The aim of the present study was to determine levels of PARP-1, cleaved PARP-1 and PARG at rest and following an acute exercise bout in order to investigate the effects of age and training status on indicators of genomic integrity and apoptotic pathways in human skeletal muscle. In this regard, we show for the first time that low physical activity levels in older people disrupts PARP-1 regulation in skeletal muscle leading to increased levels of PARP-1, a protein essential for recovery from DNA damage, and cleaved PARP-1, a characteristic of apoptosis. In support of our hypothesis, we report that lifelong training attenuates this deterioration in skeletal muscle. Additionally, we demonstrate that skeletal muscle ageing alters the response of total PARP-1 to an acute exercise bout. These findings provide novel insight into how habitual exercise training could protect the ageing genome, although further work is required to confirm this.
PARP-1 activity, predominantly by PARylation of both proteins and DNA, has key, direct and indirect, roles in the response to stress and repair of oxidative DNA damage [22, 23]. PARP-1 PARylation is also implicated in signaling to the stress granules, the proteasome, in controlling the cellular localization of key transcription factors  and in cell senescence or programed cell death responses including apoptosis, necrosis and parthanatos . Given the central role of PARP-1 in the repair of oxidative DNA damage and cell death decisions, it seemed possible that PARylation may be a key component of the protective effects of exercise. To examine this hypothesis, the levels of three proteins involved in the PARylation response to genotoxic stress, PARP-1, PARG and cleaved PARP-1 were measured. In young subjects at rest, irrespective of training, all of the three proteins were present at low levels. However, PARP-1 was significantly increased in all old subjects, irrespective of training status, presumably relating to the higher level of DNA damage in muscles from older participants . Future studies should now investigate the exercise and age-related changes in post-translational modifications of PARP-1.
When apoptosis is initiated, PARP-1 is cleaved by apoptotic caspases, and its presence is considered a characteristic feature of apoptosis. However, PARP-1 can also be cleaved by other proteins, including apoptosis independent caspase 7 which cleaves PARP-1 located on the promoters of NFĸB target genes allowing expression of pro-inflammatory genes . Although there has been some debate about the existence of caspase-dependent and independent apoptosis in skeletal muscle, it now seems likely that both pathways are indeed present [12, 26, 27]. Here, in young subjects at rest, the ratio of PARP-1:cleaved PARP-1 was low, perhaps indicating a low but not complete absence, of apoptotic and inflammatory related caspases. However, cleaved-PARP-1 was significantly elevated in the old untrained subjects, presumably indicating a highly stressed environment with myonuclear apoptosis and inflammation, compared with the much lower levels of cleaved PARP-1 observed in the trained old subjects. These data therefore suggest that exercise training slows, but does not abolish, the progress towards an age-related highly stressed state in skeletal muscle.
PARylation is NAD+ dependant, therefore regulation of PAR activity is vital to prevent depletion of NAD+ , which would affect other NAD+ dependant processes, such as the acetylation  and deacetylation (sirtuin based) pathways . Consequently, a complex array of posttranslational and automodifications of PARP-1  co-ordinate the multiple PARP-1 processes and are essential for maintaining NAD+ levels . Depleted NAD+ levels could compromise metabolism and inhibit SIRT1, a key regulator of energy metabolism and mitochondrial biogenesis [33–35]. Inhibition of SIRT1 activity leads to mitochondrial dysfunction , mimicking the aberrant mitochondrial function and homeostasis seen in the muscles of sedentary aged humans [14, 15, 17]. PARG has a central role in the regulation of NAD+ levels, primarily via the NAD+ salvage pathway, but may also regulate the expression of PARP-1 . In the present study, young participants had low levels of PARG irrespective of training status, although PARG levels were elevated in the old participants, especially the old untrained. The high PARP-1 levels seen in old untrained subjects may reflect high levels of DNA damage and will likely result in chronic PARylation causing depleted levels of NAD+. It is, therefore, proposed that the equally high PARG levels that were seen in these subjects represent an attempt to combat this PARylation-induced depletion in NAD+. The lower PARG levels in the old trained subjects may result from improved oxidative metabolism and mitochondrial biogenesis  counteracting the mitochondrial dysfunction and metabolic crisis associated with SIRT1 inhibition caused by NAD+ depletion .
We also investigated PARP-1, PARG and cleaved PARP-1 protein content immediately post and three days following an acute HIT exercise bout. A HIT model was employed due to this form of exercise being shown to be a tolerable, time-efficient, enjoyable and effective method of inducing metabolic adaptations in human skeletal muscle [36, 37]. In addition, HIT activates both type I and type II muscle fibers [38, 39], which is an important consideration when assaying a mixed fiber type muscle such as the vastus lateralis. Prior to the present study, PARP-1, PARG and cleaved PARP-1 had not been investigated in an exercise setting in humans, which consequently presented difficulties in selecting suitable biopsy time-points post-exercise. We elected to extract muscle samples at three days since our group has previously shown that stress-responsive proteins, such as heat shock proteins, are up-regulated at this time point [40, 41]. We report that there were no significant changes in PARG levels immediately post and three days following exercise in any of the subject groups, suggesting that PARG is not responsive to an acute exercise bout. Interestingly PARP-1 levels showed no change in the young trained but increased in the untrained young. These data may suggest that in young subjects, habitual exercise provides protection against the potentially damaging, exercise-induced stress response . However, in old subjects total PARP-1 expression decreased post-exercise, perhaps mediated by PARG repression of gene expression  and may be a further attempt to protect against NAD+ depletion. Gene expression and mRNA stability studies may provide further insight into this intriguing phenomenon. In young subjects, irrespective of training status, there was a trend towards an increase in cleaved PARP-1, which could indicate an increase in apoptosis following exercise but perhaps more likely reflects cleavage of PARP-1 at NFĸB target genes. In old subjects, there was a trend towards decreased cleaved PARP-1 expression following exercise which could account for the age-related attenuated induction of NFĸB stress response genes following muscle contraction . Follow-up work should examine whether this trend reaches statistical significance in a larger subject cohort and aim to further investigate the underlying mechanisms.