Journal of Neuropathology & Experimental Neurology:
Long-Term High-Level Exercise Promotes Muscle Reinnervation With Age
Mosole, Simone BS; Carraro, Ugo MD; Kern, Helmut MD; Loefler, Stefan Eng; Fruhmann, Hannah PT; Vogelauer, Michael MD; Burggraf, Samantha Eng; Mayr, Winfried PhD; Krenn, Matthias DI; Paternostro-Sluga, Tatjana PhD; Hamar, Dusan MD; Cvecka, Jan PhD; Sedliak, Milan PhD; Tirpakova, Veronika PhD; Sarabon, Nejc PhD; Musarò, Antonio PhD; Sandri, Marco MD; Protasi, Feliciano PhD; Nori, Alessandra PhD; Pond, Amber PhD; Zampieri, Sandra PhD
From the Laboratory of Translation Myology, Department of Biomedical Sciences, University of Padua, Padua, Italy (SM, UC, AN, SZ); Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria (HK, SL, HF, SB, SZ); Department of Physical Medicine and Rehabilitation, Wilhelminenspital, Vienna, Austria (HK, MV); Center of Biomedical Engineering and Physics, Medical University of Vienna, Vienna, Austria (WM, MK); Department of Physical Medicine and Rehabilitation, Medical University of Vienna, Vienna, Austria (TP-S); Faculty of Physical Education and Sport, Comenius University, Bratislava, Slovakia (DH, JC, MSedliak, VT); Science and Research Centre, Institute for Kinesilogical Research, University of Primorska, Koper, Slovenia (NS); DAHFMO-Unit of Histology and Medical Embryology, IIM, Sapienza University of Rome, Rome, Italy (AM); Venetian Institute of Molecular Medicine, Dulbecco Telethon Institute, and Department of Biomedical Science, University of Padua, Padua, Italy (MSandri); CeSI-Center for Research on Aging, and DNI-Department of Neuroscience and Imaging, University G. d’Annunzio of Chieti, Chieti, Italy (FP); and Anatomy Department, Southern Illinois University School of Medicine, Carbondale, Illinois (AP).
Send correspondence and reprint requests to: Sandra Zampieri, PhD, Laboratory of Translational Myology, Department of Biomedical Sciences, University of Padua, Viale G. Colombo, 3 I-35121 Padua, Italy; E-mail:
Simone Mosole and Ugo Carraro contributed equally.
This study was supported by the European Regional Development Fund-Cross Border Cooperation Programme Slovakia-Austria 2007–2013 (Interreg-Iva), project Mobilität im Alter, MOBIL, N_00033 (partners: Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Austria; Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria; and Faculty of Physical Education and Sports, Comenius University, Bratislava, Slovakia); Austrian national cofinancing of the Austrian Federal Ministry of Science and Research; Austrian national cofinancing of the Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Society, Vienna, Austria; and institutional funds to Ugo Carraro, Antonio Musarò, Marco Sandri, and Feliciano Protasi of Ministero dell’Istruzione, dell’Università e della Ricerca, Italy.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site ( www.jneuropath.com).
The histologic features of aging muscle suggest that denervation contributes to atrophy, that immobility accelerates the process, and that routine exercise may protect against loss of motor units and muscle tissue. Here, we compared muscle biopsies from sedentary and physically active seniors and found that seniors with a long history of high-level recreational activity up to the time of muscle biopsy had 1) lower loss of muscle strength versus young men (32% loss in physically active vs 51% loss in sedentary seniors); 2) fewer small angulated (denervated) myofibers; 3) a higher percentage of fiber-type groups (reinnervated muscle fibers) that were almost exclusive of the slow type; and 4) sparse normal-size muscle fibers coexpressing fast and slow myosin heavy chains, which is not compatible with exercise-driven muscle-type transformation. The biopsies from the old physically active seniors varied from sparse fiber-type groupings to almost fully transformed muscle, suggesting that coexpressing fibers appear to fill gaps. Altogether, the data show that long-term physical activity promotes reinnervation of muscle fibers and suggest that decades of high-level exercise allow the body to adapt to age-related denervation by saving otherwise lost muscle fibers through selective recruitment to slow motor units. These effects on size and structure of myofibers may delay functional decline in late aging.Trial registration: ClinicalTrials.gov (NCT01679977).
Aging is characterized by a gradual decline that impairs cell homeostasis and functional reserves. Degeneration, apoptosis, and death (accompanied by loss of regenerative capacity) of all cell types progressively accumulate and ultimately lead to organism death (1–4). Histologic studies of skeletal muscle have shown that denervation is among the numerous mechanisms that contribute to tissue atrophy and degeneration in aging (5, 6). The term “disseminated neurogenic atrophy” was coined to describe the progressive accumulation and clustering of small angulated fibers with aging (7–10); there is also evidence of progressive loss of α-motoneurons (11, 12). Electrophysiologic studies have confirmed that there is a decrease in the number of motor units with a concomitant increase in their size with age. These results suggest that some reinnervation events follow muscle fiber denervation (13). Further evidence supporting the occurrence of rounds of denervation and reinnervation includes the increased clustering of myofiber types in the motor units of rodents and other mammals as they age (11, 14). In adult humans, fiber types appear randomly distributed across the muscle and become increasingly grouped with age (15). Therefore, it has been proposed that, in addition to axonal disorders, apoptosis of α-motoneurons in the spinal cord, with subsequent incomplete reinnervation of fibers by surviving motor neurons, may contribute to loss of muscle strength and mass as people grow older (16). These rearrangement processes are generally accompanied by a progressive increase in the proportion of slow muscle fibers, although there is some evidence to the contrary (17). Some of the discrepancies have been dispelled by comparisons of muscle from normally active and immobile older patients that show that muscle wasting in “normally active” seniors is accompanied by a shift toward a slow-twitch phenotype, whereas inactive seniors demonstrates a shift toward fast-twitch isoform expression. This latter case is common in “unloaded” muscle undergoing atrophy, for example, during limb suspension, immobilization, paralysis, and spaceflight (18–22). To complicate the situation further, conflicting results regarding fast to slow myosin transition arise in endurance training studies using animal models and in clinical trials of humans involving either voluntary exercise or electrical stimulation—both directly to denervated muscle and indirectly to muscle through nerve stimulation (19, 21, 23–27). Furthermore, increased exercise that is sustained for decades (e.g. training as performed by track and field masters athletes) protects against age-related loss of motor units (28–30) and, thereby, of lean muscle mass (31). However, the degree to which denervation causes loss of myofibers is an open question because reinnervation events may compensate for motor neuron loss during aging as well as with spinal cord injury and/or axonal abnormalities of peripheral nerves (13–15, 32–34). Whether the aging-related shifts are under neural control or the result of the direct influence of use/disuse on myogenic processes remains to be clarified.
In the present study, we analyzed muscle biopsies harvested from the vastus lateralis of young men (aged 22–33 years), sedentary seniors (aged 67–77 years), and senior amateur athletes (aged 65–79 years). The latter routinely practiced sport activities usually more than 3 times per week up to the time of biopsy. In agreement with previous studies of masters athletes (35–37), we show that long-term high-level physical activity considerably increases the percentage of slow-type myofibers and the number of muscle fiber–type groupings. The latter provides direct evidence that long-term cycles of denervation/reinnervation occurred. In recent interim reports (38–40), we showed, and here confirm, that muscle properties of these senior recreational athletes are more similar to those of active young men than to those of sedentary seniors.
By analyzing coexpression of fast and slow myosin heavy-chain (MHC) isoforms in the muscle biopsies, we show for the first time that these events occur with recreational physical activity in seniors and that the changes may be related to selective reinnervation events. Our study supports the concept that long-term high-level exercise has beneficial effects on reinnervation of muscle fibers, resulting in preservation of muscle function, size, structure, and ultrastructure (41–43) and thereby delaying mobility decline and loss of independence that are commonly seen in aging.
MATERIALS AND METHODS
Approval from the national committee for medical ethics was obtained before the study onset (EK08-102-0608). With the exception of 2 female subjects in the sedentary group, recruited subjects were male volunteers. All subjects received detailed information on the study and gave informed consent. Three groups were enrolled: young men (n = 5; aged 22–33 years; 10 biopsies); seniors with a sedentary lifestyle (n = 6; aged 67–74 years; 10 biopsies), and seniors with a long history of high-level recreational sport activities (n = 7; aged 65 to 79 years; 10 biopsies) (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A541). All subjects were healthy and declared not to have any specific mobility impairment or disease. All of the subjects declared that they had no prescriptions for anti-inflammatory therapy related to neuropathies or myopathies. The seniors were not outpatients of any rehabilitation clinic but were located by newspaper advertisements that stressed that only healthy subjects without mobility impairment would be enrolled. Sedentary seniors were enrolled on the basis of their declaration that they had not performed any routine physical activity/training during the previous 10 years. On enrollment in the study, needle muscle biopsies were obtained from the vastus lateralis muscles through a small skin incision (6 mm); the biopsied tissue was then frozen for light microscopy or fixed for electron microscopy, as described (21, 41–43).
Light and Immunofluorescence Microscopy
Serial cryosections (8-μm thick) from frozen muscle biopsies were mounted on Polysine glass slides, air-dried, and stained with hematoxylin and eosin (42) or immunostained for either fast or slow MHC, laminin, or neural cell adhesion molecule (N-CAM), as described below.
For MHC, sections were washed with PBS and permeabilized with 0.1% Triton (Sigma-Aldrich, St. Louis, MO) in PBS for 15 minutes. After a PBS wash, nonspecific protein interactions were blocked by incubation with 10% fetal bovine serum in PBS for 30 minutes at room temperature (RT). The sections were then incubated for 1 hour at RT in primary mouse monoclonal anti-MHC fast or anti-MHC slow antibody (Novocastra, Milan, Italy) diluted 1:10 in PBS. The sections were subsequently washed in PBS and incubated with anti–mouse Cy3 secondary antibody (1:100; Sigma-Aldrich) for MHC slow and with anti–mouse FITC (1:100; Sigma-Aldrich) for MHC fast for 1 hour. The sections were washed again in PBS, and coverslips were mounted onto the glass slides using ProLong Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA). Images were acquired using a Zeiss microscope connected to a Leica DC 300F camera.
For detection of N-CAM–expressing myofibers, sections were fixed in methanol for 15 minutes at 20°C and then labeled for 1 hour at RT using rabbit polyclonal antibody directed against N-CAM (cat. no. AB5032; Chemicon, Millipore, Milan, Italy) diluted 1:200 in PBS (44, 45). Sections were rinsed 3 × 5 minutes in PBS and then incubated for 1 hour at RT with Cy3-labeled conjugate directed against rabbit IgG (Chemicon, Millipore) diluted 1:200 in 10% goat serum in PBS. Negative controls were performed by omitting the primary antibodies from sample incubations. After washes, nuclei were counterstained for 5 minutes at RT with Hoechst 33258 (Sigma-Aldrich); sections were then coverslipped using mounting medium (Dako, Glostrup, Denmark) and observed under a Zeiss microscope connected to a Leica DC 300F camera.
For coimmunolocalization of fast and slow MHC in single sections, the sections were washed, permeabilized, washed again, and incubated with blocking solution as previously described. They were then incubated for 1 hour at RT with mouse anti–MHC slow primary monoclonal antibody (Sigma-Aldrich) diluted 1:10 in PBS and secondarily with rabbit anti-laminin (Sigma-Aldrich) diluted 1:100 in PBS. Next, the sections were washed in PBS and incubated with the anti-mouse–Alexa 594 (1:200; Life Technologies) secondary antibody and the anti–rabbit FITC antibody (1:200; Sigma-Aldrich) for 1 hour. After a PBS wash, the sections were incubated for 1 hour with an anti–MHC fast primary monoclonal antibody produced in mouse (1:10; Novocastra). The sections were then washed with PBS and incubated for another hour with an anti-mouse–Alexa 488 secondary antibody (1:200; Life Technologies). After another wash with PBS, coverslips were mounted onto the glass slides using ProLong Gold antifade reagent with DAPI (Life Technologies).
Morphometric analyses of the fiber diameter and of the fiber-type distribution were performed on cryosections using Scion Image for Windows version Beta 4.0.2 (2000 Scion Corporation), as previously described (42–47).
Slow Fiber Correlations
To determine whether there was a correlation between slow fibers and the type of training undertaken by the physically active seniors (i.e. endurance, strength, or mixed training), percentages of slow muscle fibers were plotted against the probability that the value for each biopsy fell within the area under the curve. Excel equation DISTRIB.NORM(X;Mean;Dev_standard; Cumulative):
Analysis of variance (ANOVA) tests were performed with statistical algorithms of Origin (OriginLab Corp., Northampton, MA). The level of statistical significance was set at p < 0.05.
Demography and Clinical Characteristics Indicated That the Enrolled Subjects Were Healthy and Mobile
Detailed demographic and clinical characteristics of the enrolled subjects are described in (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A541). All subjects were healthy and declared not to have any specific mobility impairment or disease. Nonetheless, clinical and functional evaluations, in addition to electromyographic analyses, were performed in a few physically active seniors, resulting in detection of some neuropathic or myopathic features (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A541).
Amount of Weekly Physical Activity and Knee Extension Strength Are Significantly Higher in Physically Active Versus Sedentary Seniors
Amounts and types of physical exercise performed by the active subjects are detailed in Table, Supplemental Digital Content 2, http://links.lww.com/NEN/A542. Four of the 5 young men performed strength training; the fifth did endurance activity. They declared that they exercised 4.0 to 7.5 hours per week during the previous 5 years. The physically active seniors trained 4.5 to 24.0 hours per week. Thus, a few seniors spent more time training than the young men; however, the apparent large difference between the mean times (11.7 ± 7.3 vs 5.3 ± 1.4 hours per week ± SD, respectively) was not significant. Sedentary seniors had not performed any exercise above normal everyday living activities throughout the previous 20 years.
To permit a comparison of specific muscle strength among the groups, quadriceps force was measured (Table, Supplemental Digital Content 2, http://links.lww.com/NEN/A542). The mean (±SD) knee contraction strength in young men was 3.21 ± 0.55 Nm/kg; it was 2.17 ± 0.42 in the physically active seniors (a decrease of 32% relative to the young men); it was 1.57 ± 0.39 in sedentary seniors (a decrease of 51% vs the young men). The performances in a battery of functional mobility tests of the physically active seniors were more similar to those of young men than to those of the sedentary co-aged group (Kern et al, unpublished data). This is sound evidence that the enrolled physically active seniors are a highly active group and are likely comparable to masters athletes (35–37).
Small Angular Muscle Fibers in Both Young Men and Seniors (Sedentary and Sportsmen) Had the Size and Morphology of Denervated Muscle Fibers
Based on our experience with muscle biopsies from spinal cord–injured paraplegic patients with either disuse atrophy resulting from lesions of the central motoneuron or extreme atrophy caused by lack of innervation secondary to complete peripheral motoneuron lesions, we identify muscle fibers with a diameter less than 30 μm as denervated (20, 21, 43–47). Our interpretation of these myofibers as denervated is strengthened by the facts that half of these small fibers actually have diameters less than 25 μm and that several have angulated shapes. In the present study, serial sections of muscle biopsies from the young men reveal that myofibers having a diameter less than 30 μm are infrequent (0.4%; Table 1) and that those with a diameter less than 25 μm are even less abundant (0.2%; Table 1); the vast majority of the muscle fibers in these sections are predominantly round (Fig. 1A, B). The muscle sections from the sedentary seniors (Figs. 1C, D; 2, 3) and physically active seniors (Figs. 1E, F; 3) reveal more abundant muscle fibers having diameters less than 30 and 25 μm (Table 1), and some of these have distinct angulation (Fig. 1, white arrowheads). In addition, Figure 2 shows that, in sedentary seniors, the small angular myofibers have appreciable expression of N-CAM, an accepted marker of denervation (48). The biopsies taken from the sedentary seniors contain the highest percentage of denervated muscle fibers having a diameter less than 30 μm (6.5%) and of those having a diameter less than 25 μm (2.6%). These percentages are significantly higher than those in the other 2 groups, whereas the percentages of denervated fibers are not significantly different between the young men and physically active seniors (Table 1). Furthermore, the N-CAM staining was much less abundant in biopsies from the active seniors and even less so in sections from the young men (not shown). Indeed, in a previous study, we observed only 1 N-CAM–positive muscle fiber among the 10,000 analyzed in the biopsies from young men (49).
Percentages of Fast and Slow Myofibers in Physically Active Seniors Showed a Significant Shift Toward Slow Fibers Relative to the Young Men and the Sedentary Seniors
Immunofluorescence revealed both fast and slow MHC proteins in serial sections of muscle biopsies from the young men (Fig. 1A, B), with the fast fibers being slightly more abundant than the slow fibers in these muscle sections (Table 2). Interestingly, this pattern of fiber-type distribution was not significantly different from that observed in the matched muscle of sedentary seniors, although the latter was slightly shifted toward the slow type relative to the young men (Fig. 1C, D; Table 2). However, in the active seniors (Fig. 1E, F), slow fibers were most numerous (68.5%; Fig. 1E, F); the increase was significant versus both the young men (42% slow fibers) and the sedentary seniors (46% slow fibers) (Table 2).
Fiber-type grouping was almost absent in the young men; however, grouping was greater in the old subjects, with the physically active seniors having the greatest number; most of these were of the slow type, whereas those in sedentary seniors were mainly of the fast type. Fiber-type groupings are identified on the basis that at least 1 muscle fiber is completely surrounded by fibers of the same phenotype. Percentages of fiber-type groupings are determined by counting the number of muscle fibers in the biopsy that are surrounded by fibers of the same type and then dividing this number by the total number of fibers. To avoid problems related to the existence of many different fast MHC isoforms, we used an anti–fast MHC antibody that does not discriminate among the fast isoforms; therefore, we describe fiber-type clusters as either only “slow” or only “fast.” Muscle sections from the young men had few fiber-type groupings, and those that were detected were mainly of the fast type (1%; Fig. 1; Table 3). In the sedentary seniors, although both fast- and slow-type groupings were present, the fast type (3.0%) were more numerous than the slow type (0.5%) (Table 3). Most notable is the fact that biopsies taken from physically active seniors had the highest percentage of slow-type fiber groupings, with a mean of 7.9%, reaching almost 25% in extreme cases, in which 93% of total myofibers were of the slow type (Table 3). It is worth stressing that the physically active seniors with the most severe neuropathic or myopathic electromyographic results were not the subjects with the higher content of slow-type fibers and slow fiber–type groupings (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A541 and Table, Supplemental Digital Content 3, http://links.lww.com/NEN/A543).
Muscle Fibers Coexpressing Fast and Slow MHC
Muscle fibers coexpressing fast and slow MHCs were sparse in all of the analyzed biopsies, and there were no significant differences among these groups in terms of this parameter (Table 4). However, the serial sections from sedentary seniors that did show MHC coexpression were often small angular (denervated) muscle fibers (Fig. 1C, D, white arrows); therefore, we suggest that these are slow myofibers coexpressing fast isoforms of MHC by default myogenic programs. In contrast, the muscle fibers in the physically active seniors that were positive for both fast (green) and slow (red) MHC proteins were similar in size to the pure fast or pure slow myofibers (Fig. 4C). In Figure 4C, it is interesting to note that, although some fibers are green and others are red, not all of the fibers have the same color intensity. This might indicate that these fibers contain some variable combinations of fast and slow MHCs but not enough of both to produce the orange color of coexpression.
Slow Fiber Correlations
There was a strong correlation between the slow fiber percentages and slow fiber–type groupings in the physically active seniors (R2 = 0.82; Fig. 5), but there was no correlation between the percentages of slow fibers and the prevalent kinds of training undertaken by the physically active seniors (Fig. 6). Indeed, the marks indicating whether the subjects had performed mainly strength training, endurance training, or a mixture of both strength and endurance trainings (mixed training) were randomly distributed both among scarcely or highly transformed muscle biopsies.
The ages of the seniors were not correlated with percentages of slow fibers and slow fiber–type groupings in either group of seniors as follows: 1) Sedentary seniors, age versus percentage of slow fibers, R2 = 0.12; age versus percentage of slow-type groupings, R2 = 0.02; and 2) Physically active seniors, age versus percentage of slow fibers, R2 = 0.25; age versus percentage of slow-type groupings, R2 = 0.27. There was no correlation even when the 3 groups were pooled: age versus percentage of slow fibers, R2 = 0.15; age versus percentage of slow-type groupings, R2 = 0.56.
In the present study, we compared muscle parameters from a group of young men (training by weight lifting) with those from 2 groups of healthy mobile seniors (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A541): one composed of people leading a sedentary lifestyle and a second made up of recreational sportsmen. The muscle from the physically active seniors more closely resembled that of the younger men in terms of force generation and fiber size than that of the muscle of the sedentary seniors, indicating that long-term exercise aids in preservation of muscle health. More interestingly, the study reveals some unique characteristics of the muscle of the active seniors in terms of fiber type and fiber-type groupings that suggest that denervation/reinnervation plays a role in the maintenance of muscle health.
The knee contraction strength in the active seniors was significantly greater than that of the sedentary seniors and not significantly different from that of the younger men (Table, Supplemental Digital Content 2, http://links.lww.com/NEN/A542). Although the physically active seniors generated 32% less force than the young men on knee contraction (despite the fact that the groups had dedicated similar time to training), this is not surprising because it is well documented within the world sporting records of masters athletes that the young outperform the old (35–37, 50).
To explore the mechanisms that delayed deterioration in the muscle of the physically active seniors, we analyzed immunolabeled muscle biopsies taken from our groups and compared their relative amounts of 1) small angular myofibers (i.e. denervated muscle fibers); 2) molecular markers of fast and slow muscle fiber types (a measure of residual muscle plasticity); and 3) fiber-type grouping (representing denervated/reinnervated muscle fibers). We found that 1) biopsies from young men seldom contain denervated, reinnervated, or grouped muscle fibers; 2) biopsies from sedentary seniors contained both denervated and a few reinnervated clustered myofibers of the fast type; and 3) physically active seniors had a larger percentage of healthy slow myofibers, up to 90%, which appeared mainly clustered in slow fiber–type groups. The finding that physically active seniors have a significantly higher percentage of slow-type fibers and slow-type fiber groupings is consistent with our previous results using histochemical myosin ATPase staining in muscle biopsies from senior sportsmen (n = 15) (only 2 of those subjects are also part of the present study); 27 out of 28 biopsies had slow-type myofiber groupings (38). The increased slow-type fiber content and percentage of slow-type fiber groupings reflect the fact that immobilization drives muscle fibers toward atrophy and fast-type transformation. Because these parameters were significantly higher in the physically active versus the sedentary seniors, this is not simply a function of age. Furthermore, the lack of correlation between the kind of training and the percentages of slow-type fibers demonstrates that the type of activity is not the main determinant factor.
Interestingly, muscle fibers coexpressing fast and slow MCH proteins were seldom detected in the biopsies of any of our groups, and no statistical differences were found among the groups with respect to this parameter. It is possible that the observed coexpression is scant either because it is actually a rare event or because the denervation that occurs is promptly followed by reinnervation so that obvious coexpression of the MHCs is short lived. To our knowledge, this is the first evidence that fiber transformation cannot be the direct consequence of decades of high-level activity. It also further supports the hypothesis that these infrequent denervation events are not easily detectable with standard clinical electromyography. When these coexpressing fibers were found in the sedentary senior muscles, they were small (<30 μm) and often had the distinct angulation noted after experimental or clinical denervation; some were also positive with the anti–N-CAM antibody (Fig. 2), an accepted marker of denervation (48, 49). This type of myofiber is common in unloaded muscle (i.e. resulting from spaceflight, limb suspension, or immobilization) and with spinal cord injury and peripheral denervation (18–27, 51–53). Thus, we consider these to be denervated muscle fibers. Furthermore, because it is known that slow-type muscle fibers revert to the fast isotype when denervated during development and adulthood (18–27, 51–53), we suggest that these fibers are denervated slow-type myofibers reexpressing fast MHC through default myogenic programs.
In contrast, when myofibers coexpressing fast and slow MHCs were detected in the muscles of physically active seniors, they were similar in size and shape to the pure-type fibers in the sections and, therefore, cannot lack innervation (Fig. 4). Furthermore, their low density in these muscle sections is not in agreement with the concept that they belong to a motor unit that is undergoing exercise-driven, slow-type transformation of MHCs, a mechanism that is well known to occur in cross-reinnervation models (19, 53) but is more presumed than demonstrated in humans performing voluntary exercise (17). It is likely that the transforming myofibers (i.e. those coexpressing fast and slow MHC proteins) contribute to the increase in slow fiber–type groupings (Fig. 4); this is supported by the positive correlation between the increasing percentages of slow-type fiber number and slow-type fiber groupings in sections from the physically active seniors. Thus, we suggest that these normal-sized coexpressing fibers are very likely previously denervated fast-type fibers that have been reinnervated by sprouts from slow axons and are now temporarily coexpressing fast and slow MHC isoforms before they will finally exclusively express slow MHC. This is supported by the law of “recruitment order,” which dictates that slow motor units (and thus, muscle fibers) are activated more frequently than the fast motor units (54). In fact, the most active of the α-motoneurons are the slow type, and it may be this higher level of activity that most likely maintains motoneurons, muscle fibers, and their MHC content. Therefore, the increase in slow-type fiber groupings is evidence that some muscle plasticity still exists in both sedentary and (especially) physically active seniors. In addition, the reinnervation process may be more extensive than is obvious here because if the temporarily denervated myofibers were of the slow type, they would continue their current gene expression when reinnervated by slow-type α-motoneuron axon terminals and, thereby, escape our detection.
It is our opinion that the lack of fast-type groupings in senior sportsmen is direct evidence of selective reinnervation of denervated myofibers from slow-type α-motoneurons. In summary, our working hypothesis is that muscle fibers coexpressing fast and slow MHCs are either denervated slow myofibers also expressing fast MHC isoforms by the default myogenic program (Fig. 3A, B) (22, 24, 27) or are denervated fast fibers reinnervated by axons sprouting from slow motor neurons (19, 52, 53).
This speculation needs further study, in particular, in situ MHC expression analyses, in more numerous subjects and different muscle types. Indeed, our study has many potential confounding factors. These include the use of the fiber type–heterogeneous vastus lateralis muscle, the small size of the specimens because of the sampling method (needle biopsy), the low number of study subjects, the inherently variable genetic backgrounds of the individual subjects, and differences in the type and extent of physical activities of the physically active seniors. Moreover, muscle biopsies from that group ranged from those with scarce fiber-type transformation and grouping to those with almost fully transformed muscles. Despite these limitations, the clinical significance of our observations is confirmed by the fact that the muscle properties of the physically active senior group are more similar to those of the active young men than to those of sedentary seniors. Specifically, relative to their sedentary counterparts, the physically active seniors had greater muscle maximal isometric force, along with better-preserved muscle morphology and mobility (39, 40).
Taken together, our results suggest that, beyond the direct effects of aging on the structure and function of muscle fibers, changes occurring in the muscle tissue of the sedentary group seem to be in part a result of sparse incremental denervation. In physically active seniors, the increase in the percentage of “slow fiber groupings” is likely the result of the positive effect of long-term physical activity on the motoneuron pool, which, conceivably, has mainly spared the slow motoneurons from age-related lesion/death, thereby increasing the chance that peripheral reinnervation occurs because of sprouting of slow axons.
Certainly, numerous mechanisms contribute to long-term muscle health or deterioration, yet our study suggests that long-term exercise would allow the body to adapt to theconsequences of age-related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment of muscle fibers to different, mainly slow, motor units. We further speculate that high-level activity either by voluntary exercise or functional electrical stimulation may be applied at any age to save neurons from disorders secondary to inactivity (55) and to counteract muscle atrophy in other neuromuscular or metabolic diseases (56).
Although the subjects of this study were not masters athletes, the intensity of recreational training reported is something that the general population may achieve, particularly if properly motivated by specialists in the field. We show that recreational levels of activity are very effective in driving seniors toward improved functional performance and rearrangement of muscle fiber type. In particular, these levels of exercise seem to have beneficial effects on reinnervation of muscle fibers, resulting in preservation of muscle function, size, and structure, thereby delaying the functional decline and loss of independence that are common in late aging.
The expert assistance of Christian Hofer, PhD, is gratefully acknowledged.
1. Morrison JH, Baxter MG . The ageing cortical synapse: Hallmarks and implications for cognitive decline. Nat Rev Neurosci. 2012; 13: 240–50
2. Small SA, Schobel SA, Buxton RB, et al. A pathophysiological framework of hippocampal dysfunction in ageing and disease. Nat Rev Neurosci. 2011; 12: 585–601
3. Balistreri CR, Candore G, Accardi G, et al. NF-κB pathway activators as potential ageing biomarkers: Targets for new therapeutic strategies. Immun Ageing. 2013; 10: 24
4. Erickson KI, Voss MW, Prakash RS, et al. Exercise training increases size of hippocampus and improves memory. Proc Natl Acad Sci USA. 2011; 108: 3017–22
5. Aagaard P, Suetta C, Caserotti P, et al. Role of the nervous system in sarcopenia and muscle atrophy with aging: Strength training as a countermeasure. Scand J Med Sci Sports. 2010; 20: 49–64
6. Ohlendieck K . Proteomic profiling of fast-to-slow muscle transitions during aging. Front Physiol. 2011; 2: 105
7. Gutmann E, Hanzlikova V . Motor unit in old age. Nature. 1966; 209: 921–22
8. Tomlinson BE, Walton JN, Rebeiz JJ . The effects of ageing and of cachexia upon skeletal muscle. A histopathological study. J Neurol Sci. 1969; 9: 321–46
9. Scelsi R, Marchetti C, Poggi P . Histochemical and ultrastructural aspects of m. vastus lateralis in sedentary old people (aged 65–89 years). Acta Neuropathol. 1980; 51: 99–105
10. Urbanchek MG, Picken EB, Kalliainen LK, et al. Specific force deficit in skeletal muscles of old rats is partially explained by the existence of denervated muscle fibers. J Gerontol A Biol Sci Med Sci. 2001; 56: B191–97
11. Rowan SL, Rygiel K, Purves-Smith FM, et al. Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle. PLoS ONE. 2012; 7: e29082
12. Tomlinson BE, Irving D . The numbers of limb motor neurons in the human lumbosacral cord throughout life. J Neurol Sci. 1977; 34: 213–19
13. Doherty TJ, Vandervoort AA, Taylor AW, et al. Effects of motor unit losses on strength in older men and women. J Appl Physiol. 1993; 74: 868–74
14. Larsson LX . Motor units: Remodeling in aged animals. J Gerontol A Biol Sci Med Sci. 1993; 50: 91–95
15. Andersen JL . Muscle fibre type adaptation in the elderly human muscle. Scand J Med Sci Sports. 2003; 13: 40–47
16. Luff AR . Age-associated changes in the innervation of muscle fibers and changes in the mechanical properties of motor units. Ann N Y Acad Sci. 1998; 854: 92–101
17. Mitchell WK, Williams J, Atherton P, et al. Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength: A quantitative review. Front Physiol. 2012; 3: 260
18. Zhou MY, Klitgaard H, Saltin B, et al. Myosin heavy chain isoforms of human muscle after short-term spaceflight. J Appl Physiol. 1995; 78: 1740–44
19. Schiaffino S, Reggiani C . Fiber types in mammalian skeletal muscles. Physiol Rev. 2011; 91: 1447–531
20. Kern H, Hofer C, Mödlin M, et al. Stable muscle atrophy in long-term paraplegics with complete upper motor neuron lesion from 3- to 20-year SCI. Spinal Cord. 2008; 46: 293–304
21. Kern H, Carraro U, Adami N, et al. Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower motor neuron lesion. Neurorehabil Neural Repair. 2010; 24: 709–21
22. D’Antona G, Pellegrino MA, Adami R, et al. The effect of ageing and immobilization on structure and function of human skeletal muscle fibres. J Physiol (Lond). 2003; 552: 499–511
23. Carraro U, Catani C, Belluco S, et al. Slow-like electrostimulation switches on slow myosin in denervated fast muscle. Exp Neurol. 1986; 94: 537–53
24. Carraro U . Modulation of trophism and fiber type expression of denervated muscle by different patterns of electrical stimulation. Basic Appl Myol. 2002; 12: 263–73
25. Mayne CN, Mokrusch T, Jarvis JC, et al. Stimulation-induced expression of slow muscle myosin in a fast muscle of the rat. Evidence of an unrestricted adaptive capacity. FEBS Lett. 1993; 327: 297–300
26. Salmons S . Exercise, stimulation and type transformation of skeletal muscle. Int J Sports Med. 1994; 15: 136–41
27. Midrio M . The denervated muscle: Facts and hypotheses. A historical review. Eur J Appl Physiol. 2006; 98: 1–21
28. Meltzer DE . Age dependence of Olympic weightlifting ability. Med Sci Sports Exerc. 1994; 26: 1053–67
29. McNeil CJ, Doherty TJ, Stashuk DW . Motor unit number estimates in the tibialis anterior muscle of young, old, and very old men. Muscle Nerve. 2005; 31: 461–67
30. Leyk D, Rüther T, Wunderlich M, et al. Physical performance in middle age and old age: Good news for our sedentary and aging society. Dtsch Arztebl Int. 2010; 107: 809–16
31. Wroblewski AP, Amati F, Smiley MA, et al. Chronic exercise preserves lean muscle mass in masters athletes. Phys Sportsmed. 2011; 39: 172–78
32. Lexell J, Downham DY . The occurrence of fiber-type grouping in healthy human muscle: A quantitative study of cross-sections of whole vastus lateralis from men between 15 and 83 years. Acta Neuropathol (Berl). 1991; 81: 377–81
33. Sunnerhagen KS, Grimby G . Muscular effects in late polio. Acta Physiol Scand. 2001; 171: 335–40
34. Vetter C, Reichmann H, Pette D . Microphotometric determination of enzyme activities in type-grouped fibres of reinnervated rat muscle. Histochemistry. 1984; 80: 347–51
35. Coggan AR, Spina RJ, Rogers MA, et al. Histochemical and enzymatic characteristics of skeletal muscle in master athletes. J Appl Physiol. 1990; 68: 1896–901
36. Trappe S . Master athletes. Int J Sport Nutr Exerc Metab. 2001; 11: S196–207
37. Wright VJ, Perricelli BC . Age-related rates of decline in performance among elite senior athletes. Am J Sports Med. 2008; 36: 443–50
38. Mosole S, Rossini K, Kern H, et al. Significant increase of vastus lateralis reinnervation in 70-year sportsmen with a lifelong history of high-level exercise. Eur J Transl Myol Basic Appl Myol. 2013; 23: 117–22
39. Zampieri S, Rossini K, Carrao U, et al. Morphometry of skeletal muscle in sedentary elderly and senior sportsmen. Eur J Transl Myol Basic Appl Myol. 2012; 22: 13
40. Kern H, Loefler S, Burggraf S, et al. Electrical stimulation counteracts muscle atrophy associated with aging in humans. Eur J Transl Myol Basic Appl Myol. 2013; 23: 105–8
41. Boncompagni S, d’Amelio L, Fulle S, et al. Progressive disorganization of the excitation-contraction coupling apparatus in aging human skeletal muscle as revealed by electron microscopy: A possible role in the decline of muscle performance. J Gerontol A Biol Sci Med Sci. 2006; 61: 995–1008
42. Rossini K, Zanin ME, Podhorska-Okolow M, et al. To stage and quantify regenerative myogenesis in human long-term permanent denervated muscle. Basic Appl Myol. 2002; 12: 277–86
43. Boncompagni S, Kern H, Rossini K, et al. Structural differentiation of skeletal muscle fibers in the absence of innervation in humans. Proc Natl Acad Sci USA. 2007; 104: 19339–44
44. Zampieri S, Valente M, Adami N, et al. Polymyositis, dermatomyositis and malignancy: A further intriguing link. Autoimmun Rev. 2010; 9: 449–53
45. Zampieri S, Doria A, Adami N, et al. Subclinical myopathy in patients affected with newly diagnosed colorectal cancer at clinical onset of disease: Evidence from skeletal muscle biopsies. Neurol Res. 2010; 32: 20–25
46. Kern H, Boncompagni S, Rossini K, et al. Long-term denervation in humans causes degeneration of both contractile and excitation-contraction coupling apparatus that can be reversed by functional electrical stimulation (FES). A role for myofiber regeneration? J Neuropathol Exp Neurol. 2004; 63: 919–31
47. Kern H, Carraro U, Adami N, et al. One year of home-based daily FES in complete lower motor neuron paraplegia: Recovery of tetanic contractility drives the structural improvements of denervated muscle. Neurol Res. 2010; 32: 5–12
48. Dickson G, Gower HJ, Barton CH, et al. Human muscle neural cell adhesion molecule (N-CAM): Identification of a muscle-specific sequence in the extracellular domain. Cell. 1987; 50: 1119–30
49. Kern H, Pelosi L, Coletto L, et al. Atrophy/hypertrophy cell signaling in muscles of young athletes trained with vibrational-proprioceptive stimulation. Neurol Res. 2011; 33: 998–1009
50. Gava P, Kern H, Carraro U . Age-related decline of muscle power in track and field master athletes indicates a lifespan of 110 years. Eur J Transl Myol Basic Appl Myol. 2013; 23: 45
51. Carraro U, Catani C, Biral D . Selective maintenance of neurotrophically regulated proteins in denervated rat diaphragm. Exp Neurol. 1979; 63: 468–75
52. Carraro U, Catani C, Dalla Libera L . Myosin light and heavy chains in rat gastrocnemius and diaphragm muscles after chronic denervation or reinnervation. Exp Neurol. 1981; 72: 401–12
53. Pette D, Vrbová G . What does chronic electrical stimulation teach us about muscle plasticity? Muscle Nerve. 1999; 22: 666–77
54. Henneman E, Somjen G, Carpenter DO . Functional significance of cell size in spinal motoneurons. J Neurophysiol. 1965; 28: 560–80
55. Herrera-Rincon C, Torets C, Sanchez-Jimenez A, et al. Chronic electrical stimulation of transected peripheral nerves preserves anatomy and function in the primary somatosensory cortex. Eur J Neurosci. 2012; 36: 3679–90
56. Wang XH, Mitch WE . Muscle wasting from kidney failure—A model for catabolic conditions. Int J Biochem Cell Biol. 2013; 45: 2230–38
Aging; Coexpression of fast and slow myosin heavy chains; Denervation and reinnervation; Fiber-type grouping; Human skeletal muscle; Recreational sport activity
Supplemental Digital Content
Copyright © 2014 by the American Association of Neuropathologists, Inc.
Highlight selected keywords in the article text.