Falls are common in elderly patients because of muscle deterioration and decreased balancing ability with advancing age.6,30 Marcell reported that muscle wasting occurs in the elderly population as characterized by loss of muscle mass and strength.32 Skeletal muscle is composed of muscle fibers and connective tissues. Muscle fibers consist of fast-twitch or Type II fibers, and slow-twitch or Type I fibers. Fast-twitch Type IIA muscle fibers are responsible for repetitive, moderate-duration, burst activities; Type IIB fibers are responsible for burst of fast movements, and slow-twitch Type I muscle fibers are responsible for endurance.27 Based on the different functions of skeletal muscle fibers, we suspect Type II muscle fiber may decrease with age because of decreased physical activity. Skeletal muscle fiber changes have been reported in aging humans16,27-29,35,43 and animals such as rats and sheep.15,20,24,48
However, the age-related patterns of changes in muscle fibers and connective tissues are inconclusive. Some report an increase of slow-twitch muscle fibers (Type I) with increasing age,28,29,35,43 whereas others report insignificant changes,15,17,33 or a decreased area with increased age.14 Some studies were performed on an interventional basis such as training exercises or using electrical stimulation to show changes in muscle fibers,11,37,39,40,49 whereas studies investigating age-associated change in muscle fibers were based mostly on small sample sizes,33,35,44 narrow age ranges,3,15,33 or a comparison between two extreme age groups.3,44 Few reports included an evaluation of decreased muscle quality resulting from increased connective tissues in elderly patients,36,38 which is important for understanding muscle fibrosis affecting muscle strength.
We hypothesized that muscle fibers change with increasing age. We questioned how the ratio of Types I and II muscle fibers and connective tissues change with aging, histomorphometrically and morphologically.
MATERIALS AND METHODS
We prospectively recruited 65 patients (42 males; 23 females) aged 17 to 96 years (mean, 46 ± 25.6 years) who had elective hip surgery from January 2003 to August 2004 with a 1-day average preoperative waiting time. Patients with neuromuscular diseases or injuries or damage affecting normal muscle structure and functions in the sampling region of the lower limbs were excluded. Written consent was obtained from all patients preoperatively. Human ethics approval was obtained from the Clinical Research Ethics Committee of the Chinese University of Hong Kong (Reference number CRE-2003.295).
We obtained approximately 5 mm3 muscle biopsy specimens from areas without structural damage during surgery. The biopsy specimens were from the hamstring, vastus lateralis, gluteus, or tensor fascia lata, depending on the surgical site. The specimens were kept in Kreb's solution (20 mmol/L Hepes in pH 7.4, 145 mmol/L sodium chloride, 5 mmol/L potassium chloride, 1.3 mmol/L magnesium chloride, 1.2 mmol/L sodium hydrogen- phosphate, and 10 mmol/L D-glucose) at 4°C. The specimens then were prepared according to the modified protocol described by Nakahata et al.34 Briefly, the 5-mm3 muscle specimen was cut into approximately 1-mm3 cubes and snap-frozen in isopentane (Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany) in liquid nitrogen for 5 minutes. The frozen muscle was embedded immediately in Tissue-Tek® OCT™ Compound (Sakura Finetek USA Inc, Torrance, CA) and cryosectioned in 10-μm sections using Cryocut 1800 (Reichert-Jung, Leica, Nussloch, Germany) perpendicularly to the long axis of the muscle fibers.37 We cut 10 sections from each biopsy specimen and used the three best- quality sections for alkaline triphosphatase (ATPase) staining and analysis.
We performed ATPase staining using a modified protocol described by Dubowitz and Brooke.13 The muscle sections were incubated in preincubating solution (0.143 mol/L barbital acetate, 0.1 N HCl, and deionized water) at pH 4.4 for 5 minutes and then in ATPase solution (Sigma-Aldrich, Inc, St Louis, MO) at pH 9.4 for 25 minutes. The sections were incubated in 0.068 mol/L calcium chloride (Merck, Darmstadt, Germany) and 0.084 mol/L cobalt chloride (Merck) for 10 minutes, respectively, followed with washing in 1:20 sodium barbital (BDH Laboratory Supplies, Poole, England) and deionized water five times. The sections then were immersed in 2% (v/v) ammonium sulfide solution (Fisher Scientific International Inc, Fairlawn, NJ) for 10 to 20 seconds. After dehydration, the sections were mounted. Different muscle fibers were distinguishable with different color intensities, with the lightest colors representing Type IIA fibers, intermediate colors representing Type IIB fibers, and the darkest colors representing Type I fibers.
The ATPase-stained sections were observed by light microscope (Axioplan 2 Imaging Microscope, Zeiss, Göttingen, Germany) at ×20 magnification. Five different views were captured randomly from each section by a high-resolution, cooled CCD digital camera (SPOT-RT Sliders, Diagnostic Instruments, Inc, Sterling Heights, MI) equipped with imaging software (SPOT RT Software v3.1, Diagnostic Instruments Inc). The images then were analyzed by the Metamorph image analysis system (version 4.5, Universal Imaging Corp, Downingtown, PA) to distinguish different color intensities in the regions of interest.25 The evaluation parameters included average area percentage (average area of a muscle fiber type/total area), fiber number percentage (number of a muscle fiber type/total fiber number), mean fiber area (average area of a muscle fiber type/number of the corresponding muscle fiber type), and area percentage of connective tissues ([total area-area of all muscle fiber types]/total area), where a minimal amount of vascular or fat tissues was assumed in the sections. We then measured the aspect ratios (maximum length/maximum width) of different fiber types. We also examined and recorded the morphologic appearance.
We determined the overall distribution proportion of all skeletal muscle types and connective tissues with 65 years as the cut-off age for patients (Fig 1), as based on the clinical criteria of aging recommended by the World Health Organization (WHO) and the findings of studies related to sarcopenia.10,22,46
All quantitative results are presented as mean ± standard deviation (SD). We analyzed the relationship between the histomorphologic data and age by a liner regression model analysis using SPSS version 11.0 software (SPSS Inc, Chicago, IL). Significance was set at p < 0.05.
The ratio of Types IIA and IIB muscle fibers decreased as age increased (p < 0.05) in terms of area, number, and mean fiber area, whereas Type I fibers increased in area and number (p < 0.05) (Figs 2-4). The average area percentage of Types IIA and IIB muscle fibers decreased as age increased, whereas Type I muscle fibers increased in area percentage (Table 1) (Fig 2). For fiber number percentage (Table 1) (Fig 3), Types IIA and IIB muscle fibers decreased with age, with an opposite trend for Type I muscle fibers. Similarly, the mean fiber area of Types IIA and IIB muscle fibers decreased as age increased (Table 1) (Fig 4). However, there was no difference in the mean fiber area in Type I muscle fibers and age (Table 1). The area percentage of connective tissues also increased with increasing age (Table 1) (Fig 5).
The Types IIA and IIB skeletal muscle fibers of elderly patients appeared smaller and flattened with unsmoothed edges compared with the typical circular or polygonal fibers in younger adult muscles3 (Fig 6). The muscle fibers in younger patients were tightly packed, in contrast to the relatively loose structure with increased interstitial connective tissues in muscles of older patients. We observed increased aspect ratios of Types IIA and IIB muscle fibers in patients older than 65 years, although mean fiber area of the Type I muscle fiber did not change with age (Table 2).
Elderly patients are more prone to falls because of muscle weakness and difficulty balancing. It generally is thought that loss of Type II skeletal muscle fiber (responsible for power bursts) contributes to muscle weakness.21,39 However, the literature is conflicting regarding these changes. We wondered how the proportion of skeletal muscle fiber types change with aging.
Our study has several limitations. The muscle specimens were from only three lower-limb muscles (ham- string, vastus lateralis, and gluteus muscles). However, all three were skeletal white muscles composed predominantly of Type II muscle fibers,45 that had similar functions based on their anatomic locations. Similar proportions in types of muscle fibers from white muscles have been reported in humans and animals.9,19,23,26,41 We used three white muscles assuming other limb muscles might be affected by aging in the same way,26 therefore the regional difference in fiber composition did not affect the percentage change in the parameters. Another limitation was gender difference,42 but our study was not powered to examine this in concert with aging.
Fast-twitch muscle fibers (Types IIA and IIB) decreased with increasing age in area, number, and size. Skeletal muscle fibers in elderly patients were smaller and more flattened, which are degenerative signs.3,37 These smaller and flattened fibers may relate to poor balancing ability as fast-twitch muscles provide strength for quick response and balance. In contrast, the slow-twitch fibers (Type I) showed increases in average fiber area percentage and fiber number percentage, whereas the mean fiber areas did not change. Connective tissues also increased in elderly patients, indicating fibrosis.
Types IIA and IIB skeletal muscle fibers decreased with increasing age. Our findings were consistent with those of other cross-sectional studies which showed reduced number, area, and mean fiber area of Type II muscle fibers during aging.16,27,28,35 Animal studies on aged rats also showed declines in Type II fibers in cross-sectional areas in the soleus and plantaris muscles.2,20 Our data suggest decreases in Types IIA and IIB muscle fibers were attributable to the loss of fiber numbers and reduction of fiber sizes, as reflected from decreased fiber number percentage and mean fiber size. Both factors contributed to muscle wasting and poor balancing ability.
Histomorphologically, the Type II muscle fibers appeared flattened and small. Andersen reported that Type II muscle fibers in elderly patients were banana-shaped, crushed, and flattened.3 The differences in the size and shape of the fast-twitch fibers may be a result of decreasing muscle activity, as lack of physical activity or regular exercise47 may result in age-related disuse atrophy. Fiber loss could be compensated by hypertrophy of the remaining fibers,5,31 but our findings did not show any fiber hypertrophy. Instead, there was an increased proportion of slow-twitch muscle fibers and connective tissues.
The decrease in Type IIB fibers (ie, fiber area and number) was greater than in Type IIA fibers. This inconsistent deterioration suggests a relatively greater loss of power activities during aging, perhaps explaining the difficulty of fast motion in elderly patients because Type IIB fibers are responsible for fast movements. The change also may be attributed to the loss of motor neurons,31 particularly the larger motor neurons innervating Type IIB muscle fibers, as suggested in animal studies.7,12,18 These studies revealed that there was a preferential loss of the largest motor neurons with the lowest oxidative capacity along with the reduction of Type II muscle fibers. Usually the largest motor neurons innervate Type II fibers, especially Type IIB.
Changes in Type I muscle fibers during aging are controversial. We found the proportion of Type I muscle fibers during aging, mainly because of the increase of fiber numbers, not an increase in the size of each fiber. These data confirm those of studies in older human skeletal muscles.28,29,35,43 However, animal studies have shown no or less reduction of the cross-sectional area of Type I fibers.2,20 Clinically, Grimby et al15 and Monemi et al33 found no age-related changes in Type I fiber distribution. The discrepancy may be attributed to a narrow age range of patients (range, 66-100 years in the study by Grimby et al15; and six elderly patients with average age of 74 years in the study by Monemi et al33), as younger patients were not included and the aging effect on Type I muscle fibers already occurred. The greater proportion of Type I muscle fiber could be explained by the retired lifestyle of elderly patients. They seldom require use of the fast oxidative fibers, but regularly use slow-twitch muscles for static balance in daily life. Another possibility is the transformation from Type II to Type I fibers as fiber population has a high degree of adaptive capability.11,40 In contrast, a clinical study by Andersen et al4 showed a phenotypic shift toward fast muscle isoforms in response to detraining subsequent to resistant training. A possible explanation is that short-term inactivity leads to changes in muscle fiber types from Type I toward the faster Types IIA and IIX, whereas long-term inactivity (as in our study) leads to loss of high-threshold motor units (ie, Type II), which increases the number percentage and area percentage of Type I fibers.
Connective tissue is important for providing structural and mechanical support in skeletal muscles. Our data suggest connective or fibrous tissues increase with age. These findings support those of other clinical36,38 and animal studies1,2 which showed increases in connective tissues and fat in various skeletal muscles of elderly patients. This phenomenon also is found in disuse or inactivity-induced conditions in humans49 and animals.8,37 The increase of connective tissues or fibrosis also leads to deterioration of muscle quality in elderly patients.36,38
There were age-associated changes in skeletal muscle fiber types and connective tissues. Decreased number and size of Type II muscle fibers are common problems in elderly patients, causing decreased muscle and balancing ability.21 Evidence that increased Type I muscle fibers and connective tissues also were detected. Interventions to improve skeletal muscle quality should be explored. Our findings can be used as reference population data to evaluate the occurrence and mechanism of muscle aging. Our data and noninvasive vibromyography21 may provide information helpful in establishing a database for determining a scoring standard for muscle wasting and the risk of falls in the elderly Asian population. The scoring standard might be similar to the T score in bone mineral density measurement for assessing osteoporosis.
We thank Dr. Simon K. M. Lee for helpful discussion and professional consultancy on the muscle histology.
1. Alnaqeeb MA, Al Zaid NS, Goldspink G. Connective tissue changes and physical properties of developing and ageing skeletal muscle. J Anat
2. Alnaqeeb MA, Goldspink G. Changes in fiber type, number and diameter in developing and ageing skeletal muscle. J Anat
3. Andersen JL. Muscle fibre type adaptation in the elderly human muscle. Scand J Med Sci Sports
4. Andersen LL, Andersen JL, Magnusson SP, Suetta C, Madsen JL, Christensen LR, Aagaard P. Changes in the human muscle force- velocity relationship in response to resistance training and subsequent detraining. J Appl Physiol
5. Aniansson A, Grimby G, Hedberg M. Compensatory muscle fiber hypertrophy in elderly men. J Appl Physiol
6. Aniansson A, Hedberg M, Henning GB, Grimby G. Muscle morphology, enzymatic activity, and muscle strength in elderly men: a follow-up study. Muscle Nerve
7. Ansved T, Larsson L. Quantitative and qualitative morphological properties of the soleus motor nerve and the L5 ventral root in young and old rats. J Neurol Sci
8. Appell HJ. Muscular atrophy following immobilization: a review. Sports Med
9. Arendt EA. Muscle fiber types. Orthopedics
10. Baumgartner RN, Koehler KM, Gallagher D, Romero L, Heymsfield SB, Ross RR, Garry PJ, Lindeman RD. Epidemiology of sarcopenia among the elderly in New Mexico. Am J Epidemiol
11. Brown JM, Henriksson J, Salmons S. Restoration of fast muscle characteristics following cessation of chronic stimulation: physiological, histochemical and metabolic changes during slow-to-fast transformation. Proc R Soc Lond B Biol Sci
12. Doherty TJ, Brown WF. The estimated numbers and relative sizes of thenar motor units as selected by multiple point stimulation in young and older adults. Muscle Nerve
13. Dubowitz V, Brooke MH. Histology and histochemical stains and reactions. In: Dubowitz V, Brooke MH, eds. Muscle Biopsy: A Modern Approach
. London, England: WB Saunders; 1973:20-33.
14. Essen-Gustavsson B, Borges O. Histochemical and metabolic characteristics of human skeletal muscle in relation to age. Acta Physiol Scand
15. Grimby G, Aniansson A, Zetterberg C, Saltin B. Is there a change in relative muscle fibre composition with age? Clin Physiol
16. Grimby G, Danneskiold-Samsoe B, Hvid K, Saltin B. Morphology and enzyme capacity in arm and leg muscle in 78-81 year old men and women. Acta Physiol Scand
17. Grimby G, Saltin B. The ageing muscle. Clin Physiol
18. Hashizume K, Kanda K, Burke RE. Medial gastrocnemius motor nucleus in the rat: age-related changes in the numbers and size of motoneurons. J Comp Neurol
19. Hintz CS, Coyle EF, Kaiser KK, Chi MM, Lowry OH. Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining. J Histochem Cytochem
20. Holloszy JO, Chen M, Cartee GD, Young JC. Skeletal muscle atrophy in old rats: differential changes in the three fiber types. Mech Ageing Dev
21. Huang RP, Rubin CT, McLeod KJ. Changes in postural muscle dynamics as a function of age. J Gerontol A Biol Sci Med Sci
22. Iannuzzi-Sucich M, Prestwood KM, Kenny AM. Prevalence of sarcopenia and predictors of skeletal muscle mass in healthy, older men and women. J Gerontol A Biol Sci Med Sci
. 2002;57: M772-M777.
23. Kimura T. Correlations between feet posture and muscle fiber size in mammals: comparison of the anterior tibial muscle fiber among plantigrade type, digitigrade type and unguligrade type. Kaibogaku Zasshi
24. Konishi M, Iwamoto S, Ohara H, Shimada M. Two-dimensional changes of muscle fiber types in growing rat hind limb. Kaibogaku Zasshi
25. Leung KS, Cheung WH, Zhang C, Lee KM, Lo HK. Low intensity pulsed ultrasound stimulates osteogenic activity of human periosteal cells. Clin Orthop Relat Res
26. Lexell J. Human aging, muscle mass, and fiber type composition. J Gerontol A Biol Sci Med Sci
27. Lexell J, Downham DY. What is the effect of ageing on type 2 muscle fibers? J Neurol Sci
28. Lexell J, Henriksson-Larsen K, Winblad B, Sjostrom M. Distribution of different fiber types in human skeletal muscle: effects of aging studied in whole muscle cross sections. Muscle Nerve
29. Lexell J, Taylor CC. Variability in muscle fiber areas in whole human quadriceps muscle: effects of increasing age. J Anat
30. Lipsitz LA, Nakajima I, Gagnon M, Hirayama T, Connelly CM, Izumo H, Hirayama T. Muscle strength and fall rates among residents of Japanese and American nursing homes: an International Cross-Cultural Study. J Am Geriatr Soc
31. 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
32. Marcell TJ. Sarcopenia: causes, consequences, and preventions. J Gerontol A Biol Sci Med Sci
33. Monemi M, Eriksson PO, Eriksson A, Thornell LE. Adverse changes in fibre type composition of the human masseter versus biceps brachii muscle during aging. J Neurol Sci
34. Nakahata E, Shindoh Y, Takayama T, Shindoh C. Interleukin-12 prevents diaphragm muscle deterioration in a septic animal model. Comp Biochem Physiol A Mol Integr Physiol
35. Nikolic M, Malnar-Dragojevic D, Bobinac D, Bajek S, Jerkovic R, Soic-Vranic T. Age-related skeletal muscle atrophy in humans: an immunohistochemical and morphometric study. Coll Antropol
36. Overend TJ, Cunningham DA, Paterson DH, Lefcoe MS. Thigh composition in young and elderly men determined by computed tomography. Clin Physiol
37. Qin L, Appell HJ, Chan KM, Maffulli N. Electrical stimulation prevents immobilization atrophy in skeletal muscle of rabbits. Arch Phys Med Rehabil
38. Rice CL, Cunningham DA, Paterson DH, Lefcoe MS. Arm and leg composition determined by computed tomography in young and elderly men. Clin Physiol
39. Rogers MA, Evans WJ. Changes in skeletal muscle with aging: effects of exercise training. Exerc Sport Sci Rev
40. Salmons S, Henriksson J. The adaptive response of skeletal muscle to increased use. Muscle Nerve
41. Shimozawa A, Ishizuya-Oka A. Muscle fiber type analysis in the mouse m. digastricus, m. stylohyoideus, m. zygomaticus and m. buccinator. Anat Anz
42. Simoneau JA, Bouchard C. Human variation in skeletal muscle fiber-type proportion and enzyme activities. Am J Physiol
43. Sjostrom M, Angquist KA, Rais O. Intermittent claudication and muscle fiber fine structure: correlation between clinical and morphological data. Ultrastruct Pathol
44. Sjostrom M, Lexell J, Downham DY. Differences in fiber number and fiber type proportion within fascicles: a quantitative morphological study of whole vastus lateralis muscle from childhood to old age. Anat Rec
45. Swatland HJ. Comparison of red and white muscles by cytophotometry of their muscle fibre populations. Histochem J
. 1978;10: 349-360.
46. Tzankoff SP, Norris AH. Effect of muscle mass decrease on age- related BMR changes. J Appl Physiol
47. Westerterp KR, Meijer EP. Physical activity and parameters of aging: a physiological perspective. J Gerontol A Biol Sci Med Sci
48. White NA, McGavin MD, Smith JE. Age-related changes in percentage of fiber types and mean fiber diameters of the ovine quad- riceps muscles. Am J Vet Res
49. Zamboni M, Turcato E, Santana H, Maggi S, Harris TB, Pietrobelli A, Heymsfield SB, Micciolo R, Bosello O. The relationship between body composition and physical performance in older women. J Am Geriatr Soc