Blunted hypertrophic response in old mouse muscle is associated with a lower satellite cell density and is not alleviated by resveratrol

Highlights

• The hypertrophic response was blunted in 25-month-old compared to 9-month-old mice.

• The blunted hypertrophic response was characterized by blunted myofiber hypertrophy.

• The lower number of SCs per CSA may underlie the blunted myofiber hypertrophy.

• Resveratrol did not alleviate the age-related blunted hypertrophic response.

• Resveratrol abolished the increase in SC content after overload.

Abstract

Background

Sarcopenia contributes to the decreased quality of life in the older person. While resistance exercise is an effective measure to increase muscle mass and strength, the hypertrophic response may be blunted in old age.

Objectives

To determine 1) whether hypertrophy in the m. plantaris of old mice was blunted compared to adult and 2) whether this was related to a reduced satellite cell (SC) density and 3) how resveratrol affects hypertrophy in old mice.

Methods

In adult (7.5 months, n = 11), old (23.5 months, n = 10) and old-resveratrol-treated (n = 10) male C57BL/6J mice, hypertrophy of the left m. plantaris was induced by denervation of its synergists. The contralateral leg served as control.

Results

After six weeks, overload-induced myofiber hypertrophy and IIB–IIA shift in myofiber type composition were less pronounced in old than adult mice (P = 0.03), irrespective of resveratrol treatment. Muscles from old mice had a lower SC density than adult muscles (P = 0.002). Overload-induced SC proliferation (P < 0.05) resulted in an increased SC density in old, but not adult muscles (P = 0.02), while a decrease occurred after resveratrol supplementation (P = 0.044). Id2 and myogenin protein expression levels were higher in old than adult muscles (P < 0.05). Caspase-3 was expressed more in hypertrophied than control muscles and was reduced with resveratrol (P < 0.05).

Conclusion

The blunted hypertrophic response in old mice was associated with a lower SC density, but there was no evidence for a lower capacity for proliferation. Resveratrol did not rescue the hypertrophic response and even reduced, rather than increased, the number of SCs in hypertrophied muscles.

Introduction

During aging there is a progressive loss of skeletal muscle mass and function (Ballak et al., 2014, Degens and Korhonen, 2012). While resistance exercise is a widely used strategy to counteract the age-related loss in muscle mass and strength (Brown et al., 1990, Frontera et al., 1988, Leenders et al., 2013), the hypertrophic response to resistance training or overload may be blunted in old humans (Grimby et al., 1992, Kosek et al., 2006, Martel et al., 2006, Raue et al., 2009, Slivka et al., 2008) and rodents (Alway et al., 2002a, Blough and Linderman, 2000, Degens and Alway, 2003). It has been postulated that the age-related blunted hypertrophy is largely due to a decrease in the number of functional myofibers (Degens, 2012), but a reduced ability of old myofibers to hypertrophy may also contribute. The latter may be a consequence of a reduced mechanosensitivity of myofibers, a reduced number of satellite cells (SCs) and/or the SC's ability to proliferate and/or differentiate.

Since the activation of the AKT/mTOR pathway during overload was not diminished in muscles from old rodents, it is unlikely that a blunted rate of protein translation is a major cause of the attenuated hypertrophic response in old age (Mayhew et al., 2009). Since mechanical growth factor (MGF) and IL-4 are involved in SC activation (Aline and Sotiropoulos, 2012, Della Gatta et al., 2014), an attenuated loading-induced induction of MGF (Owino et al., 2001) and serum response factor, that controls the expression of IL-4, in old age (Lahoute et al., 2008) may result in impaired activation of SCs. This in turn could attenuate the accretion of myonuclei within the myofiber in aged muscle. SCs are thought to play an important role in the development of hypertrophy, where proliferation and differentiation of SCs prevent an excessive increase in the size of the myonuclear domain, the amount of cytoplasm controlled by a myonucleus (Van der Meer et al., 2011a). The potential importance of the addition of new myonuclei to the hypertrophying myofiber is reflected by the observation that individuals that developed the largest degree of hypertrophy in response to a training program also had the largest accretion of new myonuclei (Petrella et al., 2006) and the highest number of SCs per myofiber before training (Petrella et al., 2008). This suggests that not only an age-related decline in SC number (Chakkalakal et al., 2012), but also impaired proliferation and/or differentiation may underlie the blunted hypertrophic response in old age. That this may indeed occur is reflected by the blunted increase in myogenin and MyoD protein expression in overloaded m. plantaris in old rat (Alway et al., 2002a), indicative for an impaired SC proliferation and differentiation, respectively.

The blunted hypertrophic response in old rats was associated with an increased expression of inhibitors of differentiation (Id) proteins and apoptosis (Alway et al., 2002a). An elevated expression of Id proteins is significant, as overexpression of these proteins leads to myofiber atrophy (Gundersen and Merlie, 1994), possibly by stimulating proliferation and apoptosis (Florio et al., 1998) and inhibition of differentiation of SCs via their inhibitory action on the myogenic regulatory factors, such as MyoD and myogenin (Alway et al., 2003). Furthermore, increased oxidative stress and low-grade inflammation, often observed in aged muscle (Degens, 2010), have been linked to increased expression of Id proteins (Mueller et al., 2002). In addition, TNFα can impair the transcription of MyoD and myogenin (Guttridge et al., 2000) and stimulate their breakdown in the proteasome (Langen et al., 2004) particularly when they are dimerized with Id proteins (Abu Hatoum et al., 1998). This then would hamper the transcription of muscle specific genes, as in fact observed in old rat plantaris muscle (Alway et al., 2002b).

Treatment with an anti-inflammatory anti-oxidant may restore the SC proliferation and differentiation and thereby the hypertrophic response in old age. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenol with anti-oxidant properties that has been associated with a number of health benefits. Its effect on myoblasts in particular seems to be anti-proliferative and pro-differentiation (Kaminski et al., 2012, Montesano et al., 2013, Saini et al., 2012). This suggests that resveratrol is a potent supplement to counteract sarcopenia (Marzetti et al., 2011), enhance SC differentiation and restore the hypertrophic response to mechanical overload in old age.

Therefore, the aim of this study was threefold: 1) to test whether the hypertrophic response to a 6-week period of overload by synergist denervation was blunted in old mice compared to that in adult mice; 2) to establish whether the blunted hypertrophy was related to a reduced number of SCs and 3) to assess whether resveratrol supplementation increases the number of SCs and restores the hypertrophic response in old muscles.