with their controls. In fact, the frequency of the R allele in our centenarians (7.1 % in Spaniards and 7.6 % in Italians) was approximately twofold higher than that previously reported in the literature for – European Caucasians, i.e. 3 4 % (Corsi et al. 2002; Ferrell et al. 1999; Kostek et al. 2009). Despite the complexity of the ageing process and the fact that exceptional longevity is likely a polygenic trait with numerous candidate genes exerting an effect either individually or in complex interactions, single-gene mutations (like maybe the one we studied here) may suffice to delay the onset of age-related phenotypes in a variety of model organisms, from yeast to mammals (Bjedov and Partridge 2011). There is a strong ratio- nale for suggesting that myostatin inhibition, or alter- natively mutations in the MSTN gene affecting its gene product, could contribute to exceptional longevity. Although more research is needed, the putative effects of the K153R variation, e.g. on muscle phenotypes,

The first mechanism by which myostatin inhibition or MSTN variations could affect longevity and healthy ageing is through an increase in muscle mass/function. In effect, myostatin is a skeletal muscle-specific se- creted peptide that essentially modulates myoblast proliferation and thus muscle mass/strength (McPherron et al. 1997). Preclinical trials using myo- statin loss or inhibition have been effective in amelio- rating symptoms of weakness in several animal

Fig. 2 Genotypedistribution of the MSTN K153R poly- morphism in the Spanish and Italian cohorts. *P<0.001, for the frequency of R allele carriers in Spanish centenar- ians vs. Spanish controls; **P=0.008, for the frequen- cy of R allele carriers in Ital- ian centenarians vs. Italian controls

Spanish cohort Replication (Italian) cohort 100 90 80 70 60 50 40 30 ** 20 * 10 0 Centenarians Controls Centenarians Controls (n=156) (n=384) (n=79) (n=316) )%( ycneuqerf epytoneG

needed to clearly elucidate its influence on muscle phenotypes and sarcopenia in very old people.

models of muscle injury, atrophy and disease (Benny Klimek et al. 2010; Bogdanovich et al. 2002; Lee and McPherron 2001; Liu et al. 2008; Morrison et al. 2009; Murphy et al. 2010; Qiao et al. 2009; Siriett et al. 2006; Tsuchida 2008; Wagner et al. 2008; Zhu et al. 2007). In humans, myostatin inhibitors, such as MYO- 029, have an adequate safety margin and are able to improve the muscle strength/function or muscle con- tractile properties in some patients with muscular dys- trophy (Krivickas et al. 2009; Wagner et al. 2008). All these data are consistent with the growing interest in developing therapeutic inhibitors of myostatin for use in muscle-wasting disorders such as muscular dystro- phy, cachexia or ageing-related sarcopenia (Tsuchida 2008) and with the fact that myostatin inhibition is proposed as a potential intervention for frailty and sarcopenia in the elderly (Swan 2011).

We believe that the stronger rationale to postulate a certain beneficial effect of the infrequent R allele on exceptional longevity is based on the inhibiting role that myostatin plays in the mTOR signalling pathway. Indeed, the most robust and well-studied improve- ments in ageing are caused by dietary restriction and also by genetic down-regulation of nutrient-sensing pathways, namely mTOR (Fontana et al. 2010; Kenyon 2010). The central component of the mTOR pathway, the TOR kinase, forms two functionally dif- ferent complexes: TOR complex (TORC) 1 and TORC2, with TORC1 promoting growth by positively regulating S6 kinase (Fenton and Gout 2011) and TORC2 activating the pro-survival Akt kinase of the insulin/insulin-like growth factor signalling pathway (Cybulski and Hall 2009; Sparks and Guertin 2010). For instance, under starvation conditions, TORC1 is responsible for up-regulating autophagy, a process that generates energy by degrading portions of the cyto- plasm (Neufeld 2010; Ravikumar et al. 2010). A num- ber of studies have demonstrated that myostatin acts as a negative regulator of mTOR-directed signalling, which is consistent with its inhibitory effect on protein synthesis (Amirouche et al. 2009; Langley et al. 2002; Lipina et al. 2010; McFarlane et al. 2006; Rios et al. 2004; Sartori et al. 2009; Trendelenburg et al. 2009) and more specifically on protein translation in skeletal muscle (Trendelenburg et al. 2009). On the other hand, myostatin deletion is beneficial for bone density, insu- lin sensitivity and heart function in senescent mice (Morissette et al. 2009), and inhibition of ActRIIB, the cell membrane receptor for which myostatin has the highest affinity (Sakuma and Yamaguchi 2012), increases survival (by 17 %) in myotubularin-deficient mice, due to a delay in the point at which animals experience weight loss or complete hind limb paraly- sis (Lawlor et al. 2011) (X-linked myotubular myop- athy is a severe form of congenital myopathy which most often manifests with severe perinatal weakness and respiratory failure). An extension of lifespan (+30 %) was also observed after therapy with follista- tin (a natural antagonist of myostatin) in a mouse model of spinal muscular atrophy (Rose et al. 2009).