Current limitations and future opportunities of tracer studies of muscle ageing

The overarching question that we address here is whether current tracer approaches focused on the measurement of protein synthesis are leading us in a fruitful direction to combat muscle loss with age. Because ageing is largely a loss of protein mass over time, logically, the field has focused on improving anabolism. To assess an improvement in anabolism, tracer-based measurement of protein synthesis is often a primary outcome. It is possible that the relative ease of measuring protein synthesis in comparison to breakdown has biased research efforts towards the protein synthesis side of the equation. An additional reason is that some argue that the slowing of protein synthesis and/or anabolic resistance is the primary determinant of muscle loss with age (Burd et al., 2013; Nunes et al., 2022). The statement that ‘ageing decreases muscle protein synthesis’ often appears without citation because it is a generally accepted ‘fact’ in the skeletal muscle literature. As far as we can tell, the studies that first led to this conclusion were those studies from the Nair lab that looked at skeletal muscle in ageing humans (Balagopal et al., 1997; Rooyackers et al., 1996). A point of consideration is that these studies were performed with labelled amino acid infusions, the importance of which will be discussed here. The conclusion that ageing decreases muscle protein synthesis has led to interventions for aged muscle, such as amino acid feeding, that focus on increasing protein synthesis through activation of the mechanistic target of mTOR(definition)-inhibiting drug studied for extending healthspan and lifespan." style="text-decoration:underline dotted; text-underline-offset:2px; cursor:help;">rapamycin(definition) (mTOR). However, a rigorous evaluation of the literature indicates that there are several contradictory findings to this ‘known’. There are studies showing that protein synthesis does not decrease in skeletal muscle with age and, conversely, showing increases in protein synthesis compared to adult muscle (Fuqua et al., 2023; Kimball et al., 2004; Miller et al., 2019). Furthermore, studies in rodent models (Fuqua et al., 2023; Joseph et al., 2019; Tang et al., 2019; White et al., 2016) and humans (Guillet et al., 2004; Markofski et al., 2015) show that mTOR activity is increased in aged muscle and that treatments that inhibit mTOR or slow protein synthesis over the long-term, such as rapalogs (Joseph et al., 2019) and protein or branch chain amino acid restriction (Richardson et al., 2021), improve muscle function with age. Instead of basing interventions and future studies on the premise that protein synthesis uniformly decreases with age, an approach that focuses on proteostatic mechanisms that slow muscle ageing could open new possibilities for interventions. The current overall (but admittedly generalized) framework paints protein synthesis as good and protein breakdown as bad. In other words, it focuses the attention on increasing muscle protein synthesis in aged muscle at the same time as reducing muscle protein breakdown. However, these conclusions are based on a body of literature using approaches that may miss crucial aspects regarding protein turnover, and are also based on the foundation that accumulation of protein mass is the most important target for maintaining muscle function with age. Here, we aim to highlight some methodological considerations to shift the focus of what is important for muscle protein turnover with age. At a minimum, we hope to provide tools that help readers evaluate the literature with a critical eye. Ideally, we hope that the field will adapt some non-traditional isotope approaches to better direct therapeutic efforts. The loss of skeletal muscle function often precedes and exceeds loss of mass, indicating that protein quality is as important as protein quantity. Although muscle mass, and hence protein mass, has an impact on overall function, it is equally important to have the correct proteins for cellular tasks and for those proteins to be assembled properly and function well. The matching of well-functioning proteins to the demands of the cell is referred to as protein homeostasis, or proteostasis(definition). Proteostasis is a self-regulating process through which systems maintain protein functionality at the same time as adjusting to changing conditions. As such, maintaining proteostasis in one cell type may not be the same as in another cell type, or even at different times within a cell type. The dynamic mechanisms through which proteostasis is maintained is a network of complex interrelated cellular activities such as protein biogenesis, folding, transport and degradation that collectively determine proteome structure and function (Balch et al., 2008). This dynamic feature of proteostasis is important when assessing mechanisms because the proteome continually adjusts to the current environment. When these mechanisms fail, there is a loss of proteostasis. The mechanisms that maintain proteostasis become dysregulated with advancing age (Hipp et al., 2019; Taylor & Dillin, 2011). The accumulation of dysfunctional proteins compromises cellular function and responsiveness to cellular stresses leading to age-related deterioration, and more than 50 diseases (Walker & LeVine, 2012). Conversely, the ability to maintain proteostasis slows the ageing process (Balch et al., 2008; Jayaraj et al., 2020; Pride et al., 2015). It is well established in a variety of tissues, including skeletal muscle, that mechanisms of proteostatic quality control fail with advancing age such that decreased proteostasis is a hallmark of ageing (López-Otín et al., 2013). One well described feature of proteostatic decline is the accumulation of protein aggregates that are resistant to turnover. An additional consequence of a decline in proteostasis is increased energetic costs of maintaining the proteome. A decline of protein function in organelles such as the mitochondria compounded by increased energetic costs of maintaining the proteome can result in an increasing strain on the cell to maintain proteostasis. It is important first to distinguish proteostasis