Aging is associated with loss of skeletal muscle mass, strength and endurance. The aim of this study was to determinate age related changes in human muscles with different function and location in the body (vastus lateralis muscle and intercostal internus muscle). Our results suggest that age related muscle atrophy affect both human skeletal muscles. Also, the results showed the increase in percentage of muscle fibers with high oxidative activity during aging

Juraj Arbanas

Marina Nikolić

Olga Cvijanović

Snježana Bajek

Tamara Šoić Vranić

Nikolić, Marina

Šoić Vranić, Tamara

Arbanas, Juraj

Cvijanović, Olga

Bajek, Snježana

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Muscle Loss in Elderly

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Coll. Antropol. 34 (2010) Suppl. 2: 105–108Original scientific paperMuscle Loss in ElderlyMarina Nikoli}, Tamara [oi} Vrani}, Juraj Arbanas, Olga Cvijanovi} and Snje`ana BajekDepartment of Anatomy, School of Medicine, University of Rijeka, CroatiaA B S T R A C TAging is associated with loss of skeletal muscle mass, strength and endurance. The aim of this study was to determi-nate age related changes in human muscles with different function and location in the body (vastus lateralis muscle andintercostal internus muscle). Our results suggest that age related muscle atrophy affect both human skeletal muscles.Also, the results showed the increase in percentage of muscle fibers with high oxidative activity during aging.Key words: aging, skeletal muscleIntroductionSkeletal muscles allow the movements and accom-plishment of daily physical activities as well as sport per-formances. Indeed, muscles play important roles in livingorganisms. Skeletal muscle consists of different fibertypes characterized by their specific myosin heavy chain(MHC) isoforms. Using monoclonal antibodies specific toMHC isoforms, in adult human skeletal muscles we candistinct three major fiber types: slow contracting fibers,called type I, containing slow myosin heavy chains, andtwo different types of fast contracting fibers, called typeIIA and type IIX which express IIa and IIx myosin heavychains, respectively1.Muscle size changes across the human lifespan, ini-tially showing rapid increases due to grow and later grad-ual decreases due to aging. Whereas the initial stage ofdevelopment involves increase in the number of musclefibers before birth, later growth occurs primarily by en-largement of size in the postmitotic muscle cells. Thestage of young adulthood in the twenties involves a pe-riod of peak musculoskeletal function, middle age in theforties and fifties involves some slowing of contraction,but changes in absolute muscle strength are minor untilthe sixth decade of life2,3.Loss of muscle mass with age in humans is well docu-mented. Investigation of individual muscles shows thatafter thirties, there is decrease in cross sectional areas ofthe thigh, decreased muscle density, and increased intra-muscular fat4. Most studies on aging human muscleshave focused upon changes in the vastus lateralis muscle.Lexell has shown a loss of muscle mass in humans ofabout 10% from age 24 to 50 years, with a further 30%loss for 50 to 80 years5.The aim of this study was to determine and compareage related changes of the percentage of slow and fastmuscle fibers, fiber type distribution and fiber size in twodifferently placed human muscles (leg muscle and respi-ratory muscle). The changes in enzymatic activity of ag-ing muscles were also histochemically demonstrated.Materials and MethodsThis research was approved by the Ethical Committeeof School of Medicine, University of Rijeka.MaterialMuscle samples used for this investigation were ob-tained from thirty male subjects who had suffered sud-den accidental death. None had a history of neuromus-cular disease, nor was there evidence of pathologicalabnormalities at the post-mortem examination. Musclesamples were taken 12–40 h after death from the superfi-cial portion of vastus lateralis muscle. Also, muscle sam-ples were taken from internal intercostal muscles.Samples were divided into two age groups: fifteenyoung men (age range 20–32), and fifteen elderly men(age range 74–87).105Received for publication January 10, 2010Preparative procedureThe sample from the vastus lateralis muscle wastaken on the border between the middle and distal thirdof the femur. From the internal intercostal muscle theblock was taken in middle axillary line from 8th intercos-tal space. All samples were taken from right side of thebody.The muscle samples were quickly frozen in isopen-tane, cooled in liquid nitrogen, and stored at –80°C untilfurther analysis.Immunohistochemistry10 mm thick transverse cryosections of vastus lateralisand internal intercostal muscles were stained for slowand fast myosin heavy chain isoforms using monoclonalantibodies specific for type I (BF-F8), IIA (SC-71) and forall but type IIX (BF-35). Slides were incubated withmonoclonal antibodies (1:1000) in phosphate-bufered sa-line (PBS) for 30 minutes at room temperature. Afterthree washing steps in PBS, slides were incubated for 30minutes at room temperature with peroxidase-labelledrabbit anti human IgG (Dako A/S Copenhagen, Den-mark) in PBS (1:40). After washing in PBS, slides wereincubated in 50 ml of diaminobenzidine (DAB) solution(0.5 M Tris HCl, pH 7.6, 15 mg of DAB, 100 ml of H2O2,25 mg of imidazol) for 20 minutes at room temperature.Finally, slides were dried and mounted in Canada bal-sam6.Histochemistry10 mm thick transverse cryosections of vastus lateralisand internal intercostal muscles were stained to demon-strate the activity of nicotinamide-adenine dinucleotidetetrazolium reductase (NADH-TR)-indicator of oxidativecapacity, and a glycerophosphodehydrogenase (aGPDH)--indicator of glycolytic capacity7.Fiber typing and morphometryThe fiber type frequencies and cross sectional areaswere analyzed by the computer program for quantitativemicroscope image analysis SFORM (VAMS, Zagreb, Cro-atia). An Olympus BX50 microscope, a Pulnix 765 videocamera, and a Sony Trinitron display were used for ob-taining images. One thousand fibers of each muscle sam-ple were measured by tracing along the circumference ofthe fibers. Mean fiber size with standard deviation (SD)was calculated. Statistical evaluations were performedby t-test for independent samples. A probability of 0.05or less was accepted as statistically significant (p<0.05,p<0.001).ResultsMuscle fiber sizeThe size of fiber type I in m. vastus lateralis wasgreater than the size of fibers type IIA and IIX in youngand old subjects. In internal intercostal muscle the crosssectional area of fibers type IIA and IIX was greater thanfibers type I in young subjects. In old age, the size offibers type I compared to fibers type II became larger(Figure 1).All muscle fiber types (type I, type IIA and type IIX)in vastus lateralis and internal intercostal muscles sho-wed a reduction in fiber size with increasing age. Thisreuction was always statistically significant (p<0.001).In old age the size of fibers type IIA and type IIX weredereased more than a half size of young muscle fibers(Figure 1).Percentage of fiber type I, IIA and IIX.Human vastus lateralis muscle in young age was com-posed of type I (32.11±3%), type IIA (60.72±4%), and IIX(7.17±1%) muscle fibers. The results showed age relatedchanges in proportion of type I and IIA muscle fibers butnot in IIX. In old age the percentage of type I muscle fi-bers increased to 61.42±7%, respectively (p<0.001), andthe percentage of type IIA muscle fibers decreased to30.91±3%, respectively (p<0.001) (Figure 2).In young age m. intercostalis internus was composedof 44.65±5% of type I, 48.95±4% of type IIA and 6.40±3%of type IIX muscle fibers. In old age the percentage oftype I fibers increased to 53.60±4%, and percentage oftype IIA muscle fibers decreased to 39.80±4%. The re-sults indicated age related changes in proportion of type Iand type IIA fibers, but not in IIX muscle fibers (Figure2).Enzyme histochemistry.Results showed the increase in percentage of musclefibers with high oxidative activity during aging (Figures3 and 4).M. Nikoli} et al.: Muscle Loss, Coll. Antropol. 34 (2010) Suppl. 2: 105–108106010002000300040005000600070000500100015002000250030003500type I type IIA type IIXyoungold²crosssectionalarea(m)m²crosssectionalarea(m)mtype I type IIA type IIXyoungolda)b)*Statistically significant difference as compared to the young age group (p<0.001)Fig. 1. Relationship between age and muscle fibre cross sectionalarea in a) vastus lateralis and b) intercostalis internus muscle.DiscussionOur study focuses on the period of life from the sev-enth decade onward when muscle strength show a steadydecline, which for simple isomeric performance is on theorder of approximately 1.5% per year8. This condition ofaging has been linked to the normal process of loss ofmuscle tissue, which is related to reduce number of bothtype I and type IIA muscle fibers, plus an additional fac-tor of muscle fiber atrophy3, a process called sarcopenia9.This investigation has shown that the cross sectionalarea of all muscle fibers in examined muscles is decreas-ing from the young age group to the old age group. Com-paring the atrophy of three different muscle fiber types itwas seen the atrophy of fast contracting fibers type IIAand IIX is stronger and their size is decreased for approx-imately 60% at the old age. The atrophic changes are lessexpressed in slow contracting fibers type I.Vastus lateralis is muscle with the prevalence of fiberstype II (68%). M. intercostalis internus are not so mark-edly fast contracting muscle. Among two of muscle fiberstype II, percentage of fibers type IIX is less than 10% inall muscles. According to our results the percentage of fi-ber type I increases with increasing age and these mus-cles become slow contracting muscles that means theprevalence of fibers type I. In the same time percentageof type IIA fibers decreases proportionally. This increas-ing of percentage of type I fibers is very expressed invastus lateralis muscle. These changes are explained asconversion fibers type IIA to fibers type I because ofchanges of myosin isoforms1. The percentage of type IIXfibers remains unchanged during aging. This pattern ofchanges is less expressed in internal intercostal muscles.These results indicate that weight-bearing muscles un-dergo greater changes than respiratory muscles.Also, aging affects the metabolic capacities of skeletalmuscles; in particular there are changes in the activitiesof specific enzymes. Studies on aged human vastus late-ralis muscle showed declines in glycogenolytic and gly-colytic enzyme activities resulting in reduced respiratorycapacities10. Our results indicate that enzyme activity ofmuscle fibers was changed with aging. Results showedthe increase in percentage of muscle fibers with high oxi-dative activity.M. Nikoli} et al.: Muscle Loss, Coll. Antropol. 34 (2010) Suppl. 2: 105–10810701020304050607080type I type IIA type IIXyoungold010203040506070type I type IIA type IIXyoungoldpercentage(%)percentage(%)a)b)***** Statistically significant difference as compared to the young age group (p<0.001)Fig. 2. Relationship between age and percentage of fibre types ina) vastus lateralis and b) intercostalis internus muscle.high oxidativeactivitylow oxidativeactivityintermediatefiberyoungold010203040506070high oxidativeactivitylow oxidativeactivityintermediatefiberyoungoldpercentage(%)10203040506070percentage(%)0a)b)* Statistically significant difference as compared to the young age group (p<0.05)****Fig. 3. Relationship between age and percentage of high and lowoxidative activity of fibres in a) vastus lateralis and b) interco-stalis internus muscle.010203040506070high glycoliticactivitylow glycoliticactivityintermediatefiberpercentage(%)youngold010203040506070percentage(%)youngoldhigh glycoliticactivitylow glycoliticactivityintermediatefiber* Statistically significant difference as compared to the young age group (p<0.05)****Fig. 4. Relationship between age and percentage of high and lowglycolitic activity of fibres in a) vastus lateralis and b) interco-stalis internus muscle.Loss of muscle mass (sarcopenia) with age in humansis well documented. Computed tomography of individualmuscles shows that after age 30 there is a decrease incross sectional areas of the thigh, decreased muscle den-sity, and increased intramuscular fat. These changes aremost pronounced in women4. Muscle atrophy may resultfrom a gradual and selective loss of muscle fibers. Thenumber of muscle fibers in the section of the vastuslateralis of autopsy specimens is significantly lower inolder men compared with younger men11. The decline ismore marked in type II muscle fibers, which decreasefrom an average of 60% in sedentary young men to below30% after age of 80, and is directly related to decreases instrength12. A reduction in muscle strength is a majorcomponent of normal aging. Same studies indicate that40% of the female population aged 55–64 years, 45% ofwomen aged 65–74 years, and 65% of women aged 75–84years were unable to lift 4.5 kg13. In addition, similarlyhigh percentage of women in this population reportedthat they were unable to perform some aspects of normalhousehold work.Sarcopenia is accelerated with a lack of physical activ-ity. The amount of physical activity generally declineswith age. Physically inactive adults undergo a faster andgreater loss of muscle mass than physically activeadults14. Resistance training has been shown to be a pow-erful intervention in the prevention and treatment ofsarcopenia15.Our results indicate that age related loss of skeletalmuscle mass affected human skeletal muscles, i.e. legmuscles and respiratory muscles.R E F E R E N C E S1. SCHIAFFINO S, REGGIANI C, Physiol Rev, 76 (1996) 371. — 2.PORTER M, VANDERVOORT A, LEXELL J, Scand J Med Sci Sports, 5(1995) 129. — 3. NIKOLI] M, MALNAR-DRAGOJEVI] D, BOBINAC D,BAJEK S, JERKOVI] R, [OI]-VRANI] T, Coll Antropol, 25 (2001) 545.— 4. IMAMURA K, ASHIDA H, ISHIKAWA T, FUJII M, J Gerontol, 38(1983) 678. — 5. LEXELL J, TAYLOR CC, SJOSTROM M, J Neurol Sci,72 (1986) 211. — 6. SCHIAFFINO S, GORZA L, PITTON G, SAGGIN L,AUSONI S, SARTORE S, LOMO T, Dev Biol, 127 (1998) 1. — 7. LIN-DMAN R, ERIKSSON A, THORNELL LE, Am J Anat, 190 (1991) 385. —8. VANDERVOORT AA, McCOMAS AJ, J Appl Physiol, 61 (1986) 361. —9. NIKOLI] M, BAJEK S, BOBINAC D, [OI] VRANI] T, JERKOVI] R,Coll Antropol, 29 (2005) 67. — 10. KLEINE TO, Acta Gerontol, 6 (1976)489. — 11. LEXELL J, HENRIKSSON-LARSEN K, WIMBLOD B, SJO-STROM M, Muscle Nerve, 6 (1983) 588. — 12. LARSON L, Acta PhysiolScand, 117 (1983) 469. — 13. JETTE AM, BRANCH LG, Am J PublicHealth, 71 (1981) 1211. — 14. ROUBENHOFF R, Can J Appl Physiol, 26(2001) 78. — 15. ROTH SM, FERREL E, HURLEY BF, J Nutr HealthAging, 4 (2000) 143.M. Nikoli}Department of Anatomy, School of Medicine, University of Rijeka, Bra}e Branchetta 20, 51000 Rijeka, Croatiae-mail: nmarina@medri.hrGUBITAK MI[I]NE MASE U STAROSTIS A @ E T A KStarenje je povezano s gubitkom mi{i}ne mase, snage, te izdr`ljivosti mi{i}a. Cilj istra`ivanja bio je utvrditi dobnouvjetovane promjene u humanim skeletnim mi{i}ima koji obavljaju razli~ite funkcije i imaju razli~it smje{taj u tijelu(m. vastus lateralis i m. intercostalis internus). Rezultati dobiveni istra`ivanjem upu}uju da dobno uvjetovana mi{i}naatrofija zahva}a oba ispitivana mi{i}a. Tako|er, tijekom starenja raste broj mi{i}nih vlakana koji pokazuju visoku oksi-dacijsku aktivnost.M. Nikoli} et al.: Muscle Loss, Coll. Antropol. 34 (2010) Suppl. 2: 105–108108

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Muscle Loss in Elderly

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