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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:995-1008 (2006)
© 2006 The Gerontological Society of America

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

Simona Boncompagni, Luigi d'Amelio, Stefania Fulle, Giorgio Fanò and Feliciano Protasi

1 Interuniversity Institute of Myology, Ce.S.I. Center of Research on Aging, University G. d'Annunzio, Chieti, Italy.
2 San Liberatore Hospital, Atri, Italy.

Address correspondence to Feliciano Protasi, PhD, Associate Professor, CeSI, Centro Scienze dell'Invecchiamento, Università degli Studi G. d'Annunzio, Chieti, CH I-66013, Italy. E-mail: fprotasi{at}unich.it


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
An impairment of the mechanisms controlling the release of calcium from internal stores (excitation–contraction [EC] coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging (EC uncoupling theory). EC coupling in muscle fibers occurs at the junctions between sarcoplasmic reticulum and transverse tubules, in structures called calcium release units (CRUs). We studied the frequency, cellular localization, and ultrastructure of CRUs in human muscle biopsies from male and female participants with ages ranging from 28 to 83 years. Our results show significant alterations in the CRUs' morphology and cellular disposition, and a significant decrease in their frequency between control and aged samples: 24.4/100 µm2 (n = 2) versus 11.6/100 µm2 (n = 7). These data indicate that in aging humans the EC coupling apparatus undergoes a partial disarrangement and a spatial reorganization that could interfere with an efficient delivery of Ca2+ ions to the contractile proteins.

AGING is a complex physiological process associated with a significant decline in neuromuscular function and performance, which may affect quality of life for elderly persons (1–6) and which causes a dramatic increase in health care costs (7–9). Today, in which the average life expectancy is increasing and the number of elderly individuals continues to grow, a greater understanding of the mechanisms leading to aging is of central importance in the prevention of disability and the optimization of the independence of elderly persons.

One of the most striking effects of aging is an inevitable reduction of muscle mass that occurs in various measures in all individuals (10–12). The muscle wasting that accompanies aging represents the final result of a variety of changes, such as the loss of motor units due to progressive denervation, a shift to slower fiber types, an altered Ca2+ homeostasis, mitochondrial alterations, and oxidative stress (5,13–16). Although the impact of aging on skeletal muscle has been extensively investigated, to date the precise mechanisms that underlie the functional impairment associated to it are not yet completely understood.

Recently, it has been proposed that a reduction in the supply of Ca2+ ions available for triggering muscle contraction may be one of the key factors in explaining age-related muscle weakness (15,17–20). The reasoning for this would be an impairment in the events linking the action potential generated at neuromuscular junctions to the Ca2+ release from the sarcoplasmic reticulum (SR), that is, the excitation–contraction (EC) coupling mechanism (18,21,22). EC coupling in muscle fibers occurs in specialized structures, known as Ca2+ release units (CRUs), formed by the close apposition of two membrane systems: the transverse tubules, carrying the sarcolemmal depolarization into the fiber interior, and the SR terminal cisternae, containing the Ca2+ needed for activating muscle contraction (23–26). In mature skeletal muscle, a central transverse tubule usually forms junctions, or couplons, with two SR cisternae forming a triad (27). The depolarization coming from the neuromuscular junction enters the fiber through the transverse tubule network where it is detected by the dihydropyridine receptors (DHPRs), voltage sensors localized in the tubule membrane (28–30), which are in direct communication with the closely apposed Ca2+ release channels of the SR, the ryanodine receptors (RyRs). The RyRs are extremely large proteins, also known as feet, which physically span the junctional gap between transverse tubule and SR membrane (27,31,32). The physical coupling between DHPRs and RyRs results in the sudden opening of the RyR–Ca2+ release channels with a consequent massive efflux of Ca2+ from the SR lumen into the myoplasm (33–37).

A fairly recent theory suggests that one of the major determinants of the progressive decline in muscle strength during aging is an EC uncoupling between DHPRs and RyRs in the CRUs (18,21). This uncoupling would be caused by a significant reduction in the transverse tubule membranes of the DHPR {alpha}1S subunits, the central components of DHPRs forming the L-type Ca2+ channel and containing the voltage sensor. This lack of {alpha}1S DHPRs would lead to an increased percentage of RyRs in the SR membranes being uncoupled from the voltage sensors. Renganathan and colleagues (21) demonstrated that the main functional consequence of this uncoupling is an inefficient transmission of the sarcolemmal depolarization to the RyRs, resulting in a weakened mechanical response. However, conflicting results can also be found in the literature: A persistent expression of {alpha}1S (and {alpha}2S) DHPRs in senescent human muscle has been reported, suggesting that, at least in humans, DHPRs expression is preserved during aging of skeletal muscle fibers (38).

In the present work we have studied, using transmission electron microscopy (EM), the ultrastructure and geometry of the EC coupling apparatus in human biopsy specimens from the vastus lateralis and gluteus medius muscles of male and female participants between the ages of 28 and 83 years to determine possible structural alterations, which may explain the impaired functioning of the EC coupling mechanism in aging muscle. Our results show significant modifications of CRU ultrastructure and a drastic reduction in their frequency in elderly persons.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Collection of Human Muscle Biopsies
Biopsy specimens from either vastus lateralis or gluteus medius muscles were obtained during orthopedic surgery from patients under complete anesthesia. The patients had suffered hip fractures, and the collection of specimens was carried out after informed consent. All participants had no history of neuromuscular disorders, and all led a predominantly sedentary life. In addition, before including each participant in our study, we used the following selection criteria: (a) all patients were quite healthy individuals with a normal life and all patients reported that small daily walks were part of their daily routine; (b) none of the patients had been professional athletes or were actively exercising, neither at the time of the injury nor during life (e.g., no runners, bikers, soccer players); (c) for all patients, no more than 3 days elapsed from injury to the surgical procedure (to avoid any significant effect of muscle atrophy). Ten human muscle biopsies were collected, and the specimens were divided into two major groups (see Table 1): young (two specimens, 28 and 34 years of age, respectively) and aged (seven specimens, from 71 to 83 years of age). In addition, one sample was collected from a 58-year-old male and classified as middle-aged. Small bundles of each sample were fixed in glutaraldehyde and processed for ultrastructural examination as described below.


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  		Table 1. Quantitative and Qualitative Analysis of CRUs in Controls and Aged Specimens.

 
Preparation of Muscle Specimens for Transmission EM
Some of the biopsy specimens were fixed in 3.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer (pH 7.4, room temperature) for 2 hours followed by buffer rinse and post fixation for 1 hour in 2% osmium tetroxide. The specimens were rapidly dehydrated in graded ethanol and acetone, infiltrated with EPON-acetone (1:1) mixture for 2 hours, and embedded in EPON. For transverse tubule staining, small fiber bundles were fixed in 3.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer (pH 7.2, 4°C) containing 75 mM CaCl2 for 1–2 hours. These specimens were then postfixed in a 2% OsO4–0.8% K3Fe(CN)6 mixture for 1–2 hours followed by rinse with 0.1 M sodium cacodylate buffer with 75 mM CaCl2 (39,40). The specimens were rapidly dehydrated in graded ethanol and acetone, infiltrated with EPON–acetone (1:1) mixture for 2 hours, and embedded in EPON. Ultrathin sections (about 40 nm) were cut using a Leica Ultracut R (Leica Microsystems, Vienna, Austria) and a Diatome diamond knife (CH-2501; Diatome Ltd., Biel, Switzerland) and stained in 4% uranyl acetate and lead citrate. All sections were examined, after staining, with an FP 505 Morgagni Series 268D electron microscope (Philips, Int., Rome, Italy) at 60 kV equipped with Megaview III digital camera (Soft Imaging System, Münster, Germany).

Quantitation of CRUs
The density of junctions was determined by counting the number of junctions in EM images from seven different fibers for each specimen of the aged group (71–83 years), whereas 14 different fibers were analyzed for the 28-, 34-, and 58-year-old samples. In each fiber, 10 micrographs (all at the same magnification, x11,000, and with no overlapping regions) were randomly collected from longitudinal sections, for a total of 70 or 140 electron micrographs for each biopsy specimen. CRUs were marked and counted in each micrograph. Only those structures formed by the association of SR and transverse tubules and containing RyRs were identified as CRUs. In those cases in which electron-dense structures (feet) were not visible between the SR and transverse tubule membranes, the structures were not identified as CRUs because they do not contain the Ca2+ release channels. These structures were classified either as nonjunctional regions of the sarcotubular system or as disarranging triads, and were not considered in this analysis. The results of the CRU counting are presented in Table 1 as number of junctions per 100 µm2 of section area ± standard deviation (SD). We have calculated the SD also as a percentage of the mean frequency (column 1, in parenthesis) and standardized it for each sample by multiplying it by the mean of the sample means and dividing by its own mean (column 2). The same micrographs were used to calculate the percentage of dyads and of longitudinal junctions relative to the total number of CRUs reported in Table 1 (columns 3 and 4).

Measurement of Average Size of Junctional Arrays of RyR-Feet and Average Width of SR/Transverse Tubule Junctional Gap
The average size of feet arrays was measured in micrographs taken at x22,000 magnification. For each fiber, 10 micrographs were randomly collected from longitudinal sections for a total of 50 electron micrographs for each biopsy specimen. The length of feet arrays in the sectioned profiles of triads and dyads was determined by measuring the distances between the two external feet in each RyR array, as shown in Figure 7 (A–D, dashed lines), by using the Soft Imaging System. A total of 271–587 couplons were measured (see Table 2, column 2) in five different fibers for each of the examined specimens: two young (28 and 34 years) and three aged (71, 73, and 80 years). Results in Table 2 (column 3) are shown as nm ± standard error of the mean (SEM), and in parenthesis is reported the percentage of couplons containing more than two rows of feet, among those junctions that are sectioned transversally, that is, located between myofibrils and correctly oriented.


Figure 07
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  		Figure 7. Measurement of average size of junctional arrays of ryanodine receptor (RyR)-feet and average width of sarcoplasmic reticulum (SR)/transverse tubule (T-tubule) junctional gap. A and C show, respectively, how the length of RyR arrays was measured in electron micrographs from a young and an aged specimen, respectively (dashed lines). Length of feet arrays was determined measuring the distances between the two external feet in each RyR array as shown in B and D in all types of junctions, either triads, dyads, or longitudinal junctions. E and F, Width of the junctional gap between SR and T-tubule was determined measuring the linear distance between the two external leaflets of SR and T-tubule membranes on both sides of feet as shown in E. Bars: 0.05 µm. CSQ = calsequestrin

 

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  		Table 2. Average Junctional Length Occupied by RyR-Feet in Young and Aged CRUs.

 
In contrast, the width of the SR/transverse tubule junctional gap was calculated from measurements obtained in a different set of micrographs taken at x56,000 or higher magnification (see Figure 7F). Measurements were taken on both sides of feet (as shown in Figure 7E); the linear distance between the two external leaflets of the SR and transverse tubule membranes was measured. A total of 43–50 couplons (344–400 measurements) were measured in five different fibers for each of the five examined specimens: two young (28 and 34 years) and three aged (73, 75, and 80 years). Results of these measurements are shown in Table 3, column 3 and are shown as nm ± SEM.


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  		Table 3. Average Width of the SR/Transverse Tubule Junctional Gap.

 
Statistical Analysis
Means, SEMs, and SDs were determined from individual data values by descriptive statistics using GraphPad Prism (version 4.0; GraphPad Software Inc., San Diego, CA). To evaluate the differences between the young and the aged groups, the data were compared by analysis of variance (ANOVA) with either Tukey's test applied post hoc or Student's t test where appropriate. Values of p <.05 (95% confidence interval) were considered to be significant.

Figures were mounted and labeled using Adobe Photoshop version 7.0.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Morphology of the EC Coupling Apparatus in Young Muscle Fibers
In mature skeletal muscle (28 and 34 years, males), the EC coupling apparatuses have a distinctive ultrastructure and orientation. CRUs are located almost exclusively opposite the sarcomere I–A junctions, one on each side of the Z line (Figure 1A, arrows). These junctions, named triads, are usually formed by three elements, two SR vesicles closely apposed to a central transverse tubule that has a flat profile (Figure 1B–D). CRUs are frequent in the fibers of specimens from young individuals (see also Table 1, column 1), fairly evenly distributed (arrows in Figure 1A), and oriented transversally with respect to the long axis of myofibrils. The SR lumen contains an electron-dense matrix (calsequestrin), and the junctional gap between the SR and transverse tubule membrane is spanned by the cytoplasmic domain of RyRs (the feet, which are indicated by arrows in Figure 1D). In human skeletal muscle, as in most mammalians, feet usually are located in two parallel rows on either side of the transverse tubule. Dyads, junctions formed by a transverse tubule and only one SR vesicle, and longitudinal triads and dyads, formed by the SR and longitudinally oriented transverse tubules, are extremely rare in adult muscle. Quantitative analysis of the frequency and relative percentage of the different types of CRUs are reported in Table 1 and described below.


Figure 01
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  		Figure 1. Ultrastructure and geometry of the excitation–contraction coupling apparatus in young human skeletal muscle. A, In mature skeletal muscle, calcium release units (CRUs; arrows) are usually located at sarcomere I–A junctions, and their distribution is fairly uniform within the fiber. B–D, Higher magnification images provide a better view of the fine ultrastructure of CRUs, which in adult muscle are mostly in the form of triads. The junctional gap between the sarcoplasmic reticulum (SR) and transverse tubule (TT) membrane is spanned by the cytoplasmic domain of ryanodine receptors (RyRs), the feet (arrows in D). Bars: A, 0.5 µm; B–D, 0.1 µm

 
Morphology of the EC Coupling Apparatus in Aged Muscle
EM analysis of specimens from middle-aged (58 years, n = 1) and aged (71–83 years, n = 7) human biopsies allowed the determination of several differences and/or alterations of the EC coupling apparatus in these samples with respect to specimens from younger individuals (controls). The first important observation is that the overall number of SR/transverse tubule junctions appears to be considerably lower in the fibers of the aged participants. The two electron micrographs in Figure 2A and B show regions of fibers in which the CRUs are indicated by arrows (A, 75 years; B, 77 years). Comparison of these two panels with Figure 1A, depicting an area of similar size from a younger individual (34 years), shows a well visible difference in the CRU frequency between young and aged participants. This observation is confirmed by the determination of the average CRU density per unit area of thin section reported in Table 1, column 1. The second important observation is that, in aged skeletal muscle, the density of CRUs is quite variable even within different regions of the same fiber (compare panels A and B of Figure 4). The decrease in CRU frequency and the variability in their density results often in areas that are completely devoid of CRUs, such as the one shown in Figure 4D. By contrast, CRUs are quite uniformly distributed in fibers from younger muscles (Figure 1A). The analysis of the SD presented in Table 1 strengthens our visual observation, as both the SD calculated as a percentage of the CRU frequencies and the standardized SD (columns 1 and 2) are much higher in aged samples than in young specimens. This analysis confirms that aged samples have a variable CRU content from one area to another.


Figure 02
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  		Figure 2. Ultrastructure and geometry of the excitation–contraction coupling apparatus in aging human skeletal muscle. The two electron micrographs show fiber regions from two aged specimens in which the calcium release units (CRUs) are indicated by arrows. The frequency of CRUs in these images is low when compared to the control specimen (compare with
Figure 1A). Bar: 0.5 µm

 

Figure 04
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  		Figure 4. Uneven distribution of calcium release units (CRUs) in aged muscle. Aged skeletal muscle fibers contain areas in which CRUs are quite frequent and uniformly distributed (A), as well as other areas in which the triads are sparse (B and C) or even completely absent (D). A and B, and C and D, were (respectively) collected from the same fiber. Bar: 0.5 µm

 
In addition to the varying in frequency, the SR/transverse tubule junctions also appear more structurally variable in aging specimens than in control samples. Whereas many CRUs have the standard morphology (triads, Figure 5A and B), the frequency of (a) dyads, that is, junctions formed by the apposition of a transverse tubule with only one SR vesicle (Figure 5C and D), and (b) junctions with a large profile, that is, junctions showing more than two feet on each side of the transverse tubule (see Figures 6 and 7) is higher in aging fibers than in young specimens (see Tables 1 and 2). Junctions with a larger profile can be found also in young fibers mostly located in proximity of the I–A band transition, when the section grazes the surface of the myofibrils (i.e., transverse tubules are sectioned longitudinally). In aging specimens, however, these junctions with a large profile are also found at the classic location between myofibrils, where transverse tubules are likely sectioned transversally, and even longitudinally oriented, that is, formed by SR terminal cisternae apposed to longitudinal tubules (Figure 6). Specific staining of the transverse tubule network in aging muscle confirms that the frequency of longitudinally oriented tubules increases in aged samples when compared to controls (Figure 6C). These longitudinally oriented tubules often form junctions with the SR terminal cisternae that are located in the vicinity of the M lines of sarcomeres (Figure 6A). Whereas CRUs changes frequency, orientation, and size with increasing age, no differences were detected in the size of junctional gap between transverse tubule and SR, that in aging specimens remain unvaried (Figure 7E and F; Table 3).


Figure 05
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  		Figure 5. Altered morphology of calcium release units (CRUs) in aging muscle fibers. In the aged samples, although there are still many CRUs with a fairly normal appearance (triads, A and B), we found a higher than normal percentage of dyads (C and D), and also structures that look like junctions that are disassembling (E and F). Two observations indicate that the latter represent triads undergoing a process of disarrangement: (a) they are always localized between myofibrils in proximity of the sarcomere I–A junction, where CRUs are normally placed; and (b) they are mostly found in areas in which the density of triads is declining and/or in which normal junctions are completely missing. Bar: 0.1 µm. SR = sarcoplasmic reticulum; TT = transverse tubule; RyRs = ryanodine receptors

 

Figure 06
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  		Figure 6. Junctions with a larger profile are more frequent in aging samples, often associated to longitudinally oriented transverse tubules (TTs). Junctions with a larger profile are more frequent in aged specimens and may be found either longitudinally oriented, often localized in the vicinity of M lines (A), or even at the classic transverse location between myofibrils (B). C, Specific staining of TTs shows that, in aging muscle, the frequency of longitudinally oriented tubules increases when compared to control samples (data not shown). Arrows indicate TTs that run parallel to myofibrils. Bars: A and B, 0.1 µm; C, 0.5 µm. SR = sarcoplasmic reticulum

 
Finally, in the aged specimens we also often found structures that are quite disordered (Figure 5E and F) but are still recognizable as SR/transverse tubule junctions due to their location near the A–I junction. Such structures are never, or very rarely, found in control samples. In the aging muscles, these junctions are usually found in areas in which the density of triads is declining and/or in which normal junctions are completely missing, and we assume that they represent a stage in the disassembly of CRUs. These disassembling units were not taken into consideration for the quantitative analysis that is presented below.

Quantitative Analysis of EC Coupling Apparatus
The visual impression of a decreased frequency of CRUs with increasing age (Figures 1 and 2) has been confirmed by the quantitative analysis reported in Table 1 and Figure 3. In the young samples, the number of CRUs per unit area of thin section is significantly higher than that in the aged sample groups: 24.4/100 µm2 (average from the 28 and 34 year samples, n = 2) versus 11.6/100 µm2 (average from the 71–83 year samples, n = 7). The number of CRU profiles per section area is directly proportional to the number of CRUs per fiber volume. Only SR/transverse tubule junctions that clearly contain electron-dense structures (i.e., feet) between the two membranes were considered in this quantitative analysis (see Materials and Methods). ANOVA indicates that the difference between the two young groups and each aged group are highly significant (p <.001 in every case), whereas there are no significant differences among young and among aged specimens. The value from the middle-aged sample (58 years) is in accordance with a progressive decrease in the overall number of junctions in the fibers. We have plotted all the data collected from our specimens (each measurement obtained in every micrograph) to verify the data distribution around the average. These data are distributed around the average as a Gaussian distribution in all samples, only five of which are presented in Figure 3. In aging samples there is a clear shift to the left of the histogram peaks of the older samples indicating the decreased frequency of CRUs. The SD in Table 1 is also shown as a percentage of the calculated mean frequency (column 1) and is standardized for each sample (column 2). In both cases, the SDs are much higher in aged samples indicating a higher variability in these measurements than in those from the young specimens: 21%–43% versus 13%–20% and 1.9–2.9 versus 3.2–6.5, respectively. These analyses strengthen the visual observations presented in Figure 4, in which we noted a different frequency of CRUs among different areas even within the same fiber. The overall decrease in CRU frequencies accompanied by the high variability in CRU frequencies within the aged samples points to a disorganization of the EC coupling apparatus that affects some areas more than others.


Figure 03
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  		Figure 3. Distribution of calcium release unit (CRU) frequencies in young and aged biopsies. Data collected were plotted in histograms to verify the data distribution around the average. Although data from all 10 specimens were plotted in this manner, data from only five are presented. In all the samples, data appear to be distributed around the average as a Gaussian distribution. Changes in frequency of the CRUs between the young and aged samples are shown by the shift to the left of the histogram peaks of the older samples

 
In addition to the frequency of CRUs, we have also determined: (a) which percentage of junctions is in the form of dyads and longitudinally oriented junctions (Table 1); (b) the average size of feet arrays in transverse tubule/SR junctional domains (Table 2); and (c) the average width of the junctional gap between SR and transverse tubule (Table 3). Dyads and longitudinally oriented junctions are extremely rare in adult muscle, whereas their percentage over the total CRU number increases with age (see Table 1, columns 3 and 4). Interestingly, the percentage of dyads in the sample from the 58-year-old participant is unusually high compared to all the other samples. This observation suggests that dyads may represent an intermediate step in the disarrangement of triads. To determine the average size of feet arrays in the CRUs, we measured the RyR-feet arrays in all types of junctions, either triads, dyads, or longitudinal junctions, as shown in Figure 7A–D. This analysis allowed us to determine that the average size of couplons is higher in aging samples (see Table 2 and Figures 6 and 7). The ANOVA clearly indicates that the differences between each young and aged specimen are highly significant (p <.001 in every case). This result suggests a higher frequency of CRUs presenting more than two rows of feet on each side of the transverse tubule. These measurements could, however, be affected by the altered spatial orientation of couplons in aged fibers. To rule out this possibility, we have selected, among all the couplons, the ones in which we are confident that the transverse tubule is sectioned transversally (i.e., located at the I–A junction, between adjacent myofibrils, and likely containing a transverse tubule that is transversally oriented), and we have calculated which percentage of them presents more than two feet on each side of the transverse tubule. These percentages are reported in Table 2 (column 3, in parenthesis), and are significantly higher in aged than in young samples (11%–18% vs 5%–6%). This analysis indicates that the increased size of couplons in aged fibers is not an artefact of the orientation of triads, and it reveals the existence of a subpopulation of junctions bearing multiple rows of RyR-feet instead of the regular two rows found in the large majority of young fibers (see Figures 6 and 7). The average length of the feet arrays is directly proportional to the area occupied by Ca2+ release channels at junctional regions between SR and transverse tubules.

To determine possible alterations of the structural coupling between external and internal membranes, we measured the average width of the junctional gap between SR and transverse tubule, respectively, in two young (28 and 34 years) and three aged (73, 75, and 80 years) specimens (see Materials and Methods). The results of this analysis (Table 3, column 3; Figure 8) indicate that values of young versus aged specimens are not different. Scattergrams were plotted for each of the five samples contained in Table 3. For clarity only one control and one aged specimens are shown in Figure 8. The measurements within each sample vary over a range of values that is similar for control and aged muscle biopsies. Actually, the averages in each of the samples are extremely similar (lowest value: 12.1 nm, 75 years; highest value: 12.9 nm, 34 years). This result suggests that the structural coupling between SR and transverse tubule is quite rigid and does not change much during fixation or with age.


Figure 08
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  		Figure 8. Scattergram of junctional gap width indicates no variation in data distribution between aged and young samples. Scattergrams were plotted for each of the five samples analyzed, but (for clarity) only one control and one aged specimen are shown. Each spot represents a single measurement of the distance between the external leaflets of transverse tubule (TT) and sarcoplasmic reticulum (SR) membranes. The measurements within each sample vary over a range of values that is similar for control and aged muscle biopsies. Inset: minimum, 25% percentile, median (box), 75% percentile, and maximum

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Activation of contraction in muscle fibers is triggered by a sudden increase in intracellular Ca2+, concentration a mechanism controlled by the cross-communication between DHPRs and RyRs and known as EC coupling (33,41–43). In aged muscle, a significant reduction in the amount of Ca2+ ions available for triggering and sustaining muscle contraction has been reported and proposed to be one of the reasons for the reduction in specific force of muscle fibers (18,21,22). The results presented in this article show that aging in human skeletal muscle causes significant alterations of the membrane systems involved in EC coupling, that is, the triadic junctions formed between the SR terminal cisternae and transverse tubules, containing the two key players in the mechanism, both DHPRs and RyRs. These alterations consist primarily of a progressive disarrangement of triads resulting in a drastic reduction in the overall number of Ca2+ release sites available for triggering the sliding of contractile filaments and the consequent generation of force (Table 1; Figure 3). These findings provide a sound structural explanation for the impaired transduction of the action potential into an efficient increase in intracellular Ca2+ concentration reported in the literature (18,21,22).

Contraction of muscle fibers is mediated by sudden increases in intracellular Ca2+ known as Ca2+ transients. Ca2+ transients represent the final result of multiple Ca2+ release events arising simultaneously from multiple sites (44–46), that is, the triads, which in adult muscle are distributed in a very orderly fashion in the fiber interior, next to the I–A junction of each sarcomere (see Figure 1A). In Figure 9, we have attempted to model a possible scenario of the initial phase of muscle activation in two electron micrographs depicting a region from a young fiber (top panels) and from an aged fiber (bottom panels), respectively. Small arrows point to each visible triad in the two left panels, whereas the initial phase of Ca2+ release is represented by black circles in the two right panels. These circles appear darker in the center, mimicking a higher concentration of Ca2+ in the vicinity of release sites, that is, the triads, and fading with increasing distance from the center. This speculative model tries to show how a low number of release sites in the aged specimen could result in an inefficient activation of the contractile apparatus. The decreased supply of Ca2+ ions to the myofilaments could result in an impaired force output during muscle activity.


Figure 09
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  		Figure 9. Modeling of the initial phase of Ca2+ release in adult and aged fibers. Ca2+ release from the sarcoplasmic reticulum (SR) is modeled in two regions, respectively, from a young fiber (top panels, from
Figure 1A) and from an aged fiber (bottom panels, from
Figure 2A). The release of Ca2+ arising from all the visible release sites (arrows in left panels) are modeled by black circles in the right panels, darker in the center mimicking a higher Ca2+ in the vicinity of triads. A low number of release sites in the aged specimen could result in an inefficient delivery of Ca2+ to the contractile proteins. Bars: 0.5 µm

 
Several of the findings presented in this article support the hypothesis that the decrease in CRU frequency is due to progressive disorganization of the EC coupling apparatus. The CRU frequency data plotted in the histograms of Figure 3 show a clear shift to the left of the curve's peaks that indicates a significant increase in number of areas with fewer triads in aged samples. The high variability within data from aged samples (Table 1, columns 1 and 2) points to a disorganization of the EC coupling apparatus that affects some areas more than others. However, from these data it is difficult to determine whether these alterations are the result of other changes, such as a fast-to-slow fiber type switching of some fibers and/or nuclear domains within the same fiber (47–52). Although we have not been able to perform fiber typing in our samples, many scientific reports have dealt with fiber typing in humans, even in muscle groups that we have used in our study, that is, the vastus lateralis. Whereas in some cases a small decrease in the relative percentage of the glycolytic type II fibers has been reported (53–55), there many other articles indicating no changes in the relative percentage of slow and fast fibers (50,56–57). Although the present knowledge in the field does not completely exclude the possibility of some fiber type switching in our samples, we are quite confident that the main cause of the massive decrease in number of CRUs is a progressive degeneration of EC apparatus during aging. There are, in fact, some direct EM observations that also lead to this conclusion: (a) areas in which junctions are clearly missing, such as the one in Figure 4D, are not seen in the control/young muscle; (b) disarranging triads, such as the ones in Figure 5E and F, are found exclusively in aged fibers, always localized between myofibrils and in proximity of the sarcomere I–A junction, and are mostly found in areas in which the density of triads is declining and/or in which normal junctions are completely missing; and (c) in the middle-aged sample (58 years), the percentage of dyads is much higher than that in other samples, suggesting that dyads may represent an intermediate step in the disappearance of triads, representing indeed triads with a missing element.

Fairly recent publications have proposed that the partial impairment of the EC coupling mechanism may be due to a partial uncoupling between RyRs and DHPRs (EC uncoupling theory). This uncoupling would be caused by a significant decrease in the amount of DHPRs in the transverse tubule membrane, whereas no noticeable alterations in the quantity of RyRs were detected (18,21,22). These findings were not confirmed in biochemical studies in which it has been reported that the amount of both RyRs and DHPRs remain unvaried in aging human muscle (38). Ca2+ release channels, or RyRs, are visible in our micrographs as feet (27) at the junctional gap between SR and transverse tubule (see Figures 1D and 7F). The data presented in Table 1 indicate that the total number of SR/transverse tubule junctions containing feet is decreased on average by more than 50% in aging specimens. This observation would point to a substantial decrease in the total amount of both RyRs and DHPRs in the aging fibers, as both proteins are specifically localized at the triad. However, the decrease in total number of CRUs is accompanied by an increase in the average size of RyR-feet arrays that compensate (all or in part) for the loss of triads (Figures 6 and 7; Table 2). Although it is difficult to estimate precisely the amount of RyR-feet present in aging fibers from the electron micrographs alone, we believe that our results may be not far from the data (i.e., unvaried amount of RyRs in aging fibers) reported in the literature by Delbono, Renganathan, Wang, Ryan, and their colleagues (18,21,22,38). The measurement of the distance between SR and transverse tubule membranes (Table 3; Figure 8) adds additional information to the puzzle: If the signal transmission between transverse tubule and SR (i.e., between RyRs and DHPRs) is not efficient, this inefficiency must not be caused by an altered distance between the two proteins, but is likely due to other factors. For example, one issue that is still unsolved is whether the RyR/DHPR stechiometry is maintained in aged muscle. In fact, our micrographs do not allow us to speculate on the amount of DHPRs present in transverse tubule membranes because to visualize these proteins, a different approach would be needed (i.e., freeze fracture replicas that are extremely difficult to obtain from the transverse tubule network of mature muscle).

Interestingly, many of the abnormal features that we have described as results of the progressive disorganization and spatial rearrangement of the EC coupling apparatus during aging are similar to those features described in the literature on the developmental stages of the transverse tubule/SR networks (58–60). Similar features between developing and aging sarcotubular systems are likely due to the fact that two opposite phenomena such as formation and disorganization of the same apparatus may present similar intermediate steps, e.g., low frequency but larger size of couplons, longitudinally oriented tubules, triads not correctly targeted at the I–A band junction, and high frequency of dyads.

Conclusion
The present study proposes that the overall decrease in the number of CRUs and the uneven distribution of remaining triads in aged skeletal muscle fibers may be important factors in the age-related decline of muscle performance. A low number of CRUs could, in fact, interfere with an efficient delivery of Ca2+ ions to the contractile proteins, causing in this way an inefficient activation of the contractile apparatus and a reduction in muscle performance.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This study was supported by a research grant from MIUR (Ministero Istruzione Università e Ricerca; to Stefania Fulle) and by the University G. d'Annunzio Research Funds (to Feliciano Protasi).

We thank Diane E. Sagnella for the critical reading of the manuscript and for assistance with the English grammar and syntax, and Dante Tatone for his technical help in setting up our new laboratory in the CeSI building.


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received December 13, 2005

Accepted February 27, 2006


   References
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 Abstract
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 References
 

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