed by MitoTracker Green staining. (F) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in TBHP‐treated scrambled and si‐Prdx5 cells, assessed by Seahorse assay. (G) The levels of coprecipitated Nrf2 and UNG1 with PRDX5 in C2C12 myotubes. Data are displayed as mean ± SD, n = 6. #p < 0.05, ##p < 0.01, vehicle versus TBHP. *p < 0.05, **p < 0.01, TBHP+MY versus TBHP. &p < 0.05, &&p < 0.01, si‐Prdx5+MY versus scrambled+MY.

FIGURE 6

Cys100 of PRDX5 is critical for binding to MY. (A) Binding sites of MY–PRDX5 by virtual docking analysis. (B) The PRDX5 expression level in PRDX5‐WT and PRDX5‐C100A overexpression C2C12 cell lines, respectively. (C) ROS production. (D) ATP concentration. (E) Protein expression levels of MuRF1, UNG1 and Nrf2 in TBHP‐treated PRDX5‐WT and PRDX5‐C100A overexpression cells. β‐Actin was used as a loading control. (F) Protein expression level of nuclear Nrf‐2 in TBHP‐treated PRDX5‐WT and PRDX5‐C100A overexpression cells. Histone H3 was used as the loading control. Data are shown as mean ± SD, n = 6. #p < 0.05, ##p < 0.01, vehicle versus TBHP. *p < 0.05, **p < 0.01, TBHP+MY versus TBHP. &p < 0.05, &&p < 0.01, WT+MY versus C100+MY. $p < 0.05, $$p < 0.01, TBHP versus MY+TBHP.

FIGURE 7

Schematic diagram of molecular mechanism of MY in mitigating sarcopenia.