Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Connective tissue changes in surgically overloaded muscle

Summary The effects of overload on the connective tissue component of the soleus muscle of the rat have been investigated. Three weeks after tenotomy of its synergistic muscles the soleus underwent considerable increase in weight. This was shown to have resulted from an increase in size of the predominant fibre type. Whilst occasional groups of fibres appeared to have resulted from the splitting of large single fibres, there was no significant increase in the number of fibres in cross-section of the muscle belly. The connective tissue content of the overloaded muscles was investigated using both histological and biochemical techniques. It was found that muscle fibre hypertrophy was accompanied by an increase in the connective tissue component. Furthermore, there was an increase in the proportion of collagen to muscle fibre tissue.The author wish to acknowledge the expert technical assistance given by Mr P. Prentis. This investigation was supported by a grant from the Medical Research Council.     

Hyaluronan in human skeletal muscle of lower extremity: concentration, distribution, and effect of exercise.

The concentration and localization of hyaluronan (HYA) were determined in biopsy specimens from resting human quadriceps femoris and anterior tibial muscles. The influence of physical exercise on HYA concentrations in the quadriceps femoris muscle and in blood was also evaluated. A sensitive radioassay was used for the quantification of HYA. The distribution of the glycosaminoglycan was demonstrated using a histochemical method that involved microwave-aided fixation and an HYA-binding protein. At rest, the muscle HYA concentration was 34.9 +/- 23.6 (SD) micrograms/g muscle wet wt with a large interindividual variation. Exercise had no significant effect on the muscle HYA concentration. The serum HYA concentration increased from 35.9 +/- 22.7 to 53.4 +/- 57.1 micrograms/l during exercise, but 30 min after the exercise the HYA concentration was significantly lower (19.1 +/- 6.3 micrograms/l) than the initial preexercise value. In resting skeletal muscles of the lower extremity, HYA was heterogeneously distributed in the perimysium and endomysium. Perivascular and perineural connective tissues were distinctly HYA positive.     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.     

Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle

The appearance of collagen around individual fast twitch (FT) and slow twitch (ST) muscle fibres was investigated in skeletal muscles with different contractile properties using endurance trained and untrained rats as experimental animals. The collagenous connective tissue was analyzed by measuring hydroxyproline biochemically and by staining collagenous material histochemically in M. soleus (MS), M. rectus femoris (MRF), and M. gastrocnemius (MG). The concentration of hydroxyproline in the ST fibres dissected from MS (2.72 +/- 0.35 micrograms X mg-1 d.w.) was significantly higher than that of the FT fibres dissected from MRF (1.52 +/- 0.33 micrograms X mg-1 d.w.). Similarly, the concentration of hydroxyproline was higher in ST (2.54 +/- 0.51 micrograms X mg-1 d.w.) than in FT fibres (1.60 +/- 0.43 micrograms X mg-1 d.w.), when the fibres were dissected from the same muscle, MG. Histochemical staining of collagenous material agreed with the biochemical evidence that MS and the slow twitch area of MG are more collagenous than MRF and the fast twitch area of MG both at the level of perimysium and endomysium. The variables were not affected by endurance training. When discussing the role of collagen in the function of skeletal muscle it is suggested that the different functional demands of different skeletal muscles are also reflected in the structure of intramuscular connective tissue, even at the level of endomysial collagen. It is supposed that the known differences in the elastic properties of fast tetanic muscle compared to slow tonic muscle as, e.g., the higher compliance of fast muscle could at least partly be explained in terms of the amount, type, and structure of intramuscular collagen.     

Chronic effects of ethanol on muscle metabolism in the rat

Chronic ethanol feeding in the rat is associated with a skeletal myopathy involving primarily type-II muscle fibers, which is recognised to be mediated via a specific impairment in protein turnover. This paper investigates whether the cause of this myopathy may be related to abnormalities in carbohydrate and lipid metabolism in different muscles. [U-¹⁴C]Glucose metabolism was examined in two muscles with different fibre compsitions, the extensor digitorum longus (EDL) muscle, which contains predominantly type-II muscle fibres, and the soleus muscle, which is composed primarily of type-I muscle fibres. Feeding on the ethanol-supplemented Lieber-DeCarli liquid diet for 2 or 6 weeks was associated with profound distubances in glucose metabolism in both EDL and soleus muscles, particularly in relation to rates of glycogen and alanine formation. We discuss the importance of these metabolic changes in relation to the genesis of chronic alcoholic skeletal myopathy.     

Nitric oxide synthase in human skeletal muscles related to defined fibre types

Skeletal muscle functions regulated by NO are now firmly established. However, the knowledge about the NO synthase (NOS) expression related to a defined fibre type in human skeletal muscles necessitates further clarification. To address this issue, we examined localization of NOS isoforms I, II and III, in human skeletal muscles employing immunocytochemical labeling with tyramide signal amplification complemented with enzyme histochemistry and Western blotting. The NOS immunoreactivity was related to fibre types of different classification systems: physiological classification into slow and fast, ATPase classification into I, IIA, IIAX, IIX, and physiological-metabolic classification into slow-oxidative (SO), fast-oxidative glycolytic (FOG) and fast-glycolytic (FG). We found a correlation of NOS I–III immunoreactivity to metabolic defined fibre types with strong expression in FOG fibres. This implies that NO as modulator of muscle function is involved in oxidative metabolism in connection with fast force development, which only occurs in FOG fibres. The NOS expression showed no correlation to ATPase fibre subtypes due to the metabolic heterogeneity of ATPase fibre types. Healthy and affected vastus medialis muscles after anterior cruciate ligament rupture revealed similar NOS expression level as shown by Western blotting with, however, different expression patterns related to the fibre types in affected muscles. This suggests an altered modulation of force development in the fibres of diseased muscles.     

The fin musculature of cuttlefish and squid (Mollusca, Cephalopoda): morphology and mechanics

The lateral fins of cuttlefish and squid consist of a tightly packed three-dimensional array of musculature that lacks bony skeletal support or fluid-filled cavities for hydrostatic skeletal support. During swimming and manoeuvring, the fins are bent upward and downward in undulatory waves. The fin musculature is arranged in three mutually perpendicular planes. Transverse muscle bundles extend parallel to the fin surface from the base of the fin to the fin margin. Dorso-ventral muscle bundles extend from dorsal and ventral connective tissue fasciae to a median connective tissue fascia. A layer of longitudinal muscle bundles is situated adjacent to both the dorsal and ventral surface of the median fascia. The muscle fibres are obliquely striated and include a core of mitochondria. A zone of muscle fibres with a more extensive core of mitochondria is present in both the dorsal and the ventral transverse muscle bundles. It is hypothesized that these muscle masses include two fibre types with different aerobic capacity. A network of connective tissue fibres is present in the transverse and dorso-ventral muscle masses. These fibres, probably collagen, are oriented at 45 to the long axes of the transverse and dorsoventral muscle fibres in transverse planes.
A biomechanicayl analysis of the morphology suggests that support for fin movements is provided by simultaneous contractile activity of muscles of specific orientations in a manner similar to that proposed for other 'muscular-hydrostats'. The musculature therefore provides both the force and support for movement. Connective tissue fibres may aid in providing support and may also serve for elastic energy storage.     

Are region-specific changes in fibre types attributable to nonuniform muscle hypertrophy by overloading?

Muscle fibre composition was compared among the proximal (25%), middle (50%) and distal (75%) regions of the muscle length to investigate whether compensatory overload by removal of synergists induces region-specific changes of fibre types in rat soleus and plantaris muscles. In addition, we evaluated fibre cross-sectional area in each region to examine whether fibre recruitment pattern against functional overload is nonuniform in different regions. Increases in muscle mass and fibre area confirmed a significant hypertrophic response in the overloaded soleus and plantaris muscles. Overloading increased the percentage of type I fibres in both muscles and that of type IIA fibres in the plantaris muscle, with the greater changes being found in the middle and distal regions. The percentage of type I fibres in the proximal region was higher than that of the other regions in the control soleus muscle. In the control plantaris muscle, the percentage of type I and IIA fibres in the middle region were higher than that of the proximal and distal regions. With regard to fibre size, type IIB fibre area of the middle and distal regions in the plantaris increased by 51% and 57%, respectively, with the greater changes than that of the proximal region (37%) after overloading. These findings suggest that compensatory overload promoted transformation of type II fibres into type I fibres in rat soleus and plantaris muscles, with the greater changes being found in the middle and distal regions of the plantaris muscle.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Pathological changes in the microstructure of longissimus lumborum muscle from five breeds of pigs

The aim of the study was to determine the extent of histopathological changes in m. longissimus lumborum of PL, PLW, Duroc, Pietrain, and Pu?awska pigs (N = 30 per breed) aged 210 days. Changes in fibre size (atrophy, hypertrophy - giant fibres), changes in fibre shape (angular fibres), degenerative lesions (necrosis with phagocytosis) and connective tissue hypertrophy were evaluated. The percentage of individual pathological changes in m. longissimus lumborum of the analysed pig breeds was relatively low. Significantly more normal fibres were found in the muscles of Pu?awska compared to Pietrain pigs. Muscle fibre atrophy was the most frequent and extensive histopathological change. The muscles of Pu?awska pigs had significantly fewer atrophic, giant and angular fibres, significantly less necrosis with phagocytosis, and less animals with connective tissue hypertrophy compared to the other pig breeds. On the other hand, Pietrain pigs were characterized by a greater number of animals with giant fibres and a significantly higher proportion of giant fibres compared to the other breeds. Also the diameter of giant fibres was the largest in Pietrain, intermediate in PL and PLW, and the smallest in Duroc and Pulawska pigs. Moreover, current findings indicate that giant fibres may arise from each muscle fibre type (I, IIA and IIB). It is concluded that selection of pigs for increased leanness contributes to the incidence of histopathological changes, which may decrease pork quality.     

Fibre type specificity of haem oxygenase-1 induction in rat skeletal muscle.

The expression of the inducible haem oxygenase (HO-1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO-1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO-1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO-1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO-1 gene expression follows a fibre type-specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.     

Fibre type regionalization of forelimb muscles in two mammalian species, Galea musteloides (Rodentia, Caviidae) and Tupaia belangeri (Scandentia, Tupaiidae), with comments on postnatal myogenesis

To investigate structural differences between propulsory and antigravity muscles, the spatial distribution of slow (type I) and fast (type II) muscle fibres in forelimb muscles of two species of small mammals was studied, Galea musteloides and Tupaia belangeri. Serial sections through complete forelimbs were prepared. Following histochemical fibre typing, the forelimbs were reconstructed three-dimensionally using product design software. Most forelimb muscles of both species showed a homogenous distribution of type I fibres. In the supraspinatus and triceps brachii (capita longum et laterale) muscles, however, a segregation of fibre types into ”fast” superficial areas and ”slow” deep regions was observed. Slow regions contained at least 60% type I fibres and were positioned along intramuscular extensions of the tendons of insertion. The functional implications of fibre type regionalization are discussed. An analysis of intramuscular fibre type distribution during postnatal myogenesis revealed no significant differences in muscle fibre differentiation between altricial and precocial juveniles. Differences in locomotor ability probably arise from heterochronic development of connective tissue components (endo- and perimysium). Accepted: 10 June 1999     

Fibre operating lengths of human lower limb muscles during walking

Muscles actuate movement by generating forces. The forces generated by muscles are highly dependent on their fibre lengths, yet it is difficult to measure the lengths over which muscle fibres operate during movement. We combined experimental measurements of joint angles and muscle activation patterns during walking with a musculoskeletal model that captures the relationships between muscle fibre lengths, joint angles and muscle activations for muscles of the lower limb. We used this musculoskeletal model to produce a simulation of muscle-tendon dynamics during walking and calculated fibre operating lengths (i.e. the length of muscle fibres relative to their optimal fibre length) for 17 lower limb muscles. Our results indicate that when musculotendon compliance is low, the muscle fibre operating length is determined predominantly by the joint angles and muscle moment arms. If musculotendon compliance is high, muscle fibre operating length is more dependent on activation level and force-length-velocity effects. We found that muscles operate on multiple limbs of the force-length curve (i.e. ascending, plateau and descending limbs) during the gait cycle, but are active within a smaller portion of their total operating range.     

Characteristics of paravertebral muscles – fibre type distribution pattern in the pika, Ochotona rufescens (Mammalia: Lagomorpha)

The evolution of the locomotor apparatus in vertebrates is marked by major reorganizations in trunk's musculature. The hypothesized functions of mammalian back muscles in the literature are discussed under consideration of the distribution and proportion of oxidative, type‐I‐fibres, oxidative‐glycolytic, type‐IIa‐fibres and glycolytic, type‐IIb‐fibres in paravertebral muscles of a small mammal. The fibre type distribution was examined from a complete series of histological sections maintaining topographical relationships between the muscles as well as within the muscle, in order to establish the overall distribution pattern. The deep and short muscles showed the highest percentage of oxidative fibres. The larger, superficial paravertebral muscles contained the highest percentage of glycolytic fibres. Two muscles were intermediate in their proportion of fibre types. All epaxial muscles together can be interpreted as an antigravity muscle–complex counteracting enduringly against the rebound tendency caused by gravitation, comparable with antigravity muscles in limbs. A gradient from deep to superficial, or a clear regionalization of oxidative muscle fibres in central deep regions around a large intramuscular tendon was found in the m. spinalis and the m. quadratus lumborum, respectively. Concepts of the function of human back muscles as those of A. Bergmark (1989: Acta Orthop. Scand. 230 , 1) or S.G.T. Gibbons & M.J. Comerford (2001: Orthop. Division Rev. March/April, 21) were exposed to be more general within mammals. Functional specializations of different muscles and muscle parts are discussed under the consideration of evolutionary reorganization of the paravertebral musculature in tetrapods. Along the cranio‐caudal axis, the percentage of oxidative fibres was decreased in caudal direction within the same muscles, whereas the proportion of glycolytic fibres was increased. Therefore, classifications of muscles as ‘glycolytic’ or ‘oxidative’ based on biopsies or analyses of single cross‐sections may result in wrong interpretations. Changes in the proportions of the fibre type distribution pattern were mostly due to oxidative and glycolytic fibre types, whereas the percentage of oxidative‐glycolytic fibres had only minor influence. A significant positive correlation between the cross‐sectional area of the single fibre and its percentage in the area investigated were observed for oxidative fibres, whereby the size was positive correlated to the proportion of the oxidative fibres.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles.

Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Osteopontin,inflammation and myogenesis: influencing regeneration,fibrosis and size of skeletal muscle

Osteopontin is a multifunctional matricellular protein that is expressed by many cell types. Through cell-matrix and cell-cell interactions the molecule elicits a number of responses from a broad range of target cells via its interaction with integrins and the hyaluronan receptor CD44. In many tissues osteopontin has been found to be involved in important physiological and pathological processes, including tissue repair, inflammation and fibrosis. Post-natal skeletal muscle is a highly differentiated and specialised tissue that retains a remarkable capacity for regeneration following injury. Regeneration of skeletal muscle requires the co-ordinated activity of inflammatory cells that infiltrate injured muscle and are responsible for initiating muscle fibre degeneration and phagocytosis of necrotic tissue, and muscle precursor cells that regenerate the injured muscle fibres. This review focuses on the current evidence that osteopontin plays multiple roles in skeletal muscle, with particular emphasis on its role in regeneration and fibrosis following injury, and in determining the severity of myopathic diseases such as Duchenne muscular dystrophy.     

Lactate dehydrogenase isozymes in type I,IIA and IIB fibres of rabbit skeletal muscles

Summary Lactate dehydrogenase (LDH) isozyme patterns were analysed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I Fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M₄). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities (Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.     

Metabolic and physiologic characteristics of skeletal muscle determine its response to clenbuterol treatment

beta-Adrenoceptor agonists are reported to induce skeletal muscle hypertrophy and hence serve as valuable adjunct to the treatment of wasting disorders. In the present study, we attempted to find out whether metabolic and physiologic characteristics of fibres are important in determining skeletal muscle response to clenbuterol (an adrenergic receptor agonist) therapy, as proposed in the treatment of wasting disorders. The treatment of mice with clenbuterol (2 mg/kg body wt for 30 days) resulted in skeletal muscle hypertrophy, more common amongst fast-twitch glycolytic fibres/muscle, with increase in body mass and a parallel rise in muscle mass to body mass ratio. Measurement of fibre diameters in soleus (rich in slow-twitch oxidative fibres), ALD or anterior latissimus dorsi (with a predominance of fast-twitch glycolytic fibres) and gastrocnemius (a mixed-type of muscle) from clenbuterol-treated mice for 30 days revealed noticeable increase in the per cent population of narrow slow-twitch fibre and a corresponding decline in white-type or fast-twitch glycolytic fibres in gastrocnemius and ALD. As revealed by counting of muscle cells in soleus, narrow red fibres declined with corresponding increase in white-type glycolytic fibres population. A significant decline in the succinic dehydrogenase activity was observed, thereby suggesting abnormality in oxidative activity of skeletal muscles in response to clenbuterol therapy.     

Cardiac,skeletal muscle and serum irisin responses to with or without water exercise in young and old male rats: Cardiac muscle produces more irisin than skeletal muscle

Irisin converts white adipose tissue (WAT) into brown adipose tissue (BAT), as regulated by energy expenditure. The relationship between irisin concentrations after exercise in rats compared humans after exercise remains controversial. We therefore: (1) measured irisin expression in cardiac and skeletal muscle, liver, kidney, peripheral nerve sheath and skin tissues, as also serum irisin level in 10 week-old rats without exercise, and (2) measured tissue supernatant irisin levels in cardiac and skeletal muscle, and in response to exercise in young and old rats to establishing which tissues produced most irisin. Young (12 months) and old rats (24 months) with or without 10 min exercise (water floating) and healthy 10 week-old Sprague-Dawley rats without exercise were used. Irisin was absent from sections of skeletal muscle of unexercised rats, the only part being stained being the perimysium. In contrast, cardiac muscle tissue, peripheral myelin sheath, liver, kidneys, and skin dermis and hypodermis were strongly immunoreactivity. No irisin was seen in skeletal muscle of unexercised young and old rats, but a slight amount was detected after exercise. Strong immunoreactivity occurred in cardiac muscle of young and old rats with or without exercise, notably in pericardial connective tissue. Serum irisin increased after exercise, being higher in younger than older rats. Irisin in tissue supernatants (cardiac and skeletal muscle) was high with or without exercise. High supernatant irisin could come from connective tissues around skeletal muscle, especially nerve sheaths located within it. Skeletal muscle is probably not a main irisin source.