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Testosterone is essential for skeletal muscle rejuvenation of aged mice in heterochronic parabiosis
Purpose: In a murine model of heterochronic parabiosis, in which an old mouse is surgically joined to a young mouse and develop a shared circulation, there is an improvement of resident skeletal muscle stem cell myogenic potential in the old mouse. Serum factor(s) responsible for this improvement are not yet identified. This study examines the role of testosterone in skeletal muscle rejuvenation within the parabiotic model and of skeletal muscle stem cells in maintaining skeletal muscle mass.
Methods: We established the following heterochronic parabioses between young (Y; 4 months old) and old (O; 22-23 months old) male mice: 1) Y: O; 2) castrated Y: O; and 3) Castrated + testosterone-treated Y: O. A group of young and old mice received empty implants and were used as controls. Parabiotic pairings were maintained for 4 weeks prior to analysis. At that time, mice were sacrificed and the hind limb skeletal musculature was assessed for mass, cross-sectional fiber area, and Notch-1 levels (known to increase during skeletal muscle regeneration). Serum testosterone levels were determined as well. Electron microscopy was utilized to evaluate ultrastructural changes in hind limb skeletal musculature following parabiosis. Statistical significance was defined as p <0.05.
Results: Serum testosterone levels were 3-fold higher in young wild type mice in comparison with aged mice. Old mice paired with wild-type or testosterone-treated orchiectomized young partners demonstrated significantly elevated levels of serum testosterone. Intriguingly, parabiosis with testosterone-treated orchiectomized young mice but not with castrated young partners fully reversed the age-related changes in gastrocnemius muscle weight, muscle ultrastructure (Figure 1), and muscle fiber cross-sectional area. Testosterone supplementation further restored Notch 1 expression to levels seen in young controls.
Conclusions: These novel findings point to a critical role of testosterone in restoring a systemic environment that favors skeletal muscle rejuvenation in aging. Further refinement in our understanding of testosterone-mediated signaling changes that occur in herterochronic parabiosis is necessary for the development of pharmaceutical or stem-cell therapies for improving muscle regeneration in diseases of muscle wasting.
Figure 1. Representative electron microscopic image of gastrocnemius muscle from a young (Y) mouse exhibits normal cytoarchitecture and sarcomere organization with numerous mitochondria (arrow). Muscle from an old mouse (O) exhibits perturbed muscle ultrastructure, including IML accumulation (L) and formation of large areas of tubular aggregation (TA). Muscle from an aged parabiont (Y:O) demonstrates no IML accumulation and smaller areas of TA. In contrast, muscles from aged partners of heterochronic parabionts with castrated
young mice (ØY:O) show larger areas of TA formation and vacuolated (V) and swollen mitochondria with broken cristae (asterisk). Parabiosis with castrated plus testosterone-treated young mice (ØY+T:O) restores normal cytoarchitecture and sarcomere organization with abundant hypertrophied mitochondria in aged mice. Scale bar = 2.5 μm.