Effects of passive stretch on growth and regression of muscle from chickens of various ages

A non-invasive procedure was used to determine the effect of animal age on the growth response of muscle to passive stretch. Stretch increased patagialis muscle weight 61% in 6-week-old chicks and 34% in 10-month-old chicks, 28-month-old animals had an 18% loss of muscle mass during passive stretch. Removal of the stretch stimulus was followed by a rapid return of patagialis weight to control values in 6-week and 10-month animals, while muscle size of 28-month-old animals had not returned to control levels by 22 days, following removal of the stretch. The stretch-induced changes in muscle wet weight could, in part, be attributed to changes in muscle protein. Total muscle DNA content increased during rapid growth in 6-week- and 10-month-old chickens, and returned to control levels during muscle regression. Muscle hydroxyproline content increased in parallel with increases in muscle mass but did not return to control levels during muscle regression in 6-week-old animals. Results of the present study indicate that there was an effect of animal age on stretch-induced hypertrophy and regression of the patagialis muscle.     

Regional injury and the terminal differentiation of satellite cells in stretched avian slow tonic muscle.

In the avian stretch model, the application of a weight overload to the humerus induces enlargement of the anterior latissimus dorsi (ALD) muscle and an increase in muscle fiber number which is accompanied by satellite cell activation. Myofiber injury may be an important stimulus to muscle fiber hyperplasia; therefore, light and electron microscopic evaluation was undertaken to determine if myofiber injury occurs in the stretch-enlarged ALD muscle of the adult quail. Autoradiographic studies were used to determine the terminal differentiation of labeled myogenic cells. A weight equal to 10% of body mass was attached to one wing of 27 adult quail and 3 birds were euthanized at 9 intervals of stretch, from 1 to 30 days. Birds were injected with tritiated thymidine at intervals ranging from 1 hr to 3 days prior to euthanization. Labeled nuclei were detected by light microscopic examination and identified by electron microscopy of a serial section. Three regions of the muscle were examined for disorganization of contractile elements, presence of cytoplasmic vacuoles, and/or phagocytic cell infiltration. The percentage of fibers exhibiting one or more of these criterion was significantly greater in the stretched ALD by Days 5 and 7 and declined at Day 10, reaching near control values by Day 14. Myofiber necrosis and phagocytic cell infiltration were only observed in the middle and distal regions of the stretched ALD muscle. Traditional signs of regeneration and repair were observed, including clusters of labeled myoblast-like cells and myotube formation within an existing basal lamina. New myotube formation with labeled central nuclei was also noted in the interstitial space, outside of basal lamina of persisting fibers. Labeled myonuclei were observed in the stretched fibers. These results demonstrate that chronic stretch produces regional injury and fiber degeneration and resultant regeneration in the ALD muscle of the adult quail. This may be an important stimulus for new fiber formation in this model.     

Stretch-induced growth in chicken wing muscles: role of soluble growth-promoting factors

The involvement of soluble growth-promoting factors in stretch-induced hypertrophy of the Patagialis muscle (PAT) in the chicken wing was investigated. Soluble extracts were prepared from young chicken PAT muscles made hypertrophic by passive stretch and from unstretched contralateral controls. Extracts were tested for their ability to stimulate cell proliferation and creatine phosphokinase (CPK) activity in primary monolayer cultures of chick embryo muscle cells. Factors were present in muscle extracts which showed a dose-dependent stimulation of cell proliferation and CPK activity in vitro. Passive stretch for 5 days produced a rapid hypertrophy of the PAT which was accompanied by a dramatic increase in the activity of the growth factor(s). Release of stretch resulted in an arrest of growth and an immediate fall in growth factor activity. The difference in growth-stimulating activity between control and stretched PAT extracts could be demonstrated in chicken transferrin-sensitive chick myoblast cultures. Stretch thus induces an increase in a class-specific growth factor, possibly Transferrin, in the PAT. Stretched PAT extracts stimulated: (a) chick myoblast proliferation to a greater extent than an optimum concentration of chick embryo extract, and (b) CPK activity in vitro to a greater extent than excess Transferrin. Both control and stretched PAT extracts supported the growth of rat myoblasts. We conclude that PAT muscle extracts also contain unknown growth factor(s) which are different from Transferrin.     

The contribution of muscle progenitor cells to maintaining morphological characteristics of unweighted rat soleus muscle during passive stretch

Skeletal muscle work hypertrophy is usually connected with muscle progenitor SC (satellite cells) activation with subsequent incorporation their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and be accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by gamma-irradiation would partly preavent stretched muscle fiber capability to maintain their size under condition of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposure to ionizing radiation (2500 Rad) and then subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myo-nuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We concluded that satellite cells did not make essential contribution to passive stretch preventive action under condition of simulated weightlessness.     

Age-related apoptotic responses to stretch-induced hypertrophy in quail slow-tonic skeletal muscle

In the present study, we examined the responses of apoptosis and apoptotic regulatory factors to muscle hypertrophy induced by stretch overload in quail slow-tonic muscles. The wings from one side of young and aged Japanese quails were loaded by attaching a tube weight corresponding to 12% of the bird's body weight for 7 or 21 days. Muscle from the contralateral side served as the intraanimal control. Relative to the intraanimal contralateral control side, the muscle wet weight increased by 96% in young birds, whereas the muscle weight gain in aged birds was not significant after 7 days of loading. After 21 days of loading, muscle weight significantly increased by 179% and 102% in young and aged birds, respectively. Heat shock protein (HSP)72 and HSP27 protein contents in the loaded sides were higher than on the control sides exclusively in young birds after 7 days of loading. Compared with the contralateral control muscle, the extent of apoptotic DNA fragmentation and the total cytosolic apoptosis-inducing factor protein content were reduced in all loaded muscles except for the 7-day-loaded muscles from the aged birds. Bax protein content was diminished in the loaded muscle relative to the control side from all groups, whereas Bcl-2 protein content was reduced in the young and aged muscles after 21 days of loading. The total cytosolic cytochrome c protein content was decreased and the X chromosome-linked inhibitor of apoptosis protein content was elevated in 7- and 21-day-loaded muscles relative to the intraanimal control muscle from young birds. Furthermore, after 7 days of loading the muscles of aged birds, H₂O₂ content and the total cytosolic protein content of second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point were elevated compared with the intraanimal control side. These data suggest that stretch overload-induced muscle hypertrophy is associated with changes in apoptosis in slow-tonic skeletal muscle. Moreover, discrepant apoptotic responses to muscle overload in young and aged muscles may account in part for the age-related decline in the capability for muscle hypertrophy. aging; sarcopenia; Bcl-2; Bax; heat shock proteins; apoptosis-inducing factor     

Orientation and length of mammalian skeletal myocytes in response to a unidirectional stretch

Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch.     

Muscle mechanosensitive reflex is suppressed in the conscious condition: effect of anesthesia.

To test the hypothesis that a muscle mechanosensitive reflex is suppressed in the conscious condition, we examined the effect of anesthesia on the cardiovascular responses to passive mechanical stretch of the hindlimb triceps surae muscle in six conscious cats. The triceps surae muscle was manually stretched for 30 s by extending the hip and knee joints and subsequently by dorsiflexing the ankle joint; the lateral gastrocnemius muscle was lengthened by 19 +/- 2.6 mm. Heart rate (HR) and mean arterial blood pressure (MAP) did not change significantly during passive stretch of the muscle in the conscious condition. At 10-40 min after intravenously administering pentobarbital sodium (20-25 mg/kg), the identical passive stretch of the triceps surae muscle was able to induce the cardiovascular responses; HR and MAP were increased by 14 +/- 1.3 beats/min and 14 +/- 1.4 mmHg, respectively, and the cardiovascular responses were sustained throughout the passive stretch. In contrast, stretching skin on the triceps surae muscle evoked no significant changes in HR and MAP in the anesthetized condition. When anesthesia became light 40-90 min after injection of pentobarbital and the animals started to show spontaneous body movement, the cardiovascular response to passive muscle stretch tended to be blunted again. It is therefore concluded that passive mechanical stretch of skeletal muscle is capable of evoking the reflex cardiovascular response, which is suppressed in the conscious condition but exaggerated by anesthesia.     

Thyroxine-induced cardiomegaly in rats of different age

In the present study the effect of thyroxine treatment on the development of cardiomegaly was compared in young (10-day-old) and adult (12-week-old) rats. L-thyroxine was administered subcutaneously in a dose of 1 mg per kg b.w. for 5 days. In young thyroxine-treated rats the heart weight increased by 79% in comparison with the control rats. The number of blood capillaries and muscle fibres per mm2 remained unchanged. The concentration of hydroxyproline was even lower than in control animals. The number of 3H-thymidine-labelled muscle cell nuclei was significantly higher both in the left and right ventricles of thyroxine treated rats. The density of capillaries and muscle fibres was significantly lower in adult rats than in the group of young animals. In adult thyroxine-treated animals the heart weight was higher by 36%, the number of capillaries and muscle fibres as well as the concentration of hydroxyproline was unchanged. Thyroxine induced significant increase in the number of DNA synthesizing nuclei of muscle cells in the left ventricle while the change in the right ventricular myocardium was not statistically significant. The present data indicate that a hyperplastic response of cardiac muscle cells to thyroxine occurs in both ventricles of young rats and also in the left ventricle of adult animals.     

Subcellular responses of p53 and Id2 in fast and slow skeletal muscle in response to stretch-induced overload.

Tumor suppressor p53 and inhibitor of DNA-binding/differentiation Id2 were examined after 7 or 21 days of wing weighting in fast patagialis (PAT) and slow anterior latissimus dorsi (ALD) wing muscles of young adult and old Japanese quails. The contralateral wing served as the intra-animal control. Seven days of loading increased PAT and ALD muscle weight by 28 and 96%, respectively, in young birds. PAT and ALD muscle weight was 49 and 179% greater, respectively, than control muscles after 21 days of loading in young birds. In aged birds, no PAT or ALD hypertrophy was found after 7 days of loading; however, PAT and ALD muscle weight increased by 29 and 102%, respectively, after 21 days of loading. Id2 protein in the nuclear muscle fraction increased in both PAT and ALD muscles from young adult and old birds that were loaded for 7 days and in ALD muscles after 21 days of loading relative to contralateral control muscles. Nuclear p53 protein was greater in 7- or 21-day loaded PAT and ALD muscles relative to control muscles in both age groups. Cytosolic Id2 and p53 protein contents were not changed in loaded PAT or ALD muscles relative to control muscles at any time point. These data suggest that nuclear, but not cytosolic, Id2 and p53 are responsive to stretch-induced muscle overload. Moreover, the attenuated ability of the aged skeletal muscle to achieve hypertrophy does not appear to be explained by the subcellular changes in Id2 and p53 content with overload.     

Neutrophil accumulation following passive stretches contributes to adaptations that reduce contraction-induced skeletal muscle injury in mice.

Skeletal muscles can be injured by their own contractions, especially when the muscle is stretched during a lengthening contraction. Exposing a muscle to a conditioning protocol of stretches without activation (passive stretches) before lengthening contractions reduces contraction-induced injury. Although passive stretching does not damage muscle fibers, neutrophils are elevated in the muscle after passive stretches. Our purpose was to investigate the relationship between neutrophil accumulation following passive stretches and the protection from subsequent contraction-induced injury provided by the passive stretches. Our hypothesis was that passive stretch conditioning would not provide protection from subsequent lengthening contraction-induced injury under circumstances when the increase in muscle neutrophils in response to the conditioning was prevented. Extensor digitorum longus muscles of mice were conditioned with passive stretches 14 days before exposure to a protocol of damaging lengthening contractions. Mice were either untreated or treated with an antibody (RB6-8C5) that reduced the level of circulating neutrophils by over 95% before administration of passive stretches. Neutrophil levels recovered in treated mice by the time lengthening contractions were performed. Lengthening contractions were also administered to muscles with no prior exposure to passive stretches. Maximum isometric force, number of damaged fibers, and muscle neutrophil concentration were measured 3 days after lengthening contractions. Compared with nonconditioned control muscles, the severity of contraction-induced injury was not reduced by prior passive stretch conditioning when mice were treated with RB6-8C5 before conditioning. We conclude that neutrophils contribute to adaptations that protect muscles from injury.     

Regionalized adaptations and muscle fiber proliferation in stretch-induced enlargement

The relative contribution of increases in fiber area to stretch-induced muscle enlargement was evaluated in the slow tonic fibers of the anterior latissimus dorsi of adult Japanese quails. A weight corresponding to 10% of the bird's body mass was attached to one wing. Thirty days of stretch in 34 birds averaged 171.8 +/- 13.5% increase in muscle mass and 23.5 +/- 0.8% increase in muscle fiber length. The volume density of noncontractile tissue increased in middle and distal regions of stretch-enlarged muscles. Mean fiber cross-sectional area increased 56.7 +/- 12.3% in the midregion of stretched muscles. Further analysis indicated slow beta-fiber hypertrophy occurred in proximal, middle, and distal regions; however, fast alpha-type fiber hypertrophy was limited to middle regions of stretched muscles. Stretched muscles had a significant increase in the frequency of slow beta-fibers that were less than 500 microns 2 in all regions and fast alpha-type fibers in middle and distal regions. Total fiber number was determined after nitric acid digestion of connective tissue in 10 birds. Fiber number increased 51.8 +/- 19.4% in stretched muscle. These results are the first to clearly show that muscle fiber proliferation contributes substantially to adult skeletal muscle stretch-induced enlargement, although we do not know whether the responses of the slow tonic anterior latissimus dorsi might be similar or different from mammalian twitch muscle.     

Cardiac vagal and sympathetic efferent discharges are differentially modified by stretch of skeletal muscle

We directly measured cardiac vagal efferent nerve activity (CVNA) and cardiac sympathetic efferent nerve activity (CSNA) in cats decerebrated at the level of the precollicular-premammillary body while the hindlimb or the triceps surae muscle was passively stretched. CVNA gradually decreased during passive stretch of the hindlimb, and this decrease was sustained throughout the stretch. CSNA increased at the onset of passive stretch, but this increase was not sustained. CVNA and CSNA responded differentially to graded passive stretches of the triceps surae muscle as well as the hindlimb. The sustained decrease in CVNA but not the initial increase in CSNA became greater depending on muscle length and developed tension. The time course and direction of the cardiac autonomic responses to muscle stretch were not affected by partial sinoaortic denervation, although the magnitude of the CSNA response was augmented. We conclude that the muscle mechanoreflex contributes to differential regulation of cardiac parasympathetic and sympathetic efferent discharges during passive stretch of skeletal muscle irrespective of arterial baroreceptor input.     

Glycogen utilization in isolated frog muscle: an effect of passive mechanical stretch

The effect of passive mechanical stretch on glycogen utilization and total protein maintenance in paired sartorii muscles from small male frogs has been investigated, in vitro. Glycogen content of stretched muscles decreased 72.6% over a 24-hr incubation period, compared to a 23.1% decline in unstretched control muscles. Stretched muscles retained 54.7% of their total protein content over a 24-hr period, compared to 38.4% in the unstretched control muscles. Unstretched muscles demonstrated a significant (11.1%) increase in wet weight following 24 hr of incubation, presumably due to increased water retention. There were no significant changes in the wet wts of stretched muscles.     

Development of isoproterenol-induced cardiac hypertrophy

The development of cardiac hypertrophy was studied in adult female Wistar rats following daily subcutaneous injections of isoproterenol (ISO) (0.3 mg/kg body weight). A time course was established for the change in tissue mass, RNA and DNA content, as well as hydroxyproline content. Heart weight increased 44% after 8 days of treatment with a half time of 3.4 days. Ventricular RNA content was elevated 26% after 24 h of a single injection and reached a maximal level following 8 days of therapy. The half time for RNA accumulation was 2.0 days. The total content of hydroxyproline remained stable during the first 2 days of treatment but increased 46% after 4 days of therapy. Ventricular DNA content was unchanged during the early stage (1-4 days) of hypertrophic growth but increased to a new steady-state level 19% above the controls after 8 days of treatment. Intraventricular pressures and coronary flow measures were similar for control and experimental animals following 4 days of developed hypertrophy. However, dP/dt in the ISO-treated hearts was slightly but significantly (P less than 0.05) elevated. These data indicate that the adaptive response to ISO shows an early hypertrophic phase (1-4 days) characterized by a substantial increase in RNA content and cardiac mass in the absence of changes in DNA. However, prolonged stimulation (8-12 days) appears to represent a complex integration of both cellular hypertrophy and hyperplasia within the heart.     

Satellite cells are increasingly refractory to activation by nitric oxide and stretch in aged mouse-muscle cultures

Age-related muscle atrophy or sarcopenia results in progressive loss of muscle function and satellite cells in aging muscle are increasingly refractory to activation that could mitigate atrophy. We know that nitric oxide release triggered by mechanical stretch of skeletal muscle, initiates satellite cell activation in vitro in single fiber, single cell and whole-muscle cultures, and in vivo in animals. This study examined muscle cell activation using tritiated-thymidine incorporation into the DNA of muscle cells in cultured muscles from female mice between 6 weeks and 18 months-of-age. Experiments examined age-related changes in activation by mechanical stretch and/or NO treatments (with the substrate of nitric oxide synthase (l-arginine), a nitric oxide donor (isosorbide dinitrate) and/or nitric oxide synthase inhibition). Activation without stretch was highest at 8 months. Stretching muscles by 10% more than doubled activation in muscles at 6 weeks of age and only a 20% stretch similarly activated cells in cultured 6-month-old muscles. Only treatment with ISDN in combination with a 20% stretch activated cell proliferation in muscles from 8-month-old mice. A nitric-oxide donor drug rescued muscle satellite cells in adult, 8-month-old mice from being refractory to mechanical stretch, apparently by overcoming an ineffective release of nitric oxide during stretch. Results suggest that treatment with nitric oxide has the potential to enhance the effectiveness of exercise in preventing the onset of age-related muscle atrophy in adult muscle.     

Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles

A state of hypokinesia and hypodynamia has been induced in the hindlimb muscles of the rat (100 g) using a suspension model. The ensuing muscle atrophy was assessed by reference to muscles in fully mobile control animals, which were either fed ad libitum or fed the same lower food intake of the suspended animals. Over a total of 7 days of suspension the slow-twitch postural soleus muscle underwent a much greater atrophy than the fast-twitch phasic extensor digitorum longus. Changes with respect to the position of the suspended foot, and hence muscle length, necessitate caution in comparing the extent of the atrophy between different muscle types. After 3 days of inactivity the atrophy of the soleus muscle was explained by a 21% decrease in the fractional rate of synthesis (measured in vivo) and a 100% increase in the rate of protein breakdown. The reduction in the synthetic rate was associated with a net loss (23%) of RNA and hence muscle ribosomes. In contrast when this inactive soleus muscle was permanently stretched the RNA content (44%) and protein synthetic rate increased (59%) markedly above control values. Although protein breakdown remained elevated in this stretched muscle, the extent of the atrophy in response to hypokinesia and hypodynamia was greatly reduced.     

Stretch-induced myoblast proliferation is dependent on the COX2 pathway

Skeletal muscle increases in size due to weight bearing loads or passive stretch. This growth response is dependent in part upon myoblast proliferation. Although skeletal muscles are responsive to mechanical forces, the effect on myoblast proliferation remains unknown. To investigate the effects of mechanical stretch on myoblast proliferation, primary myoblasts isolated from Balb/c mice were subjected to 25% cyclical uniaxial stretch for 5 h at 0.5 Hz. Stretch stimulated myoblast proliferation by 32% and increased cell number by 41% 24 and 48 h after stretch, respectively. COX2 mRNA increased 3.5-fold immediately poststretch. Prostaglandin E2 and F2alpha increased 2.4- and 1.6-fold 6 h after stretch, respectively. Because COX2 has been implicated in regulating muscle growth and regeneration, we hypothesized that stretched myoblasts may proliferate via a COX2-dependent mechanism. We employed two different models to disrupt COX2 activity: (1) treatment with a COX2-selective drug, and (2) transgenic mice null for COX2. Treating myoblasts with a COX2-specific inhibitor blocked stretch-induced proliferation. Likewise, stretched COX2-/- myoblasts failed to proliferate compared to controls. However, supplementing stretched, COX2-/- myoblasts with prostaglandin E2 or fluprostenol increased proliferation. These data suggest that the COX2 pathway is critical for myoblast proliferation in response to stretch.     

Thyroidal influence on body growth

This review of thyroid influence on body growth in poultry is organized around the following parameters of growth: increase in body weight and skeletal size, muscle growth, and growth of cartilage and bone. The greatest effect of goitrogens on growth of embryos occurs during late embryogenesis at a time when normal thyroid hormone levels are increasing. Posthatching growth is reduced in severely hypothyroid animals, and body weight gain is affected more than bone growth. Thyroid hormone replacement restores body growth of thyroidectomized chickens, but supplemental hormone in normal animals has no beneficial effect on growth. Excessive T3 (fed at 1 ppm) is detrimental to growth and feed efficiency. No clear correlation between thyroid hormone concentration and growth rate of normal chickens has been identified. Growth depression in sex-linked dwarf birds is at least partially reversed by supplemental T3. Muscle growth is reduced in goitrogen-treated chickens and the growth reduction is reversed by supplemental thyroxine. Total DNA accumulation is reduced in hypothyroid chickens, but muscle mass relative to DNA content is normal following long-term treatment; this suggests some regulation of muscle mass relative to DNA content. T3 increases the number of muscle fiber nuclei in hypothyroid chickens and the uptake of 3H-thymidine into nuclei within the basal lamina. T3 directly stimulates growth and maturation of embryonic chick cartilage and enhances the in vitro action of somatomedins on cartilage growth. There is little information concerning the role of the thyroid in posthatching cartilage and bone growth in poultry.