Use of a Mitochondrially Targeted Antioxidant Fails to Reduce Sarcopenia

Regular readers will be at least passingly familiar with the commercial development of mitochondrially targeted antioxidant compounds that has taken place over the past decade. This initially attracted attention in this community because the evidence suggests that these molecules can modestly slow the progression of aging, though unfortunately not to the same degree as interventions like calorie restriction, which to my eyes at least means that serious life extension efforts are better directed elsewhere. The furthest advanced of these mitochondrially targeted antioxidants is the SkQ series of plastinquinones, currently being developed as a treatment for a few different conditions by Mitotech. It turned out that the effects on inflammatory eye conditions were considerably larger than the effects on aging, so that is the direction presently taken. Another of the compounds under development by a different set of researchers is SS-31; you'll find an introduction in the Fight Aging! archives. In the paper noted below, SS-31 is used to demonstrate that reducing oxidative stress in muscle cells doesn't slow the age-related loss of muscle mass and strength known as sarcopenia. This is a nice piece of work that might help focus future research efforts in more productive directions.

Given that straightforward antioxidants of the sort purchased in a supplement store either do nothing for health and aging or actually somewhat harm long term health, why would be expect antioxidants targeted to the mitochondria within the cell to be a different proposition? It helps to start by noting that the roles of oxidative molecules and antioxidants in the cell are manifold and complicated. Oxidative molecules cause damage by reacting with important protein machinery, but they are also used as signals. Too much is bad, and too little is bad. Exactly where there is too much or too little is also of great importance. Globally suppressing oxidative signaling via high levels of ingested antioxidants has negative effects like blocking the benefits of exercise, which depend upon that signaling. Mitochondria are central to all discussions of oxidative signaling, as they generate oxidative molecules as a result of their primary role as power plants, supplying energy store molecules to power the cell. Many of the genetic alterations and other interventions that modestly slow aging in laboratory species change mitochondrial function, either increasing or reducing the flux of oxidative molecules.

There are several likely ways in which altered mitochondrial output of oxidative molecules can affect long term health, that alteration achieved either by changing the rate at which such molecules are created, or by applying targeted antioxidants that soak them up immediately. Firstly there is hormesis: a slightly higher than usual output and the resulting damage can trigger greater and lasting repair and maintenance activities in the cell, leading to a net benefit. Or, alternatively, a lower level of output of oxidative molecules might just lead directly to less damage. Further, there are many other aspects of cellular metabolism that might run in ways better suited to a slower pace of aging if mitochondria generate either more or less oxidative molecules; that is poorly mapped and highly dependent on species, tissue, and circumstances. Lastly there is the important matter of mitochondrial DNA damage in aging. Mitochondrial dysfunction appears to be an important contribution to aging, and it is likely driven by mutational damage to DNA in the mitochondria caused by their own generation of oxidants. This DNA damage can produce mitochondria that are both dysfunctional and resistant to quality control. They quickly overtake a cell, and over the course of a lifetime ever more cells fall into this state. Their behavior contributes to degenerative aging in a range of ways, starting with the export of much larger amounts of oxidizing molecules into the surrounding tissues.

Thus rising levels of oxidative stress are considered important in many aspects of the progression of aging, but it is far from the only type of change, damage, and dysfunction taking place. So it shouldn't be a complete surprise to find that some conditions are not affected in the slightest by adjusting mitochondrial oxidant output, even when the indications suggested that outcome to be plausible enough to try. Metabolism is a ferociously complex business, incompletely understood, which is why bypassing its alteration in favor of identifying and fixing fundamental forms of damage should be a much more efficient approach to the treatment of aging and age-related conditions. We have a metabolism that works well when young, even it isn't fully understood, and researchers have a list of the fundamental, first cause differences between young and old tissues, so the goal should be to revert those differences and maintain the working system, rather than try to adjust it.

Mitochondrial ROS regulate oxidative damage and mitophagy but not age-related muscle fiber atrophy

Skeletal muscle is a major site of metabolic activity and is the most abundant tissue in the human body. Age-related muscle atrophy (sarcopenia) and weakness, characterized both by loss of lean muscle mass and reduced skeletal muscle function, is a major contributor to frailty and loss of independence in older people. Studies of humans indicate that by the age of 70, there is a ~25-30% reduction in the cross sectional area (CSA) of skeletal muscle and a decline in muscle strength by ~30-40%. Age-dependent loss of muscle mass and function has a complex aetiology and the primary biochemical and molecular mechanisms underlying this process have not been fully determined.

Oxidative stress has been suggested to be a key factor contributing to the initiation and progression of the muscle atrophy that occurs during aging. Consistent with a role of oxidative stress as a contributor to sarcopenia, studies have shown that genetic manipulations of redox regulatory systems can alter the aging process in muscle. Skeletal muscle decline with advancing age has been linked to an altered oxidative status of redox-responsive proteins and a number of studies have indicated a positive correlation between tissue concentration of oxidized macromolecules and life span including an increase in DNA damage, accumulation of oxidized proteins and increased levels of lipid peroxidation with age. In support of these findings recent quantitative proteomic approaches have further provided evidence that muscle aging is associated with a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, implying age-related redox changes as an underlying cause of age-related muscle atrophy.

Skeletal muscle produces reactive oxygen and nitrogen species (RONS) from a variety of subcellular sites and there is evidence that isolated skeletal muscle mitochondria exhibit an age-related increase in hydrogen peroxide (H2O2) production. Furthermore, muscle aging is associated with reduced mitochondrial oxidative-phosphorylation, reduced mitochondrial DNA (mtDNA) content, accumulation of mutated mtDNA, impaired mitophagy and increased mitochondrial permeability transition pore sensitivity, which are all proposed to contribute to the sarcopenic phenotype. Although cumulative oxidative stress has been proposed to induce age-associated reductions in mitochondrial function, this remains a controversial topic.

We and others have recently reported that pharmacological application of the mitochondria-targeted SS31 tetrapeptide can attenuate mitochondrial superoxide production in intact mitochondria of skeletal muscle fibers. This pharmacological approach complements genetic approaches, including those using targeted overexpression of the human catalase gene to mouse mitochondria. Such pharamacological agents may have substantial translational implications for the use and/or development of mitochondria-targeted antioxidants for treatment of human mitochondrial myopathies as well as mitochondrial reactive oxygen species (mtROS) mediated muscular dysfunctions. The purpose of the present study was to determine the effect of the mitochondria-targeted SS31 peptide on redox homeostasis in muscles of old mice, including mtROS and oxidative damage, mitochondrial content and mitophagy and on age-related muscle atrophy and weakness. Through this approach we aimed to determine the role of modified mitochondrial redox homeostasis on age-related loss of muscle mass and function.

Our findings demonstrated that a reduction in mtROS in response to SS31 treatment prevented age-related mitochondrial oxidative damage and improved mitophagic potential, but further demonstrated that changes in mitochondrial redox environment towards a more reduced state failed to rescue the sarcopenic phenotype associated with muscle fiber atrophy and loss of muscle mass and strength. This work has therefore identified that the age-related changes in mitochondrial redox potential play a key role in the loss of mitochondrial organelle integrity that occurs with aging, but are not involved in the processes of age-related muscle fiber atrophy.

Comments

My experience is that mitoq does significantly reduce delayed onset muscle soreness after exercise. However, I'm not sure this is a good thing in the long term because it could mitigate the training effect from hormetic stress.

Posted by: Chris at October 1st, 2016 6:20 AM

This is a weird result, given the SENS RF hypothesis that mutant mitochondria quickly clonally expand to take over the area of a muscle fiber near a nucleus, which then results in the death of the fiber. Surely a less oxidative environment in the mitochondria would lead to a lower chance of gene deletion through oxidative damage, and a slower rate of loss of muscle fibers?

Posted by: Jim at October 2nd, 2016 2:12 AM

There was a recent paper I read which pointed at replication error as the most probable cause for mtDNA mutations rather than ROS.

http://nar.oxfordjournals.org/content/early/2016/08/17/nar.gkw716.full.pdf+html

"Byproducts of mitochondrial metabolism, reactive
oxygen species are recurrently associated with organismal
aging and mtDNA mutagenesis (4,6,83,84). Recent ap-
praisals of the mitochondrial mutation spectrum in aging
and in models of attenuated oxidative damage repair, al-
though, have concluded that oxidative damage imparts min-
imal contributions to mtDNA mutation frequency (
85,86)."

Posted by: Anonymoose at October 2nd, 2016 2:26 AM

This is a very strange result; how do you square it with the positive result in the treatment of rats with SkQ1?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3969282/

This study clearly showed benefits for both OXYS and Wistar rats in terms of preserved volume of skeletal muscle.

Posted by: Mark at October 3rd, 2016 3:38 AM

C60 decreased my DONS considerably but had to be discontinued due to the toxic restproduct concern.

Anyway I doubt Mito stuff will be enough to prevent something like Sarcopenia. I always assumed a true fix for Sarco to include at least two or three fundamental damage types.

MitoQ is what I'm using now thanks to FA for the original tip. I think it makes me a lot more actionable. Waiting for SkQ1 and obviously waiting for true rejuvenation medicine.

Posted by: Arren Brandt at October 3rd, 2016 3:42 PM

So, a few things about this new study.

First, and most importantly: to be effective in reducing those aspects of sarcopenia driven by mitochondrial superoxide (O2-) generation, SS-31 would have to be able to quench enough O2- or other mtROS in situ within the mitochondria in order to substantially reduce the initiation of large deletions in mtDNA. It is the clonal expansion of such deletions to take over a postmitotic cell (or, in the case of skeletal muscle, a muscle fiber segment) that have been shown by Judd Aiken to drive age-related muscle fiber breakage.

This study hasn't really shown any of that. First and foremost, they haven't even measured SS-31's effect on the accumulation of mtDNA deletions, which is the key outcome you would want from interdicting mt O2-.

And to the extent that they've shown a reduction in mt O2-, they've only shown that it "attenuated the age-related change in mtROS ... in myofibers of 28 mo old mice to comparable levels to those of fibres of adult [10-11 mo old] mice" - that is, mice in early middle age, similar to 38- to 41-year-old humans. But of course, from birth until treatment began at age 24 months, the mitochondria in the muscle fibers of these mice had been producing mtROS at levels typical for their age, and accumulating mtDNA deletions accordingly.

Indeed, much of the damage had already been done at this point: by 18-19 months of age (early seniority, similar to a 56- to 60-year-old human), these mice had already suffered a 37% loss of muscle fibers in their shin muscle as compared with young mice, and even the oldest tested mice "only" suffered a further 6% loss (from 37% at 18-19 mo to 43% at 28 mo). So by the time SS-31 was administered at age 24 mo, there wasn't really much further loss remaining for them to prevent, even if they had a highly effective intervention against mt O2-.

And while restoring the "excessive" production of O2- in these old mice to levels similar to middle-aged mice is surely better than doing nothing, it still leaves them generating new mtDNA deletions at the same "normal" rate that middle-aged mice do.

It bears mentioning, too, that (despite what is widely believed) most studies report that the age-related increase in mtROS production is actually very modest, if it happens at all. So restoring the older mice's mtROS production to levels typical of middle-aged mice is not really even a very large effect. We don't actually know quantitatively how large it was in this study, because they simply didn't report any data in adult (and more importantly young adult) mice: they do state in the text that SS-31 "attenuated the age-related change in mtROS ... in myofibers of 28 mo old mice to comparable levels to those of fibres of adult [10-11 mo old] mice," but they don't provide any actual data for the latter group: they only provide a graph (Figure 3(b)) showing that treatment with SS-31 reduced O2- levels in the older treated animals as compared to untreated animals of the same age, without actually revealing the level in middle-aged and (more importantly) young adult animals. From data reported in other studies, this difference is likely very modest.

By contrast, what makes Calorie restriction (CR) protective against mtDNA deletion accumulation and ensuing muscle fiber breakage and loss - and thus to attenuate the age-related loss of muscle-specific force - is not that it simply holds the line of mtROS production at levels typical of middle-aged mice. Rather, CR actively forces down mtROS production to "abnormally" low levels. In muscle, in particular, 40% CR typically pushes mtROS generation down to levels ≈30-40% below those of age-matched ad libitum-fed mice or rats, even in young animals, and in one study did so by as much as 74%.

Additionally, CR works by reducing the generation of O2- in the first place, rather than quenching it post facto as SS-31 does. We can't be sure that SS-31 is "catching" O2- in a location and at a time that will effectively prevent it from hitting the mtDNA (rather than at some other, unprotective location within the mt where it is unlikely to come into contact with genetic material), but we can be sure that, by definition, mtROS that are simply never produced in the first place have zero chance of doing damage to mtDNA.

The profound reduction in the rate of mtROS production that is enforced by CR (again, reducing it to levels well below those seen even in healthy young ad libitum-fed mice or rats) can continue to slow the rate of accumulation of cells overtaken by deletion-bearing mitochondria and of muscle fiber loss at relatively advanced ages. For instance, in this study, rats switched to severe CR at age 17 mo suffered 14-38% fewer mtDNA deletions in different muscle groups going forward, and lost 25% fewer muscle fibers in the main quadriceps muscle, by age 30-32 months (similar to an 80-year-old human).

And CR still has some effect on muscle loss even later in life than the age of initiation of SS-31 therapy in this study: in this study, CR initiated at age 27 months allowed mice to retain 5-10% more mass of different muscles by just a month and a half later, and more if you adjusted this for the CR animals' lighter weights.

Now, if SS-31 had really managed to interdict a very large percentage of mtROS (from available data, it did not), and if it were to do so at a time and location suitable to have a significant protective effect against mtDNA deletions (and again, we just don't have any data on this) - under those conditions, SS-31 might be expected (like CR) to reduce the rate of mtDNA deletion accumulation in such segments. Even then, its effects would be limited because it would not be expected to do anything to retard the spread of such mitochondria throughout the muscle fiber, since CR itself does not.

Aaren is also correct in his hunch that many different kinds of damage are involved in sarcopenia, and not just mtDNA deletions and ensuing fiber breakage, so "a true fix for Sarco" would have to repair or replace all of these fundamental damage types. This is actually a confounder to CR's effects on mtDNA deletions in muscle: as the Wikipedia page notes, CR's anti-sarcopenic "Mechanisms include reduced muscle cell apoptosis and inflammation; protection against or adaptation to age-related mitochondrial abnormalities; and preserved muscle stem cell function."

And, of course, even this is ultimately not adequate. Merely retarding the rate of accumulation of all of this damage slows down the rate of age-related decline, but does not prevent it entirely - and as is evidenced by the studies quoted, becomes progressively less effective as therapies are intiated at later and later ages, as it cannot undo the damage that aging has already inflicted in their muscles and across all their organs and systems. To truly deal with the ill-health, misery, frailty, and death inflicted by the degenerative aging process, we need a comprehensive panel of rejuvenation biotechnologies that will actually remove, repair, replace, or render harmless the damage that has already been inflicted when therapy begins, and that will not itself make the organism vulnerable in other ways (like the ironic non-age-related loss of muscle mass imposed by CR itself).

Again, Sakellariou et al didn't measure the effect of SS-31 on mtDNA deletions (or muscle fiber segments showing histological signs of having been taken over by deletion-bearing mt) for SS-31, but for reasons aforesaid you wouldn't necessarily expect that it would have had much of a protective effect, and therefore it's not really all that surprising that it didn't have any effect on muscle fiber loss.

Re: the SkQ1 study: if you read the abstract carefully (or, better, the full text), you'll see although the investigators do claim that SkQ1 treatment did ameliorate some of the abnormalities in mitochondrial morphology in aging normal (Wistar) rats, they actually don't claim that it had any effect on sarcopenia. They did find that it prevented some sarcopenia-like changes OXYS rats, but these are very, very screwed-up animals even before aging gets ahold of them: developed by inbreeding brother-sister pairs and selecting for those offspring that were most susceptible to forming cataracts when fed a galactose-rich diet, subsequent studies revealed that they suffer from abnormally high oxidative stress, hypertension, accelerated thymic involution, and early-onset osteoporosis, cognitive dysfunction and retinopathy.

As part of this, the mitochondria of OXYS mice were already abnormal in young (3 month old) animals, and not just in ways that simulate what occurs in otherwise-normal aging rats but in ways that are abnormal at *any* age. Unsurprisingly, this is not good for a mouse. And also unsurprisingly, when you make an animal abnormally susceptible to oxidative stress, giving it an antioxidant makes it healthier - but still not as healthy even as otherwise-normal but aging mice.

Similarly, it's worth noting that SkQ1 has never been shown to extend the life of genetically normal, healthy, well-cared-for, non-toxin-administered animals of any species. All of the controls animals in experiments showing beneficial effects were significantly (and in some cases miserably) short-lived for their species, for reasons either of strain or husbandry, and SkQ1 partly normalized those abnormally-shortened life expectancies (and in a couple of cases didn't even do this).

When reading about results of putative aging interventions, one must always bear in mind Michael Rose's fine maxim:

"Until you show me that you can postpone aging, I don't know that you've done anything," sniffs Michael R. Rose, geneticist at the University of California. "A lot of people can kill things off sooner, by screwing around with various mechanisms, but to me that's like killing mice with hammers - it doesn't show that hammers are related to aging."

Anonymoose: I suggest that you should re-read the section you quoted in its original context: from the material immediately preceding and especially that following the material you quoted, it seems clear that the authors don't find that line of argument convincing, and are instead generally supportive of the view of mitochondrially-generated ROS as a driver of age-related mutations in mitochondria, although calling for more studies to verify it:

the majority of mutations induced in mtDNA are likely consequences of endogenous sources of error (78). Indeed, the most reliable models of increased mtDNA mutation frequency employ functional mutants of pol γ ... Curiously, the mutation spectrum of mtDNA in mutator mice is inconsistent with the expected spectra of pol γ misincorporation on undamaged template DNA (46,81), and expression of a mitochondrial-targeted human catalase in these mice, which reduces the ROS hydrogen peroxide, also reduced their mutation frequency (46). Thus ... naturally-occurring mitochondrial ROS may contribute to the elevated spontaneous mutation frequency of the mitochondrial genome (60).

Byproducts of mitochondrial metabolism, reactive oxygen species are recurrently associated with organismal aging and mtDNA mutagenesis (4,6,83,84). Recent appraisals of the mitochondrial mutation spectrum in aging and in models of attenuated oxidative damage repair, although, have concluded that oxidative damage imparts minimal contributions to mtDNA mutation frequency (85,86). Importantly, these assertions rely upon a narrowly-defined, unverified consensus signature of oxidative damage and induced mutagenesis in mitochondria. The lesions generated by reactive oxygen species range in severity from the subtle, 8-oxo-dG, to the obvious, strand breaks (87); consequently, the imputed mutation 'signature' of oxidative DNA damage has developed as the amalgam of results derived from mutagenesis studies using defined lesions, often pursued in vitro, and not necessarily in the context of the mitochondrial replisome (88-92). Given the varied lesions formed by oxidative DNA damage (87), a direct assessment of mutation frequency and spectrum in mtDNA following oxidative damage is warranted

Additionally, of the two studies they cite as concluding "that oxidative damage imparts minimal contributions to mtDNA mutation," neither actually looks at their contribution to large mtDNA deletions, which are the specific mutation that actually accumulates (rather than simply occurring sporadically) in the mitochondria of aging cells; the Drosophila paper is explicit about this:

further work will be required to determine whether superoxide contributes to the frequency of mtDNA deletions, and to assess the influence of other forms of ROS, such as hydroxyl radicals, on mtDNA mutation frequency.

The human study just doesn't go into the question, being focused on point mutations. And in any case, fruit flies are a very poor model of mammalian aging in any case and studies conducted therein should generally be ignored until replicated in mammals.

Posted by: Michael at October 5th, 2016 6:41 PM

Great answer, thanks Michael. I admit I wasn't sure when reading the SKQ1 report whether any improvement in Sarco was actually reported, or whether it was simply a case on mitochondrial improvement - but as you state, probably not enough to prevent MtDNA deletions altogether, which is what counts.

So would any amount of Mito targeted antioxidants be able to do the job, even to slow down the accumulation of deletions?

Posted by: Mark at October 6th, 2016 7:04 AM

Posted by: Mark at October 6th, 2016 7:04 AM: Great answer, thanks Michael. I admit I wasn't sure when reading the SKQ1 report whether any improvement in Sarco was actually reported, or whether it was simply a case on mitochondrial improvement - but as you state, probably not enough to prevent MtDNA deletions altogether, which is what counts.

As with the SS-31 study, there are no data on mtDNA deletions in the SKQ1 paper, so we actually don't know whether it had any protective effect on mtDNA deletions, let alone preventing them altogether. They showed that SkQ1 normalized some of the age-related changes in mitochondrial morphology in the normal (Wistar) rats, as well as some of the additional abnormalities in the OXYS rats.

Posted by: Mark at October 6th, 2016 7:04 AM: So would any amount of Mito targeted antioxidants be able to do the job, even to slow down the accumulation of deletions?

It would depend, first, on whether such agents actually acted as antioxidants in situ: with the exception of spin traps like PBN, antioxidants almost never truly "quench" a free radical, but rather donate an electron to complete the unpaired valence electron in the reactive species and then themselves adopt a pro-oxidant form (such as conversion of GSH and NAC into thiyl radicals, vitamin E to tocoperoxyl radical, beta-carotene to beta-apo-carotenals, etc). What makes a given molecule an "antioxidant" in meaningful terms is that its radicalized form is less toxic than the original radical species em>in the biological context in which it occurs, which is not always straightforward: perverse examples may include beta-carotene in the lung in the presence of tobacco smoke and tocopherol-mediated peroxidation in atherosclerosis.

The other question is, again, where exactly a mitochondrially-targeted antioxidant localizes inside the mt. To interdict mtROS in time to prevent them from hitting the mtDNA, a molecule of a mt-targeted antioxidant would have to be localized between the electron transport chain in the mitochondrial inner membrane and the mtDNA loop in the matrix. This could occur either if it were present in the matrix itself, or at a well-placed location inside the electron transport chain where mt O2- is generated. If an mt-targeted antioxidant never penetrates the matrix, or rapidly diffuses out into the intermembrane space, or is not close to any of the "hotspots" of superoxide prodiction in the ETS, it can't be expected to have any effect on mtDNA deletions because it won't be there to catch it.

The report on which this blog post is based says that "SS-31 has previously been shown to reduce mitochondrial matrix superoxide in intact mitochondria of single isolated skeletal muscle fibers and C2C12 myotubes," but it's not clear to me that either of the cited papers show that. In fact, SS-31 is positively charged, and thus not delivered into the mitochondrial matrix, so such a straightforward role in O2- interdiction is not possible for it.

On the other hand, SS-31 has been shown to selectively target the inner mitochondrial membrane, binding to cardiolipin and facilitating the interaction of different components of the electron transport chain, and it has been hypothesized to "promot[e] electron transfer at the rate-limiting step of the ETC, where cytc must transfer electrons efficiently from complex III to complex IV in order to prevent electron leak at complex III ... [and] modulat[e] the interaction between cardiolipin and the heme iron of cytc ... These peptides also inhibit cytc peroxidase activity by protecting the reactive heme iron from H2O2, thus preventing cardiolipin peroxidation and destruction of cristae membranes." Any of this might reduce mtDNA damage in a less obvious way, but again, we just don't have any data on this point.

Similarly, SkQ1 has been shown to bind to cardiolipin and protect it from oxidation by hydroxyl radicals (which might explain its effects on mitochondrial cristae and enlargement in the Wistar rat study above), and has been calculated from first principles to also accumulate in the matrix - but the latter has not been empirically shown, and again, as there is no evidence that in any of these roles it prevents accumulation of mtDNA deletions with age.

And then, as with all putative antioxidants, there is the question of the potential off-target biological effects of the molecule beyond its role in detoxifying free radicals, which might moot the question of whether it prevents mtDNA deletions or not. For instance, although promoted as a neuroprotective mitochondrially-targeted antioxidant, in vitro studies examining its mechanism of action later suggested that Idebenone actually enhances the production of superoxide, and it has a range of complex non-antioxidant effects as well. Although never found to be neurotoxic (as you would expect if it really did increase mt O2-), it never lived up to its neuroprotective hype, either.

What, where, when, and how, a "mitochondrially-targeted antioxidant" actually behaves in vivo then, are all key to whether it will prevent mtDNA deletions from occurring (and to what degree) - and whether that benefit results in real value in slowing aging.

And, whatever the answers to such questions, we can't expect miracles out of a small molecule, or even as much of a benefit as rodents get from CR: if we are going to really take on aging, we need new therapies that repair the damage of aging.

Posted by: Michael at October 6th, 2016 2:15 PM

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