Localization and quantitation of oxidative damage in skeletal muscle from aging rhesus monkeys: Attenuation by caloric restriction

Sarcopenia refers to an involuntary loss of skeletal muscle mass, which consequently results in loss of muscle strength and an increase in physical frailty in older persons. Applied to sarcopenia, the oxidative stress/damage hypothesis of aging would predict that loss of mass and functional performance of skeletal muscle with age is correlated with increased levels of oxidative stress and subsequent damage. In laboratory rodents, caloric restriction (CR) extends maximum lifespan and retards the appearance of a broad spectrum of age-associated pathophysiological changes. In skeletal muscle, CR retards several age-dependent physiological and biochemical changes, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. To date no studies have characterized oxidative damage in aging mammalian skeletal muscle from a histological standpoint. The well-established ferric nitrilotriacetate (Fe-NTA) model of iron-induced free radical injury was utilized as a positive control for markers of age-related oxidative stress and damage by demonstrating that the intensity of immunolabeling at both the light and EM levels correlated with increased biochemically measured lipid peroxidation products and protein carbonyl content. Furthermore, the data suggested that the mechanism of acute nephrotoxicity of Fe-NTA involves mitochondrial and nuclear oxidative damage. We employed immunogold light and electron microscopic (EM) techniques utilizing antibodies raised against 4-hydroxy-2-nonenal (HNE)-modified proteins, dinitrophenol (DNP), and nitrotyrosine (NT) to quantitate and localize oxidative damage in aging rhesus monkey vastus lateralis skeletal muscle. In animals ranging in age from 2--34 years old, a 4-fold maximal increase in levels of HNE-modified proteins was observed. Likewise, carbonyl levels increased ∼2-fold with aging. Comparing 17--23 year old normally fed to age-matched monkeys subjected to CR for 10 years, levels of HNE-modified proteins, carbonyls, and nitrotyrosine in skeletal muscle from the CR group were significantly less than control group values. Oxidative damage specifically localized to myofibrils. Accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-alkenals (4-HDA), and protein carbonyls were measured biochemically and confirmed the morphological data. This study is the first to quantitate morphologically and localize the age-dependent accrual of oxidative damage in mammalian skeletal muscle and to demonstrate that oxidative damage in primates is lowered by CR.