Abstract
Sarcopenia, defined as the loss of skeletal muscle mass and strength, contributes to disability and health-related conditions with aging. In vitro studies indicate that age-related mitochondrial dysfunction could play a central role in the development and progression of sarcopenia, but because of limitations in the methods employed, how aging affects muscle mitochondrial function in vivo is not fully understood. We use muscle-targeted fluorescent proteins and the ratiometric ATP reporter, ATeam, to examine changes in muscle mitochondrial mass and morphology, and intracellular ATP levels in C. elegans. We find that the preserved muscle function in aging daf-2 mutants is associated with higher muscle mitochondrial mass, preserved mitochondrial morphology, and higher levels of intracellular ATP. These phenotypes require the daf-16/FOXO transcription factor. Via the tissue-specific rescue of daf-16, we find that daf-16 activity in either muscle or neurons is sufficient to enhance muscle mitochondrial mass, whereas daf-16 activity in the muscle is required for the enhanced muscle function and mobility of the daf-2 mutants. Finally, we show through the use of drugs known to enhance mitochondrial activity that augmenting mitochondrial function leads to improved mobility during aging. These results suggest an important role for mitochondrial function in muscle aging.
During aging all people lose muscle mass and strength with the peak strength and muscle mass being observed between the late 20’s and early 40’s (reviewed in [1]). The term sarcopenia is often used to refer to these losses, and the study of sarcopenia is of interest both at a scientific and clinical level. In particular, the loss of muscle mass and muscle strength is connected with the development of geriatric phenomena including reduced gait speed, mobility limitations, need for assistance with daily activities, and falls. As a result of the intertwined losses of muscle mass and strength, and the associated functional declines, definitions of the condition have been proposed that focus on from one to all of these aspects [1]. While the exact percentages vary depending on the definition used, the population sampled, and the technique used for assessment, clinically meaningful levels of sarcopenia are thought to affect roughly 30% of individuals over 60 years of age and up to more than 50% of people over 80 years [2]. Unfortunately, the adverse effects of muscle aging produce both human and financial costs. For example, one study estimated that if 45% of the older U.S. population is affected by sarcopenia, then the estimated direct healthcare costs attributable to sarcopenia in the United States was $18.5 billion in 2000, which is approximately 1.5% of the total healthcare expenditures for that year [3]. Conversely, if the prevalence of sarcopenia could be reduced by just 10% then this could lead to healthcare cost savings of $1.1 billion (in 2000) per year in the U.S. alone [3]. Given growing numbers of older people in both the U.S. and worldwide, both the numbers of individuals who develop sarcopenia and the impact on health and wellness are likely to increase in the coming years.
Despite the fairly high prevalence of sarcopenia, our knowledge of the mechanisms involved is incomplete, and consequently there are few proven treatment approaches for the condition other than regular weight-bearing exercise. Both clinical and basic research has provided evidence for multiple potential mechanisms ranging from losses of motor neurons, hormonal changes, and alterations in the function of muscle stem cells, called satellite cells, that alone or together could cause or worsen the age-related changes in the muscle (for recent review see [4]). Among these mechanisms, multiple studies have suggested that mitochondrial dysfunction could either be a hallmark of muscle aging or even play a causal role in the process (for review see [5,6]). The adverse effects of aging on muscle mitochondria include changes such as a reduction in mitochondrial DNA and increase in mitochondrial DNA damage [7–9]; a reduction in the coupling of mitochondrial electron transport activity to ATP production [10,11]; declines in mitochondrial quality control due to decreased removal of mitochondria by mitophagy [10]; changes in the number, size, and orientation of the mitochondria in the myocytes [12]; and an increased tendency of older mitochondria to promote myocyte apoptosis [10]. However, the relative importance of each of these adverse changes in the development and progression of sarcopenia is unclear. Another limitation of these results is that the data are generated from samples removed from either animals or human subjects and either the removal or subsequent processing might alter the structure or function of the mitochondria compared to their in vivo properties. For example, the subcellular fractionation of muscle tissue to isolate mitochondria has been reported to selectively damage the mitochondria from older individuals [13].
The non-parasitic nematode Caenorhabditis elegans has been shown to also develop declines in muscle mass and function during aging [14,15]. The cause of these declines is unclear, but adverse effects of the aging process on both the muscle and motor neurons may play a role [14,16]. The use of C. elegans as a model of muscle aging is attractive due to the short lifespan of the animals, the amenability to genetic manipulation and RNAi treatment, and the optical transparency of the animals which allows the muscles to be observed in vivo via non-invasive imaging approaches. We and others have shown that mutations affecting the daf-2/IGFR insulin/insulin-like growth factor receptor both extend lifespan and delay the onset of age-related declines in muscle function in these worms [14,15,17]. Hence, the study of muscle aging in C. elegans permits the examination of aging effects on components of the muscle, such as the mitochondria, and also permits the parallel examination of each component in animals with altered rates of muscle aging.
In this work, we examine the effects of aging on in vivo muscle mitochondrial mass and function. We adapted the ATeam ratiometric reporter for use in C. elegans to examine mitochondrial function by assessing the relative ATP levels in myocytes in vivo [18]. We find that mitochondria dysfunction might play a causal role in muscle aging as we find that the treatment of worms with both riboflavin [19] and methylene blue [20], which can enhance the activity of dysfunctional mitochondria, increase muscle function. We then use the daf-2/IGFR mutant, which also exhibits preserved mobility [14,15,17] and enhanced mitochondrial function during aging [21], to examine the interplay between the muscle and nervous system with regards to mitochondrial activity and muscle function during aging. Via the use of tissue-specific daf-16 transgenes, we unexpectedly find that restoring daf-16/FOXO in either body wall muscle or neurons is able to attenuate the loss of muscle mitochondria whereas daf-16 only reduces age-related declines in muscle mitochondrial activity and muscle function when restored in the muscle. These findings suggest that both cell autonomous and non-cell autonomous mechanisms could contribute to the beneficial effects of daf-2/IGFR mutations on muscle mitochondria and muscle function during aging.
Results
Mitochondria enhancing drugs promote mobility during aging
Mitochondria are the primary producers of intracellular energy, which can be used for a number of processes in myocytes, including muscle contraction, maintaining membrane polarization, protein synthesis, or maintaining proteostasis, and hence dysfunctional mitochondria could play a key role in age-related declines in muscle function. During aging, C. elegans exhibits declines in mobility with regards to both crawling on solid media and thrashing-like swimming movement in liquid [14,17,22–24]. To evaluate whether enhanced mitochondrial activity can improve muscle function, we tested the effects of the mitochondrial activators, riboflavin and methylene blue on the age-related declines in muscle function. Both of these drugs have been shown to enhance the respiratory activity of dysfunctional mitochondria due to aging (methylene blue [20]), or due to genetic mutations affecting the electron transport chain (riboflavin [19]). When we treated wild-type animals with these drugs starting on day 1 of adult life, we found that the mobility of treated day 5 animals, as measured by the number of body bends made by the animals during 30 seconds of observation, was increased compared to untreated control animals (Figure 1A). This finding suggests that enhancing mitochondrial function can enhance muscle function in older animals though this could be mediated by effects either in the muscle or another tissue.
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