Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P17 docx

This notion is consistent with the aforementioned increase in mitochondrial ROS generation in aged skeletal muscles Section 2.2, and observations indicating greater accumulation of non-h

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burdens of mtDNA damage at the whole muscle level and very much higher fractions

of muscle fibers exhibiting complex IV enzyme activity deficiency, and yet in these patients neither individual muscle fibers lacking complex IV activity (Fig 6) nor their muscles as a whole are grossly atrophied relative to healthy individuals of the same age (Jacobs 2003) As such, the degree to which this phenomenon might contribute to sarcopenia remains an important area of investigation

As suggested above, one specific manner in which mitochondria are proposed to

be involved in sarcopenia involves apoptosis (Pollack and Leeuwenburgh 2001; Chabi et al 2008; Seo et al 2008) Mitochondria play a key role in regulating apoptosis, via the mitochondrial permeability transition pore (mPTP) which regu-lates the release of cytochrome c into the cytoplasm A variety of stimuli, such as high Ca2+ and high ROS exposure, can lead to opening of the mPTP, allowing cyto-chrome c to leak out of the mitochondria and into the cytoplasm Once released into the cytoplasm, cytochrome c binds with Apaf-1 and caspase 9, leading to the formation of an apoptosome, activation of caspase 9 and subsequent commitment

of the apoptotic pathway via activation of caspase 3 In support of a role for apoptosis in age-related muscle atrophy, many studies have reported an increase in pro-apoptotic signaling in aged muscles (Alway et al 2002; Dirks and Leeuwenburgh 2002; Giresi et al 2005; Baker and Hepple 2006; Rice and Blough 2006; Chabi

et al 2008) On the other hand, differences in the degree of muscle atrophy between

Fig 6 Succinate dehydrogenase and complex IV doubly-stained cross-section of muscle from a patient with heteroplasmic mtDNA mutation Note that the complex IV deficient fibers (blue fibers) are no different in size than fibers with normal complex IV activity (brown-orange fibers) (Reproduced from Taivassalo and Haller [2005], with permission from The American College of Sports Medicine)

individuals in senescent animals do not track well with differences in expression of apoptotic transcripts (Baker and Hepple 2006) In addition, although the pro-gression of muscle atrophy with aging correlates generally with an increase in number of apoptotic nuclei in both fast-twitch and slow-twitch muscles, it is strik-ing that there are markedly more apoptotic nuclei in the slow-twitch soleus muscle than the fast-twitch extensor digitorum longus muscle, despite very similar amounts

of atrophy (Fig 7; data taken from (Rice and Blough 2006)) This difference may relate to the fact that muscle fibers are multi-nucleated and, therefore, apoptotic loss of a nucleus within a given myocyte does not need to result in loss of the myo-cyte entirely As such, a difference in the incidence of apoptotic nuclei between muscles having the same amount of atrophy could reflect differences in the ability

of these muscles to regenerate and repair, e.g., via recruitment of satellite cells Whether this or another explanation applies awaits further investigation

Notwithstanding some uncertainty about the degree to which apoptosis directly explains the degree of muscle atrophy with aging, recent data suggests that accu-mulation of non-heme iron in skeletal muscle mitochondria may be one mechanism leading to an increased incidence of mitochondrial-mediated apoptosis in aged skeletal muscle Specifically, accumulation of non-heme iron with aging is hypoth-esized to exacerbate mitochondrial ROS generation (and thus oxidative damage) via the Fenton reaction, wherein the increased mitochondrial damage leads to an increased probability of mPTP opening (Seo et al 2008) This notion is consistent with the aforementioned increase in mitochondrial ROS generation in aged skeletal muscles (Section 2.2), and observations indicating greater accumulation of non-heme iron in mitochondria isolated from aged skeletal muscle (Seo et al 2008) In addition, mitochondria from aged muscles exhibit a greater release of cytochrome

Fig 7 Muscle mass in the fast-twitch extensor digitorum longus (EDL) muscle and slow-twitch soleus muscle (Sol) versus the density of TUNEL-positive nuclei (a marker of apoptotic nuclei)

as sarcopenia progresses with aging (Data reproduced from Rice et al [2006])

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c in response to ROS-induced stress (Chabi et al 2008), which may in part explain the increased susceptibility to mitochondrial-driven apopotosis in aging muscle Thus, collectively, there is substantial evidence that apoptosis increases in aged muscles and that age-related changes in mitochondria are likely to be involved

4.2 Involvement of Mitochondria in Age-Related Muscle

Dysfunction

In addition to the potential involvement of mitochondria in the age-related loss of muscle mass, there is considerable support for the involvement of mitochondria in impaired muscle function with aging For example, there is a progressive decline in skeletal muscle aerobic function with aging that is not due to loss of capillaries (Hepple and Vogell 2004; Mathieu-Costello et al 2005), but rather correlates with

a progressive loss of mitochondrial oxidative capacity in aging muscles (Hagen

et al 2004) (Fig 8) As noted in Section 3, a decline in muscle mitochondrial oxi-dative capacity may be caused by a reduction in the expression of PGC-1a in aged muscles (Baker et al 2006; Chabi et al 2008) In this context, it is important to note that aged muscles, particularly in senescence, are characterized by an accumulation

of very small muscle fibers Although this area requires further study, it seems likely that a large proportion of these small fibers are denervated (Hepple et al 2004b) and that a sub-fraction of these may be attempting to regenerate The reason this is relevant here is that these small fibers have lower levels of markers of

Fig 8 Muscle maximal oxygen uptake (VO2max) in pump-perfused rat hindlimb versus the flux capacity of complex I–III in homogenates of gastrocnemius muscle The figure shows that the age-related decline in VO2max parallels the decline in flux capacity through a key part of the mitochondrial electron transport chain (Reproduced from Hagen et al [2004], with permission from The Gerontological Society of America)

mitochondrial content (e.g., complex IV activity) (Fig 9), and because of this they contribute significantly to the lower muscle oxidative capacity Furthermore, den-ervation, or perhaps failure to reinnervate, may be constraining the mitochondrial content of these fibers, secondary to the aforementioned reduction in drive on mito-chondrial biogenesis that occurs in denervated muscle (Adhihetty et al 2007) (Section 3.1) Thus, the reduction of muscle mitochondrial oxidative capacity with aging may have an important neurological involvement This point needs further consideration in the experimental literature

As noted in Section 3.2, aged muscles are also characterized by mitochondria that emit higher levels of ROS This increase in mitochondrial ROS generation in aging skeletal muscles can exacerbate oxidative damage to proteins, which has been shown to inhibit the biological activity of enzymes, particularly those contain-ing iron-sulfur centers (Bota et al 2002; Ma et al 2009) In addition, several pro-teins involved in muscle contraction are known to be specifically targeted by oxidative stress, and thus, likely contribute to the impairment in muscle contractile function with aging Prochniewicz et al (2005) previously showed using in vitro motility assays that although actin function was unaltered with aging, the catalyti-cally active portion of myosin (heavy meromyosin) was impaired in muscles of aged versus young adult rats In addition, this difference in actin versus myosin function with aging corresponded to differences in the susceptibility of actin versus

Fig 9 Senescent rat gastrocnemius muscle cross-section stained for complex IV activity Note that the very small fibers have a lower complex IV activity than the larger fibers, showing that the accumulation of these very small fibers in aged muscle, particularly in senescence, contributes to the overall decline in muscle oxidative capacity with aging (R.T Hepple [unpublished])

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myosin to accumulate oxidative damage to cysteine molecules (Prochniewicz et al 2005) Similarly, there is an increase in oxidative damage, particularly nitrotyrosine damage, to the sarcoplasmic reticulum ATPase in aged muscles (Fugere et al 2006; Thomas et al 2009), and this is thought to contribute to decreases in maximal SERCA activity in aged muscle (Thomas et al 2009) As such, the collective evi-dence suggests that oxidative damage to various proteins within skeletal muscle, and the mitochondria therein, can lead to functional deterioration in aging skeletal muscle

5 Plasticity of Mitochondria in Aging Muscles

Given the above evidence of reduced mitochondrial oxidative capacity and increased ROS generation with aging, both of which have been attributed in part to accumulation of damaged mitochondria secondary to reduced mitochondrial renewal, an obvious question is whether aged muscle simply loses the capacity to increase its mitochondrial content The majority of what we know about this ques-tion has been obtained from experiments examining changes in muscle mitochon-drial oxidative capacity in response to exercise training or chronic electrical stimulation Significantly, an emerging concept is that the capacity for mitochon-drial biogenesis in response to muscle activation, while relatively preserved in the younger of the old, becomes severely impaired in the oldest old

There are many studies showing that aged muscles can respond favorably by increasing markers of mitochondrial content in response to endurance exercise training in both the human (Orlander and Aniansson 1980; Hagberg et al 1989; Meredith et al 1989; Short et al 2003) and animal model (Cartee and Farrar 1987; Rossiter et al 2005; Betik et al 2008) literature However, it is important to realize that these prior studies have not considered potential differences in the endurance training responses between late middle age versus the senescent period (i.e., when survival rates drop below 50%), and it is the senescent period when the consequences

of aging for skeletal muscle become most severe To address this issue, we recently examined the effect of aging on the responses of the skeletal muscle aerobic machinery to endurance training in rat skeletal muscles Interestingly, whereas skeletal muscle aerobic function (in situ maximal oxygen consumption) and mitochondrial enzyme activities increased significantly when endurance exercise training was imposed in late middle age and continued for 7 weeks (Betik et al 2008) (Fig 10), the skeletal muscles completely lost this positive adaptation when the training was continued for 7 months into the senescent period (Betik et al 2009) (Fig 11) Further to this, the normally robust response of PGC-1a expression to endurance exercise training seen in studies of rodents (Baar et al 2002; Terada

et al 2002) and young adult humans (Norrbom et al 2004) was abolished in senescent rat skeletal muscles following 7 months of endurance exercise training in both the slow-twitch soleus muscle and the fast-twitch plantaris muscle (Fig 12) (Betik et al 2009) On the basis of these results, therefore, it appears that senescent

muscle in particular has a markedly diminished capacity to increase mitochondrial biogenesis in response to an endurance training stimulus, and that this is due in part

to an impaired ability to up-regulate PGC-1a This finding of reduced adaptability with endurance training in senescence is consistent with studies demonstrating that skeletal muscle from the oldest old also has a diminished plasticity in response to resistance exercise training (Slivka et al 2008; Raue et al 2009) and functional overload (Blough and Linderman 2000)

The aforementioned results indicate that senescent skeletal muscle loses its abil-ity to generate new mitochondria in advanced age, suggesting that the reduced

Fig 10 Muscle oxygen uptake during incremental muscle contractions in distal rat hindlimb

muscles pump-perfused in situ (top) and the activity of complex IV in homogenates of plantaris (Plan) and gastrocnemius (Gas) muscle (bottom) in sedentary late middle aged rats and late middle

aged rats exercise-trained for 7 weeks (Reproduced from Betik et al [2008], with permission from The Physiological Society [London])

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mitochondrial turnover rate with aging is secondary to this diminished capacity to make new mitochondria However, an important question remains: is it that senes-cent muscle loses its adaptive plasticity per se, or is the limitation the result of the much lower exercise stimulus that can be sustained in very old age To help address this issue, a recent study examined the response of young adult versus senescent skeletal muscle to an acute bout of low frequency electrical stimulation Interestingly, these experiments revealed that whereas the cell signaling pathway, including molecules involved in driving mitochondrial biogenesis (e.g., adenosine monophosphate protein kinase [AMPK] activation), was relatively intact in the highly oxidative region of the tibialis anterior muscle, there was a blunted response

Fig 11 Muscle maximal oxygen uptake (VO2max) during incremental muscle contractions in

distal rat hindlimb muscles pump-perfused in situ (top) and the activity of complex IV in homo-genates of plantaris (Plan) and Soleus (Sol) muscle (bottom) in sedentary senescent rats and

senescent rats trained for 7 months beginning in late middle age (Data reproduced from Betik

et al [2009])

in the highly glycolytic region of this muscle in senescence (Ljubicic and Hood 2009) These data are generally consistent with another study showing that AMPK activation is markedly blunted in aged muscles following either pharamacological stimuli or an acute exercise bout (Reznick et al 2007) What is not yet clear, how-ever, is the degree to which an attenuated mitochondrial biogenesis response is a general property of all muscle fibers in an aged muscle, versus there being an increasing proportion of muscle fibers which cannot contribute to the whole muscle mitochondrial biogenesis response (e.g., those that have become denervated and/or which are undergoing regeneration) Irrespective of this point, the growing consen-sus is that aging muscle, particularly in senescence, displays an impaired ability to up-regulate mitochondrial biogenesis and this in turn plays an important role in the attenuated benefits of endurance exercise training for skeletal muscle aerobic capacity in senescence Future studies need to address whether this loss of adaptive plasticity is an immutable consequence of aging, or if other interventions yet to be identified can help restore the adaptive response to increased muscle use

6 Conclusions

Mitochondrial changes in aging skeletal muscles, and the implications these have for the decline in both muscle mass and its function with aging, have constituted an intensive area of study The aforementioned chapter provides some context for the current knowledge in this area and areas that will be refined through further study Given the central importance of mitochondrial biology to so many facets of normal

Fig 12 PGC-1 protein expression in plantaris (Plan) and soleus (Sol) muscles of sedentary

senes-cent rats and senessenes-cent rats trained for 7 months beginning in late middle age *P < 0.05 versus

Sedentary group (Data reproduced from Betik et al [2009])

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cell function, particularly in tissues with a wide metabolic scope like skeletal muscle, new discoveries about the significance of changes in mitochondria for aging skeletal muscles, and their potential remedy through lifestyle modification (e.g., exercise training, diet) and/or medical intervention (e.g., pharmaceuticals, gene therapy), will remain at the forefront of our quest to promote healthy aging

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