Elsevier

Experimental Gerontology

Volume 47, Issue 12, December 2012, Pages 913-918
Experimental Gerontology

Severe atrophy of slow myofibers in aging muscle is concealed by myosin heavy chain co-expression

https://doi.org/10.1016/j.exger.2012.07.013Get rights and content

Abstract

Although slow myofibers are considered less susceptible to atrophy with aging, slow fiber atrophy may have been underestimated previously. First, the marked atrophy of the aging rat soleus (Sol) muscle cannot be explained by the atrophy of only the fast fibers, due to their low abundance. Second, the increase in small fibers co-expressing both fast and slow myosin heavy chains (MHC) in the aging rat Sol is proportional to a decline in pure MHC slow fibers (Snow et al., 2005), suggesting that these MHC co-expressing fibers represent formerly pure slow fibers. Thus, we examined the size and proportion of MHC slow, MHC fast, and MHC fast-slow co-expressing fibers in the Sol and mixed region of the gastrocnemius (Gas) muscle in young adult (YA) and senescent (SEN) rats. Our results suggest that formerly pure MHC slow fibers are the source of MHC co-expressing fibers with aging in both muscle regions. Accounting for the atrophy of these fibers in calculating MHC slow fiber atrophy with aging revealed that MHC slow fibers atrophy on average by 40% in the Sol and by 38% in the mixed Gas, values which are similar to the 60% and 31% atrophy of pure MHC fast fibers in the Sol and mixed Gas, respectively. Probing for the atrophy-dependent ubiquitin ligase, MAFbx (atrogin 1), it was suggested that former slow fibers acquire atrophy potential via the up-regulation of MAFbx coincident with the co-expression of fast MHC. These results redefine the impact of aging on slow fiber atrophy, and emphasize the necessity of addressing the atrophy of fast and slow fibers in seeking treatments for aging muscle atrophy.

Highlights

► The incidence of MHC co-expressing fibers is characterized in aging muscle. ► A 14-fold increase with age is observed in soleus muscle. ► This increase is found to be at the expense of slow type muscle fibers. ► MHC co-expressing fibers demonstrate a significant and severe degree of atrophy. ► The belief that slow type muscle fibers are protected from atrophy is refuted.

Introduction

Sarcopenia is the age-related loss of skeletal muscle mass and function (Hepple, 2003). It is characterized by atrophy and loss of individual muscle fibers (Lexell et al., 1988, Lushaj et al., 2008), as well as shifts in myosin heavy chain (MHC) expression (Andersen, 2003, Caccia et al., 1979, Monemi et al., 1999). One of the most widely accepted tenets of muscle aging is that there is preferential atrophy of fast twitch myofibers, with rather convincing evidence in the literature (Caccia et al., 1979, Lexell et al., 1988, Lushaj et al., 2008). As a result, the majority of intervention strategies for sarcopenia, from strength training (Rolland et al., 2011) to the use of anabolic pharmacological agents (Glass and Roubenoff, 2010), are based upon this belief. Despite the acceptance of this view, more recent evidence raises the possibility that the atrophy of slow twitch fibers with aging may have been dramatically under-estimated heretofore.

Specifically, the rat soleus muscle (Sol; predominantly slow) exhibits a strikingly similar rate of atrophy between late middle age (28–30 months) and senescence (SEN; 35–36 months) as the gastrocnemius muscle (Gas; predominantly fast) (Rowan et al., 2011). The substantial atrophy observed in the SEN Sol muscle cannot be explained by the atrophy of only fast twitch fibers, due to their very small proportion in this muscle. In addition, serial sections immunolabeled for fast and slow MHC show the shift toward an increase in fast MHC expression in the aging rat Sol muscle (Carter et al., 2010) is due to a marked increase in fibers that co-express both fast and slow MHC isoforms, and a proportional decline in fibers expressing slow MHC only (Snow et al., 2005). Although the presence of MHC fast-slow co-expressing fibers in aging muscle has been reported previously in several studies (Andersen et al., 1999, Ansved and Larsson, 1989, Edstrom and Larsson, 1987, Rowan et al., 2011, Snow et al., 2005), no prior study has accounted for the possibility that a fraction of these co-expressing fibers are derived from formerly pure slow fibers when ascribing the degree of atrophy in slow versus fast myofibers with aging.

To this end, we examined the size and proportion of fibers expressing only slow MHC, only fast MHC, and both slow and fast MHC (co-expressing fibers) by immunofluorescence in two muscle types; the Sol muscle and the Gas muscle of YA and SEN rats. We hypothesized that accounting for the origins of co-expressing fibers in aging muscle would reveal a greater severity of slow fiber atrophy with aging than has been previously indicated.

Section snippets

Ethics

All experimental procedures on animals were made with prior approval of the University of Calgary Animal Care Committee, protocol ID BI09R-11.

Experimental animals and tissue collection

5 young adult (YA; 8–10 months of age) and 6 senescent (SEN; 36 months of age) male Fisher 344 × Brown Norway F1 (F344BN) rats were obtained from the colony maintained by the National Institute on Aging (NIA; Baltimore, MD). They were housed in the Biological Sciences vivarium at the University of Calgary in single cages (12/12 h light/dark cycle, 21 °C) and

Muscle mass

In the soleus muscle, there was 15% lower muscle mass in SEN versus YA samples (YA = 155 ± 6 mg, SEN = 132 ± 6 mg, p < 0.05). In the gastrocnemius muscle, there was 35% lower muscle mass in the SEN versus YA samples (YA = 2004 ± 78 mg, SEN = 1309 ± 50 mg, p < 0.05).

Fiber type expression and size

Note that the fiber size in YA animals documented here is similar to that seen in another cohort of YA animals, as we have reported in the supplemental data of a prior report (Rowan et al., 2011). Similarly, our current results on fiber type alterations in

Discussion

The purpose of this study was to take into account the atrophy behavior of MHC fast-slow co-expressing fibers in computing the degree of MHC slow fiber atrophy with aging. We hypothesized that this approach would reveal a substantially greater degree of slow fiber atrophy than has been previously indicated in the literature. Our analysis showed that in both the Sol muscle and the mixed region of the Gas muscle, MHC fast-slow co-expressing fibers appeared to originate primarily from formerly

Conclusions

The most significant aspect of our findings is that they contradict the widely accepted notion that slow twitch fibers are protected from atrophy with aging. In explaining how this point has escaped notice previously, as noted above, we believe that the phenomenon of MHC co-expression has obscured the actual degree of atrophy of MHC slow fibers with aging in previous studies. Although several prior studies have commented upon the occurrence of MHC co-expression in both human (Andersen et al.,

Acknowledgments

The authors thank Dr. Melissa Thomas for her help in collecting tissues and Ms. Rui Mi for sectioning the tissues. This work was supported by operating grants from the Canadian Institutes of Health Research to Dr. R.T. Hepple (MOP 57808 and IAO 84673).

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