Abstract
Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.
Introduction
Sarcopenia, the age-related loss of skeletal muscle function, is a major contributor to the decline in the quality of life in the elderly for which there are few successful interventions. Age-related skeletal muscle degeneration, including both muscle atrophy and reduced performance of existing muscle, leads to exercise intolerance and is considered a key risk factor along the path to frailty, loss of independence, and poor quality of life. The associated costs were estimated to be 18 billion in the United States alone in 2000 and are expected to continue to increase due to the aging population [1]. Thus, there is a desperate need for interventions that will improve the quality of life for elderly individuals. Despite extensive effort there are few effective interventions, beyond exercise, that can prevent or restore skeletal muscle metabolic and contractile health in the elderly.
Mitochondria play a central role in skeletal muscle health. Sarcopenia is a complex pathology involving diverse age-related changes in metabolism and proteostasis, signaling pathways, chronic inflammation, disruption of neuromuscular junctions, and excitation-contraction defects [2]. However, mitochondrial dysfunction is involved in all of these processes as either a cause or consequence of these age-related changes. This central role of mitochondria in skeletal muscle health is due to their function as both the primary source of adenosine triphosphate (ATP) to meet the sustained energetic demand for muscle contraction and cell maintenance and their role as one of the main sources for cellular reactive oxygen species production. Reduced quality of mitochondria in aging skeletal muscle is manifested by a decline in ATP production accompanied by elevated oxidant production that disrupts both energy and redox homeostasis [3], [4].
Exercise tolerance and fatigue resistance depend on sustained ATP production by the mitochondria to fuel cross-bridge dynamics and calcium cycling that underlie muscle force production. Mitochondrial ATP production decreases with age in rodents and humans [5], [6]. When ATP or respiratory capacity is expressed per unit mitochondria there is a greater deficit [7], [8], indicating that both mitochondrial capacity and quality decline with age. Not all studies have found a decrease in maximal mitochondrial ATP production in aging skeletal muscle [9], despite consistent agreement on the decline in exercise capacity and muscle health with age. In addition to their role in ATP production, mitochondria are also a significant source of superoxide from multiple sites within the electron transport system [10]. Thus, mitochondria are the main integrators of cell bioenergetics, redox biology, and cell signaling affecting muscle performance and health.
An important aspect of mitochondrial quality is the ability of mitochondria to produce ATP efficiently while minimizing oxidant production. Mitochondria from aged skeletal muscle produce elevated levels of superoxide and hydrogen peroxide [11] leading to increased redox stress. An important role for oxidative stress in muscle aging is supported by mice lacking Cu/Zn superoxide dismutase, which is found in the cytoplasm and inner mitochondrial space [12]. The absence of this enzyme results in elevated mitochondrial oxidant production and early development of sarcopenia due to the interaction between elevated mitochondrial oxidative stress in the neuromuscular junction and myofibers. Many studies have focused on oxidative damage to proteins, lipids, and DNA as the link between mitochondrial oxidative stress and tissue pathology, however, the concept of redox stress suggests that more subtle or reversible disruptions in redox signaling may play an important role in tissue pathology [13]. Increased redox stress can further inhibit mitochondrial ATP production and alter muscle contractility through direct effects on redox sensitive post-translational modifications (PTMs) on protein thiols [14], [15]. For example, a mild oxidative stress in vivo with low dose paraquat treatment induced energy deficits in young mice that mimicked those observed with age. Additionally, mitochondrial energetics in old mice was more sensitive to this mild stressor [6]. These results are consistent with work demonstrating reversible inhibition of mitochondria in cells under redox stress [16]. Thus, elevated oxidant production due to poor mitochondrial quality in aged muscle can not only inhibit ATP production, but also contribute to sarcopenia through disruption of redox homeostasis and chronic activation of aberrant cell signaling.
The central role of mitochondria in skeletal muscle health points to direct manipulation of mitochondrial oxidative stress as a potential strategy to attenuate sarcopenia. We have previously shown that a single treatment with the mitochondrial targeted peptide SS-31 (elamipretide) improves mitochondrial coupling and maximal ATP production in aged mouse muscle [4]. SS-31 has also been shown to protect against mitochondrial related pathologies and injuries such as cardiac failure [17], disuse atrophy [18], Parkinson’s disease [19], ischemia-reperfusion injury [20] and severe burn trauma [21], [22]. The ability of SS-31 to target to the mitochondria [23] and associate with cardiolipin [24] while also enhancing ATP production [25] makes SS-31 an ideal therapeutic to test for long-term skeletal muscle health in aging tissue. In this study we test whether reducing mitochondrial oxidative stress with sustained treatment with SS-31 will lead to remodeling of the aged skeletal muscle resulting in functional improvement beyond those observed with a single acute treatment.
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Section snippets
SS-31 reverses energetic deficits in aged mitochondria
We delivered SS-31 (3 mg/kg/day) or saline for 8 weeks to young (5 mo) and old (26 mo) female mice using osmotic pumps. Simultaneous optical and 31P magnetic resonance spectroscopy revealed that aging leads to decreased maximum ATP production (ATPmax) (Fig. 1A) and coupling of oxidative phosphorylation (P/O ratio) (Fig. 1B). Decline of in vivo P/O ratio in aged animals was largely driven by decreased ATPase activity rather than increased oxygen consumption with age (Supplemental Fig. 1A). The
Discussion
Directly targeting age-related mitochondrial dysfunction with the mitochondrial targeted peptide SS-31 improves skeletal muscle energetics and exercise tolerance. We have previously shown that acute SS-31 treatment improves in vivo bioenergetics and leads to increased skeletal muscle function in the form of greater treadmill endurance capacity and resistance to fatigue. [4]. This result led us to test the hypothesis that longer-term treatment would further improve mitochondrial quality and
Animals
This study was reviewed and approved by the University of Washington Institutional Animal Care and Use Committee. Female C57BL/6 mice were received from the National Institute of Aging aged mouse colony. All mice were maintained at 21 °C on a 14/10 light/dark cycle and given standard mouse chow and water ad libitum with no deviation prior to or after experimental procedures.
Surgery and SS-31
Osmotic minipumps (Alzet #1004) were loaded with isotonic saline or SS-31 in isotonic saline. SS-31 pumps were primed to
Acknowledgments
This work was supported by the National Institutes of Health, USA (R21 AG042637, P01 AG001751, T32 AG000057, P30 ES007033). The University of Washington Nathan Shock Center, USA (P30 AG013280) and the American Federation for Aging Research, USA. Elamipretide (SS-31) was provided by Stealth BioTherapeutics, Inc.
References (70)
et al.
Impaired adaptability of in vivo mitochondrial energetics to acute oxidative insult in aged skeletal muscle
Mech. Ageing Dev.
(2012) R.L. Goncalves et al.
Sites of superoxide and hydrogen peroxide production by muscle mitochondria assessed ex vivo under conditions mimicking rest and exercise
J. Biol. Chem.
(2015) R.S. Sohal et al.
The redox stress hypothesis of aging
Free Radic. Biol. Med.
(2012) B.G. Hill et al.
Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation
Biochim Biophys. Acta
(2010) D.F. Dai et al.
Mitochondrial targeted antioxidant Peptide ameliorates hypertensive cardiomyopathy
J. Am. Coll. Cardiol.
(2011) K. Zhao et al.
Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury
J. Biol. Chem.
(2004) E.J. Lesnefsky et al.
Enhanced modification of cardiolipin during ischemia in the aged heart
J. Mol. Cell Cardiol.
(2009) I. Dalle-Donne et al.
S-glutathionylation in protein redox regulation
Free Radic. Biol. Med.
(2007) J. Vina et al.
Mitochondrial biogenesis in exercise and in ageing
Adv. Drug Deliv. Rev.
(2009) A.P. Gomes et al.
Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging
Cell
(2013)
1
Current address: Frances Payne Bolton School of Nursing, Case Western Reserve University, Cleveland, OH, USA.View full text
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