Rutin Restores Circadian Rhythms and Mitochondrial Health in Aging Muscle

Skeletal muscle aging—culminating in sarcopenia—involves a convergence of oxidative stress, mitochondrial dysfunction, impaired myogenic differentiation, and disrupted circadian rhythms. These processes are interconnected: circadian clock disruption impairs antioxidant defense and mitochondrial biogenesis, while oxidative damage feeds back to destabilize clock gene oscillations. Despite growing interest in flavonoid-based interventions, whether rutin could simultaneously address all these axes in muscle had not been systematically examined.

This study tested rutin (20 μM in vitro; 100 mg/kg orally in vivo) in a D-galactose (D-gal) model of accelerated aging—D-gal at 20 g/L for C2C12 myotubes and 150 mg/kg i.p. for C57BL/6 mice. D-gal is well-established for inducing oxidative stress, mitochondrial dysfunction, and circadian disruption that closely mimics physiological aging. The dual in vitro/in vivo design with time-series sampling (every 4 hours across 48 hours) to capture circadian oscillations is a notable methodological strength.

In cell culture, D-gal caused marked cellular senescence (confirmed by SA-β-galactosidase staining), impaired myotube formation, and flattened circadian oscillations of core clock genes (Bmal1, Per2, Rorc) and myogenic regulatory factors (MyoD, myogenin). Rutin restored myotube architecture, enhanced differentiation indices, and reinstated robust rhythmic expression of these genes—critically correcting an aberrant antiphase relationship between Per2 and Rorc induced by D-gal. On the oxidative stress front, rutin significantly reduced intracellular ROS and malondialdehyde (MDA) while boosting SOD, CAT, and GPx activities. Mitochondrial function improved across multiple metrics: ATP production increased, membrane potential was restored, and pMitoTimer red/green fluorescence ratios (a marker of mitochondrial oxidative aging) decreased. Rhythmic expression of oxidative phosphorylation proteins and Pgc1α—a master regulator of mitochondrial biogenesis—was also reinstated.

In vivo, rutin-supplemented aged mice showed significantly prolonged hanging time, particularly during the active nighttime phase (ZT14–ZT16), indicating enhanced grip strength and neuromuscular performance aligned with circadian biology. Muscle oxidative capacity improved without changes in fiber-type composition, suggesting functional rather than structural remodeling. Core clock gene rhythmicity was normalized in muscle tissue, corroborating the circadian modulatory mechanism observed in vitro.

These findings position rutin as a multi-target agent acting on the circadian–oxidative–mitochondrial axis in aging skeletal muscle. The circadian angle is particularly novel: rather than simply acting as an antioxidant, rutin appears to restore the temporal architecture of muscle gene expression programs, which may be prerequisite for sustained improvements in oxidative defense and mitochondrial quality control. However, the translational path to humans requires further investigation given rutin's modest oral bioavailability (~20%) and the artificial nature of D-gal aging models.