Unbiased proteomics, histochemistry, and mitochondrial DNA copy number reveal better mitochondrial health in muscle of high-functioning octogenarians
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Curated by eLife
Evaluation Summary:
This study analyzed muscle protein differences between octogenarian master athletes and non-athletes. The data showed that high physical function in octogenarians was associated with the increased mitochondrial proteome, reduced number of muscle fibers impaired by oxphos, and higher mtDNA copy number. The authors propose that this is one of the mechanisms contributing to better performance in master athletes compared with non-athletes, suggesting that mitochondrial health in skeletal muscle is a key feature in inducing improved physical function in the elderly. This article has the potential to generate a significant impact within the field and will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.
Methods:
We performed muscle biopsies in 15 octogenarian world-class track and field MAs and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mitochondrial DNA (mtDNA) copy number, and proteomics by liquid-chromatography mass spectrometry.
Results:
MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage-dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and that are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA.
Conclusions:
We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with underrepresentation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging.
Funding:
Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, USA.
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Author Response
Reviewer #1 (Public Review):
Masters athletes are viewed as a useful model to study the effects of human ageing that can be somewhat disassociated from the combined effects of increased inactivity, and the current study provides data on specific differences in the muscle proteome compared to those less active older people. Notably, the MA were successfully competing at a high level and are of an age were neuromuscular decrements would expected to be most severe (80yrs). The authors have employed a range of methods of which the most prominent is proteomic analyses of muscle biopsies, and although in a subset of participants only, this should not be considered a small study. Primary outcomes reveal a range of proteins which are differentially observed in MA, a large portion of which relate to mitochondrial function. …
Author Response
Reviewer #1 (Public Review):
Masters athletes are viewed as a useful model to study the effects of human ageing that can be somewhat disassociated from the combined effects of increased inactivity, and the current study provides data on specific differences in the muscle proteome compared to those less active older people. Notably, the MA were successfully competing at a high level and are of an age were neuromuscular decrements would expected to be most severe (80yrs). The authors have employed a range of methods of which the most prominent is proteomic analyses of muscle biopsies, and although in a subset of participants only, this should not be considered a small study. Primary outcomes reveal a range of proteins which are differentially observed in MA, a large portion of which relate to mitochondrial function. These findings are further underpinned to a certain extent by histochemically assessed muscle fibre sections and mitochondrial DNA copy numbers. New insights into an extremely rare cohort are provided which are highly relevant to an ageing human population.
Mass spectrometry analyses employing tandem mass tagging is a robust method to study the human muscle proteome and the methodological description and supplementary data represent a significant body of analysis. Confidence in these outcomes are also further unpinned by previous work from members of the group, again relating to human muscle proteomics and ageing. Specific proteins relating to nuclear pore complex and spliceosome activity reported, for the first time in aged human athletes. There are, however, a number of points that require greater clarification and/or discussion.
An association of enhanced mitochondrial function in highly exercised individuals, which is greater than those less active, is not overly surprising. Nevertheless, additional analyses within the MA may further reveal the potential role of over represented proteins relevant to mitochondrial function and individual performance, such as VO2MAX and peak cycle workrate. Of particular interest here is the training and competition history of the MA which appears to be fairly short, and the majority would have been considered as aged/old prior to competing in their respective disciplines. As non lifelong exercisers the implication here is they have reversed mito decrements normally observed with ageing (alluded to throughout), or, the MA were predisposed to higher physical function prior to engaging in competition. The limitations of cross-sectional design commonly preclude such insights, but this point does deserve further discussion.
This issue is further addressed in reference to the comment made about Line 558 below. Briefly, the athletes would have been between 55 y (Endurance Athletes) and 65 y (Sprint/Power Athletes) at the start of their competitive careers as Masters Athletes (see Table 2). In addressing the potential for reversing aging-related mitochondrial impairment through the training done by MA, it could be that the mitochondrial adaptations to training overshot the mild mitochondrial impairment due to aging when training was initiated and that this built a “buffer” which contributed to their higher mitochondrial protein levels in advanced age. To address this idea, we now add text on lines 518-528 of the Discussion, as follows:
“Although we can only speculate on this point, one contributing factor to the higher abundance of mitochondrial proteins in MA may relate to mitochondrial adaptations incurred at the initiation of training in the MA group. Noting that the athletes in our MA group started training between 55 y of age (Endurance athletes) and 65 y (Sprint/Power athletes) (see Table 2), the nature of the mitochondrial adaptations were likely in excess of the mild age-related impairment that would have been present at the age training was initiated. Thus, perhaps this training built in a ‘buffer’, such that even similar rates of age-related decrements in mitochondrial proteins between both MA and NA would still yield the higher levels of mitochondrial proteins that we observed in MA versus NA at the participants’ age when the muscle was sampled. Unfortunately, the cross-sectional nature of our study limits conclusions regarding this and other possibilities.”
We hope this addresses your point.
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Evaluation Summary:
This study analyzed muscle protein differences between octogenarian master athletes and non-athletes. The data showed that high physical function in octogenarians was associated with the increased mitochondrial proteome, reduced number of muscle fibers impaired by oxphos, and higher mtDNA copy number. The authors propose that this is one of the mechanisms contributing to better performance in master athletes compared with non-athletes, suggesting that mitochondrial health in skeletal muscle is a key feature in inducing improved physical function in the elderly. This article has the potential to generate a significant impact within the field and will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
This study analyzed muscle protein differences between octogenarian master athletes and non-athletes. The data showed that high physical function in octogenarians was associated with the increased mitochondrial proteome, reduced number of muscle fibers impaired by oxphos, and higher mtDNA copy number. The authors propose that this is one of the mechanisms contributing to better performance in master athletes compared with non-athletes, suggesting that mitochondrial health in skeletal muscle is a key feature in inducing improved physical function in the elderly. This article has the potential to generate a significant impact within the field and will be of interest to a broad audience.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Masters athletes are viewed as a useful model to study the effects of human ageing that can be somewhat disassociated from the combined effects of increased inactivity, and the current study provides data on specific differences in the muscle proteome compared to those less active older people. Notably, the MA were successfully competing at a high level and are of an age were neuromuscular decrements would expected to be most severe (80yrs). The authors have employed a range of methods of which the most prominent is proteomic analyses of muscle biopsies, and although in a subset of participants only, this should not be considered a small study. Primary outcomes reveal a range of proteins which are differentially observed in MA, a large portion of which relate to mitochondrial function. These findings are …
Reviewer #1 (Public Review):
Masters athletes are viewed as a useful model to study the effects of human ageing that can be somewhat disassociated from the combined effects of increased inactivity, and the current study provides data on specific differences in the muscle proteome compared to those less active older people. Notably, the MA were successfully competing at a high level and are of an age were neuromuscular decrements would expected to be most severe (80yrs). The authors have employed a range of methods of which the most prominent is proteomic analyses of muscle biopsies, and although in a subset of participants only, this should not be considered a small study. Primary outcomes reveal a range of proteins which are differentially observed in MA, a large portion of which relate to mitochondrial function. These findings are further underpinned to a certain extent by histochemically assessed muscle fibre sections and mitochondrial DNA copy numbers. New insights into an extremely rare cohort are provided which are highly relevant to an ageing human population.
Mass spectrometry analyses employing tandem mass tagging is a robust method to study the human muscle proteome and the methodological description and supplementary data represent a significant body of analysis. Confidence in these outcomes are also further unpinned by previous work from members of the group, again relating to human muscle proteomics and ageing. Specific proteins relating to nuclear pore complex and spliceosome activity reported, for the first time in aged human athletes. There are, however, a number of points that require greater clarification and/or discussion.
An association of enhanced mitochondrial function in highly exercised individuals, which is greater than those less active, is not overly surprising. Nevertheless, additional analyses within the MA may further reveal the potential role of over represented proteins relevant to mitochondrial function and individual performance, such as VO2MAX and peak cycle workrate. Of particular interest here is the training and competition history of the MA which appears to be fairly short, and the majority would have been considered as aged/old prior to competing in their respective disciplines. As non lifelong exercisers the implication here is they have reversed mito decrements normally observed with ageing (alluded to throughout), or, the MA were predisposed to higher physical function prior to engaging in competition. The limitations of cross-sectional design commonly preclude such insights, but this point does deserve further discussion.
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Reviewer #2 (Public Review):
The study authors conclude that preservation of mitochondrial structure/function proteins underlies the better performance of octogenarian master athletes than non-athletes. I do concur with the authors that this most likely is an important factor that contributes to the exceptional performance of the master athletes. One thing to consider is perhaps that these things may be just normal training adaptations that can even be elicited in octogenarians who start training. In fact, there is ample evidence that even when one starts training later in life (see for instance Hepple et al, JAP, 1997) still gains in maximal oxygen uptake and muscle mass/strength can be achieved. The point is therefore that perhaps these are not so much signs of 'preservation' but rather 'training-induced' adaptations. It would be …
Reviewer #2 (Public Review):
The study authors conclude that preservation of mitochondrial structure/function proteins underlies the better performance of octogenarian master athletes than non-athletes. I do concur with the authors that this most likely is an important factor that contributes to the exceptional performance of the master athletes. One thing to consider is perhaps that these things may be just normal training adaptations that can even be elicited in octogenarians who start training. In fact, there is ample evidence that even when one starts training later in life (see for instance Hepple et al, JAP, 1997) still gains in maximal oxygen uptake and muscle mass/strength can be achieved. The point is therefore that perhaps these are not so much signs of 'preservation' but rather 'training-induced' adaptations. It would be interesting to see whether training of non-athlete octogenarians results in the same pattern of structural/functional mitochondrial proteins as seen in the octogenarian master athletes. So, I have some problems with the term 'preservation' as they may just have what is expected after training.
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