J Comp Physiol B DOI 10.1007/s00360-014-0857-5
Original Paper
Aging alters contractile properties and fiber morphology in pigeon skeletal muscle Emidio E. Pistilli · Stephen E. Alway · John M. Hollander · Jeffrey H. Wimsatt
Received: 12 February 2014 / Revised: 6 August 2014 / Accepted: 10 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force– frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force–frequency relationship between the
age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middleaged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of agingrelated alterations in skeletal muscle function and structure. Keywords Sarcopenia · Contraction time · Fiber area · Isometric force
Communicated by G. Heldmaier. E. E. Pistilli (*) · S. E. Alway · J. M. Hollander Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, WV 26506, USA e-mail: [email protected] S. E. Alway e-mail: [email protected] J. M. Hollander e-mail: [email protected] E. E. Pistilli · S. E. Alway · J. M. Hollander Center for Cardiovascular and Respiratory Sciences, West Virginia University School of Medicine, Morgantown, WV 26506, USA J. H. Wimsatt Office of Laboratory Animal Resources, West Virginia University School of Medicine, Morgantown, WV 26506, USA e-mail: [email protected]
Introduction Aging is associated with a progressive loss of skeletal muscle mass, a condition known as sarcopenia (Kamel 2003; Rosenberg 1989; Roubenoff and Hughes 2000; Visser and Schaap 2011). In humans, decreases in muscle mass up to 40 % can occur by age 60 and are correlated with decrements in muscle strength (Doherty 2003; Faulkner et al. 2007). Specifically, maximal voluntary strength can be decreased approximately 20–40 % by age 70 (Alway et al. 1996; Doherty 2003; Larsson et al. 1979; Murray et al. 1985; Young et al. 1984, 1985). These reductions in muscle mass and maximal strength are associated with an increased incidence of falls and a general increase in frailty in the elderly (Wickham et al. 1989). A number of underlying mechanisms have been proposed to contribute
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to sarcopenia, including: denervation of skeletal muscle which preferentially occurs in type II fibers (Doherty et al. 1993); an altered hormonal environment that does not promote maintenance of muscle mass (Tenover 1997); and increases in circulating inflammatory mediators (Bruunsgaard et al. 2001, 2003). Regardless of the relative contributions of these individual mechanisms to the sarcopenic process, the eventual result is a loss of functional muscle mass with advancing age. The functional assessment of skeletal muscle contractile properties reveals characteristic changes associated with aging, although differences among animal models have been observed. In rodents, advanced age (i.e., >30 months of age) is associated with loss of muscle mass, reductions in absolute maximal isometric force, a slowing of twitch contraction durations (i.e., contraction time, 1/2 relaxation time), and a decrease in the velocity of contraction (Degens and Alway 2003). Similar alterations in speed-related contractile properties have been observed in skeletal muscles from birds. Specifically, the slow fiber anterior latissimus dorsi (ALD) muscle from 90-week-old Japanese quail displayed longer contraction and relaxation times, decreases in contraction velocity, and a leftward shift of the force–frequency relationship as compared to younger adult birds (Alway 1995; Carson et al. 1995). Interestingly, there were no differences in ALD muscle size or isometric force output, suggesting a specific effect of age on speed-related parameters in these quail muscles (Alway 1995; Carson et al. 1995). The maximal lifespan potential (MLSP) of rodents and quail are relatively similar, while the MLSP of pigeons is approximately 6–7 times greater (genomics.senescence. info). This difference in lifespan could provide unique insights into the mechanisms underlying age-related alterations in muscle contractile properties in the pigeon model. Therefore, the purpose of this study was to examine contractile and morphological characteristics in skeletal muscles from young, middle-aged, and aged pigeons. We hypothesized that tensor propatagialis pars biceps muscles from pigeons would display age-related alterations in isometric force and contractile parameters (i.e., twitch and tetanic force output; contraction and relaxation durations; force–frequency relationship) as well as a shift of the fiber cross-sectional area (CSA) distribution toward smaller fiber sizes, consistent with other animal models of aging. Our data show that skeletal muscles from pigeons display characteristic alterations in muscle function consistent with aging-related studies in other animal species. Interestingly, minimal differences were observed in muscle function and morphology in muscles from middle-aged and aged pigeons. These novel data provide insights into the aging process in the pigeon, which may be a suitable model to study the effects of aging on skeletal muscle function and structure.
13
J Comp Physiol B
Materials and methods Experimental animals Domestic pigeons (Columba livia) were obtained from the Palmetto Pigeon Plant (Sumter, SC, USA) and utilized for experiments at three different ages; young (3 years old, n = 6), middle-aged (18 years old, n = 7) and aged (30 years old, n = 2). Pigeons were housed in a temperature controlled vivarium under a 12:12 h light:dark cycle, and were maintained within this facility as they aged to the specific time-points of the study. Each pigeon was housed individually in a cage with the following dimensions: 0.36 m long, 0.32 m wide, and 0.33 m high. This cage size allowed the pigeons to walk along the length of the cage; however, the size restricted their ability to spread their wings and fly. Food was restricted to maintain the pigeons at approximately 80 % of their free-feed body weight. All experiments and procedures were approved by the Animal Care and Use Committee of West Virginia University (ACUC #11-1206). Muscle contractile properties Pigeons were euthanized by breathing carbon dioxide gas and immediately after death, the tensor propatagialis pars biceps muscle was removed for ex vivo contractile analyses. This muscle is active during the last third of the upstroke and the first two-thirds of the down stroke of the wing stroke in free living birds (Dial 1992); however, it is presumed to be relatively sedentary in caged animals and therefore it is representative of the effects of aging on muscle function in sedentary avian muscle. Nylon suture was tied around the distal tendon of the muscle, which was isolated by blunt dissection. At the proximal end of the muscle the tendon was left attached to a piece of bone and nylon suture was tied around the bone. The muscle was then transferred to a tissue bath that contained oxygenated Ringer solution (100 mM NaCl, 4.7 mM KCl, 3.4 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, and 5.5 mM d-glucose) that was maintained at 22 °C using a circulating water bath. Contractile parameters were determined using a commercially available ex vivo muscle physiology system (Aurora Scientific, Ontario, CA) with data acquired using a PowerLab A/D convertor and associated Chart software (ADInstruments, Colorado Springs, CO). Muscle length was adjusted to obtain the maximal twitch force response, and this muscle length was measured and reported as optimal length (Lo). Contraction time, 1/2 relaxation time, and maximal force output were obtained from isometric twitch contractions with muscle at optimal length (i.e., Lo). Stimulation frequencies ranging from 5 to 125 Hz were used to obtain the force–frequency relationship and maximal isometric tetanic force was
J Comp Physiol B
determined from the peak force output of this relationship. After contractile assessment, muscles were flash frozen in isopentane cooled to the temperature of liquid nitrogen and stored at −80 °C for further analyses. Muscle morphology and fiber area Frozen muscle sections (10 μm thickness) were obtained from the mid-belly portion of the muscle using a cryostat maintained at −20 °C and placed onto glass slides (Superfrost/Plus, Fisher Scientific, Pittsburgh, PA). Muscle sections were fixed in methanol at −20 °C for 5 min and then processed for histological examination using Mayer’s hematoxylin (Sigma-Aldrich) and eosin-Y (Sigma-Aldrich, St. Louis, MO) solutions. Digital images were acquired using an Olympus BX51 microscope and Magnafire digital camera. Morphometric measurements of fiber CSA were made from images acquired at a 20× objective magnification using the Image J image processing software (http://rsbweb.nih.gov/ij). The fiber CSA distribution was determined from a total of 2,739 fibers from young muscles, 2,000 fibers from middle-aged muscle, and 3,000 fibers from aged muscles. Analysis was performed after fiber CSA values were grouped per 200 μm. Fluorescent stain for slow and fast myosin Frozen muscle sections (10 μm thickness) were obtained from the mid-belly portion of the muscle using a cryostat maintained at −20 °C and placed onto glass slides (Superfrost/Plus, Fisher Scientific). Muscle sections were fixed in methanol at −20 °C for 5 min and were blocked in 5 % fetal calf serum in PBS for 1 h at 4 °C. Sections were incubated with a mouse monoclonal anti-chicken slow myosin primary antibody (ALD-58; Developmental Studies Hybridoma Bank, Iowa City, IA) for 1 h in a humidified chamber maintained at room temperature. The tissue sections were washed in PBS and then incubated in goat anti-mouse fluorescent secondary antibody (Alexa-Fluor 546, Life Technologies, Grand Island, NY). Sections were covered with mounting media containing Dapi (Vector Labs, Burlingame, CA), and visualized on an Olympus MVX10 MacroView microscope at an objective magnification of 25.2×. This magnification is a product of the specific objective on the microscope, the zoom, and the magnification changer. Fluorescent microscope images were acquired via a Hamamatsu ORCA camera and cellSens 1.9 Dimension Acquisition software.
effects of age and stimulation frequency as well as the age × stimulation frequency interaction. Tukey’s post hoc test was used to determine differences between groups. The fiber CSA distribution was evaluated using nonlinear regression fitted to a Gaussian equation to obtain R2 values. Data were analyzed using the GraphPad Prism software package and are presented as mean ± SD.
Results Contractile properties of the tensor propatagialis pars biceps muscle There were no significant differences in wet weights of the tensor propatagialis pars biceps muscle among groups of pigeons (young 399.4 ± 160.8 mg, middle-aged 348.4 ± 61.36 mg, aged 335.9 ± 27.39 mg, p = 0.631). Similarly, optimal muscle length was not significantly different among groups (young 27.1 ± 2.1 mm, middle-aged 24.3 ± 3.3 mm, aged 24.9 ± 0.7 mm, p = 0.287). Representative isometric twitch force traces for the tensor propatagialis pars biceps muscle are presented in Fig. 1a. Maximal twitch force was significantly lower in muscles from middle-aged and aged pigeons compared to young pigeons. Specifically, twitch force in muscles from middle-aged and aged pigeons was 40 and 47 % less than twitch force in muscles from young pigeons, respectively (Fig. 1b). Representative maximal tetanic force traces for the tensor propatagialis pars biceps muscle are presented in Fig. 1c. Maximal tetanic force was significantly lower in muscles from middle-aged and aged pigeons compared to young pigeons. Specifically, tetanic force in muscles from middle-aged and aged pigeons was 52 and 67 % less than tetanic force in muscles from young pigeons, respectively (Fig. 1d). Isometric twitch contractions were analyzed for contraction time, measured as the time from the start of force production to peak force, and 1/2 relaxation time, measured as the time from peak force to halfway down the relaxation phase of the force trace. Contraction time was significantly longer in the tensor propatagialis pars biceps muscle with aging, as depicted in the representative force traces (Fig. 2a). Specifically, contraction time was 29 % longer and 48 % longer in muscles from middle-aged and aged pigeons, respectively, when compared to muscles from young pigeons (Fig. 2b). There were no significant differences in the 1/2 relaxation time in the tensor propatagialis pars biceps muscle with aging (Fig. 2c).
Statistics Force–frequency relationship Isometric force data were analyzed using a one-way ANOVA. Data from the force–frequency relationship were analyzed using a two-way ANOVA to examine the main
Isometric tetanic force was evaluated at different stimulation frequencies to establish the force–frequency
13
J Comp Physiol B Young Middle-Aged Aged
500
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400 300 200 100 0
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Fig. 1 Isometric force production of twitch and tetanus contractions. a Representative twitch contraction force traces in muscles from young, middleaged, and aged pigeons. b Maximal absolute twitch force was greatest in muscles from young pigeons compared to muscles from middle-aged and aged pigeons. c Representative tetanus contraction force traces in muscles from young, middleaged, and aged pigeons. d Maximal absolute tetanus force was greatest in muscles from young pigeons compared to muscles from middle-aged and aged pigeons. mN millinewtons, *p
Original Paper
Aging alters contractile properties and fiber morphology in pigeon skeletal muscle Emidio E. Pistilli · Stephen E. Alway · John M. Hollander · Jeffrey H. Wimsatt
Received: 12 February 2014 / Revised: 6 August 2014 / Accepted: 10 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force– frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force–frequency relationship between the
age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middleaged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of agingrelated alterations in skeletal muscle function and structure. Keywords Sarcopenia · Contraction time · Fiber area · Isometric force
Communicated by G. Heldmaier. E. E. Pistilli (*) · S. E. Alway · J. M. Hollander Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, WV 26506, USA e-mail: [email protected] S. E. Alway e-mail: [email protected] J. M. Hollander e-mail: [email protected] E. E. Pistilli · S. E. Alway · J. M. Hollander Center for Cardiovascular and Respiratory Sciences, West Virginia University School of Medicine, Morgantown, WV 26506, USA J. H. Wimsatt Office of Laboratory Animal Resources, West Virginia University School of Medicine, Morgantown, WV 26506, USA e-mail: [email protected]
Introduction Aging is associated with a progressive loss of skeletal muscle mass, a condition known as sarcopenia (Kamel 2003; Rosenberg 1989; Roubenoff and Hughes 2000; Visser and Schaap 2011). In humans, decreases in muscle mass up to 40 % can occur by age 60 and are correlated with decrements in muscle strength (Doherty 2003; Faulkner et al. 2007). Specifically, maximal voluntary strength can be decreased approximately 20–40 % by age 70 (Alway et al. 1996; Doherty 2003; Larsson et al. 1979; Murray et al. 1985; Young et al. 1984, 1985). These reductions in muscle mass and maximal strength are associated with an increased incidence of falls and a general increase in frailty in the elderly (Wickham et al. 1989). A number of underlying mechanisms have been proposed to contribute
13
to sarcopenia, including: denervation of skeletal muscle which preferentially occurs in type II fibers (Doherty et al. 1993); an altered hormonal environment that does not promote maintenance of muscle mass (Tenover 1997); and increases in circulating inflammatory mediators (Bruunsgaard et al. 2001, 2003). Regardless of the relative contributions of these individual mechanisms to the sarcopenic process, the eventual result is a loss of functional muscle mass with advancing age. The functional assessment of skeletal muscle contractile properties reveals characteristic changes associated with aging, although differences among animal models have been observed. In rodents, advanced age (i.e., >30 months of age) is associated with loss of muscle mass, reductions in absolute maximal isometric force, a slowing of twitch contraction durations (i.e., contraction time, 1/2 relaxation time), and a decrease in the velocity of contraction (Degens and Alway 2003). Similar alterations in speed-related contractile properties have been observed in skeletal muscles from birds. Specifically, the slow fiber anterior latissimus dorsi (ALD) muscle from 90-week-old Japanese quail displayed longer contraction and relaxation times, decreases in contraction velocity, and a leftward shift of the force–frequency relationship as compared to younger adult birds (Alway 1995; Carson et al. 1995). Interestingly, there were no differences in ALD muscle size or isometric force output, suggesting a specific effect of age on speed-related parameters in these quail muscles (Alway 1995; Carson et al. 1995). The maximal lifespan potential (MLSP) of rodents and quail are relatively similar, while the MLSP of pigeons is approximately 6–7 times greater (genomics.senescence. info). This difference in lifespan could provide unique insights into the mechanisms underlying age-related alterations in muscle contractile properties in the pigeon model. Therefore, the purpose of this study was to examine contractile and morphological characteristics in skeletal muscles from young, middle-aged, and aged pigeons. We hypothesized that tensor propatagialis pars biceps muscles from pigeons would display age-related alterations in isometric force and contractile parameters (i.e., twitch and tetanic force output; contraction and relaxation durations; force–frequency relationship) as well as a shift of the fiber cross-sectional area (CSA) distribution toward smaller fiber sizes, consistent with other animal models of aging. Our data show that skeletal muscles from pigeons display characteristic alterations in muscle function consistent with aging-related studies in other animal species. Interestingly, minimal differences were observed in muscle function and morphology in muscles from middle-aged and aged pigeons. These novel data provide insights into the aging process in the pigeon, which may be a suitable model to study the effects of aging on skeletal muscle function and structure.
13
J Comp Physiol B
Materials and methods Experimental animals Domestic pigeons (Columba livia) were obtained from the Palmetto Pigeon Plant (Sumter, SC, USA) and utilized for experiments at three different ages; young (3 years old, n = 6), middle-aged (18 years old, n = 7) and aged (30 years old, n = 2). Pigeons were housed in a temperature controlled vivarium under a 12:12 h light:dark cycle, and were maintained within this facility as they aged to the specific time-points of the study. Each pigeon was housed individually in a cage with the following dimensions: 0.36 m long, 0.32 m wide, and 0.33 m high. This cage size allowed the pigeons to walk along the length of the cage; however, the size restricted their ability to spread their wings and fly. Food was restricted to maintain the pigeons at approximately 80 % of their free-feed body weight. All experiments and procedures were approved by the Animal Care and Use Committee of West Virginia University (ACUC #11-1206). Muscle contractile properties Pigeons were euthanized by breathing carbon dioxide gas and immediately after death, the tensor propatagialis pars biceps muscle was removed for ex vivo contractile analyses. This muscle is active during the last third of the upstroke and the first two-thirds of the down stroke of the wing stroke in free living birds (Dial 1992); however, it is presumed to be relatively sedentary in caged animals and therefore it is representative of the effects of aging on muscle function in sedentary avian muscle. Nylon suture was tied around the distal tendon of the muscle, which was isolated by blunt dissection. At the proximal end of the muscle the tendon was left attached to a piece of bone and nylon suture was tied around the bone. The muscle was then transferred to a tissue bath that contained oxygenated Ringer solution (100 mM NaCl, 4.7 mM KCl, 3.4 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, and 5.5 mM d-glucose) that was maintained at 22 °C using a circulating water bath. Contractile parameters were determined using a commercially available ex vivo muscle physiology system (Aurora Scientific, Ontario, CA) with data acquired using a PowerLab A/D convertor and associated Chart software (ADInstruments, Colorado Springs, CO). Muscle length was adjusted to obtain the maximal twitch force response, and this muscle length was measured and reported as optimal length (Lo). Contraction time, 1/2 relaxation time, and maximal force output were obtained from isometric twitch contractions with muscle at optimal length (i.e., Lo). Stimulation frequencies ranging from 5 to 125 Hz were used to obtain the force–frequency relationship and maximal isometric tetanic force was
J Comp Physiol B
determined from the peak force output of this relationship. After contractile assessment, muscles were flash frozen in isopentane cooled to the temperature of liquid nitrogen and stored at −80 °C for further analyses. Muscle morphology and fiber area Frozen muscle sections (10 μm thickness) were obtained from the mid-belly portion of the muscle using a cryostat maintained at −20 °C and placed onto glass slides (Superfrost/Plus, Fisher Scientific, Pittsburgh, PA). Muscle sections were fixed in methanol at −20 °C for 5 min and then processed for histological examination using Mayer’s hematoxylin (Sigma-Aldrich) and eosin-Y (Sigma-Aldrich, St. Louis, MO) solutions. Digital images were acquired using an Olympus BX51 microscope and Magnafire digital camera. Morphometric measurements of fiber CSA were made from images acquired at a 20× objective magnification using the Image J image processing software (http://rsbweb.nih.gov/ij). The fiber CSA distribution was determined from a total of 2,739 fibers from young muscles, 2,000 fibers from middle-aged muscle, and 3,000 fibers from aged muscles. Analysis was performed after fiber CSA values were grouped per 200 μm. Fluorescent stain for slow and fast myosin Frozen muscle sections (10 μm thickness) were obtained from the mid-belly portion of the muscle using a cryostat maintained at −20 °C and placed onto glass slides (Superfrost/Plus, Fisher Scientific). Muscle sections were fixed in methanol at −20 °C for 5 min and were blocked in 5 % fetal calf serum in PBS for 1 h at 4 °C. Sections were incubated with a mouse monoclonal anti-chicken slow myosin primary antibody (ALD-58; Developmental Studies Hybridoma Bank, Iowa City, IA) for 1 h in a humidified chamber maintained at room temperature. The tissue sections were washed in PBS and then incubated in goat anti-mouse fluorescent secondary antibody (Alexa-Fluor 546, Life Technologies, Grand Island, NY). Sections were covered with mounting media containing Dapi (Vector Labs, Burlingame, CA), and visualized on an Olympus MVX10 MacroView microscope at an objective magnification of 25.2×. This magnification is a product of the specific objective on the microscope, the zoom, and the magnification changer. Fluorescent microscope images were acquired via a Hamamatsu ORCA camera and cellSens 1.9 Dimension Acquisition software.
effects of age and stimulation frequency as well as the age × stimulation frequency interaction. Tukey’s post hoc test was used to determine differences between groups. The fiber CSA distribution was evaluated using nonlinear regression fitted to a Gaussian equation to obtain R2 values. Data were analyzed using the GraphPad Prism software package and are presented as mean ± SD.
Results Contractile properties of the tensor propatagialis pars biceps muscle There were no significant differences in wet weights of the tensor propatagialis pars biceps muscle among groups of pigeons (young 399.4 ± 160.8 mg, middle-aged 348.4 ± 61.36 mg, aged 335.9 ± 27.39 mg, p = 0.631). Similarly, optimal muscle length was not significantly different among groups (young 27.1 ± 2.1 mm, middle-aged 24.3 ± 3.3 mm, aged 24.9 ± 0.7 mm, p = 0.287). Representative isometric twitch force traces for the tensor propatagialis pars biceps muscle are presented in Fig. 1a. Maximal twitch force was significantly lower in muscles from middle-aged and aged pigeons compared to young pigeons. Specifically, twitch force in muscles from middle-aged and aged pigeons was 40 and 47 % less than twitch force in muscles from young pigeons, respectively (Fig. 1b). Representative maximal tetanic force traces for the tensor propatagialis pars biceps muscle are presented in Fig. 1c. Maximal tetanic force was significantly lower in muscles from middle-aged and aged pigeons compared to young pigeons. Specifically, tetanic force in muscles from middle-aged and aged pigeons was 52 and 67 % less than tetanic force in muscles from young pigeons, respectively (Fig. 1d). Isometric twitch contractions were analyzed for contraction time, measured as the time from the start of force production to peak force, and 1/2 relaxation time, measured as the time from peak force to halfway down the relaxation phase of the force trace. Contraction time was significantly longer in the tensor propatagialis pars biceps muscle with aging, as depicted in the representative force traces (Fig. 2a). Specifically, contraction time was 29 % longer and 48 % longer in muscles from middle-aged and aged pigeons, respectively, when compared to muscles from young pigeons (Fig. 2b). There were no significant differences in the 1/2 relaxation time in the tensor propatagialis pars biceps muscle with aging (Fig. 2c).
Statistics Force–frequency relationship Isometric force data were analyzed using a one-way ANOVA. Data from the force–frequency relationship were analyzed using a two-way ANOVA to examine the main
Isometric tetanic force was evaluated at different stimulation frequencies to establish the force–frequency
13
J Comp Physiol B Young Middle-Aged Aged
500
Twitch Force (mN)
400 300 200 100 0
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Fig. 1 Isometric force production of twitch and tetanus contractions. a Representative twitch contraction force traces in muscles from young, middleaged, and aged pigeons. b Maximal absolute twitch force was greatest in muscles from young pigeons compared to muscles from middle-aged and aged pigeons. c Representative tetanus contraction force traces in muscles from young, middleaged, and aged pigeons. d Maximal absolute tetanus force was greatest in muscles from young pigeons compared to muscles from middle-aged and aged pigeons. mN millinewtons, *p
