Satellite cells of the rat soleus muscle in the process of compensatory hypertrophy combined with denervation

Compensatory hypertrophy was induced in the rat soleus muscle by sectioning the tendon of the ipsilateral gastrocnemius and plantaris muscle. Seven days after tenotomy of synergistic muscles, when soleus hypertrophy attains about 40%, the number of satellite cells (expressed as percentage of all muscle nuclei found in the same cross-sections) as revealed by electron microscopy, was increased from 5.8+/-0.06% in the normal soleus muscle to 16.6+/-1.26%. After four days' denervation of the soleus muscle the percentage of satellite cells was increased to 7.2+/-0.62%. In experiments where hypertrophy of the soleus muscle was combined with denervation three days after tenotomy of synergists, and examined after another four days (during which time it loses, as has previously been shown, over 40% of its predenervation weight), the number of satellite cells was greatly increased to 29.9+/-3.42%. This increase is apparently due to two independent processes which take place during the first postoperative period: a) mitotic division of satellite cells during the early stages of compensatory hypertrophy and b) pinching off of muscle nuclei from rapidly atrophying muscle fibres due to subsequent denervation. Activation of satellite cells was mainly manifested by expansion of smooth and especially of rough endoplasmic reticulum, a rich Golgi complex, high pinocytotic activity, increased number of ribosomes and by nuclear changes. Concomitantly with the increased number of satellite cells, proliferation of fibroblasts, macrophages and mast cells could be observed.     

Regulation of subcellular localization of muscle FBPase in cardiomyocytes. The decisive role of calcium ions

Glyconeogenesis, the synthesis of glycogen from carbohydrate precursors like lactate, seems to be an important pathway participating in replenishing glycogen in cardiomyocytes. Fructose-1,6-bisphosphatase (FBPase), an indispensible enzyme of glyconeogenesis, has been found in cardiomyocytes on the Z-line, in the nuclei and in the intercalated discs. Glyconeogenesis may proceed only when FBPase accumulates on the Z-line. Searching for the mechanism of a FBPase regulation we investigated the effects of the calcium ionophore A23187, a muscle relaxant dantrolene, glucagon, insulin and medium without glucose on the subcellular localization of this enzyme in primary culture of neonatal rat cardiomyocytes. Immunofluorescence was used for protein localization and the intracellular calcium concentration was measured with Fura. We found that the concentration of calcium ions was the decisive factor determining the localization of muscle FBPase on the Z-line. Calcium ions had no effect on the localization of the enzyme in the intercalated discs or in the nuclei, but accumulation of FBPase in the nuclei was induced by insulin.     

The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD-/- mice, has provided evidence for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we compared satellite cells from MyoD-/- and wildtype mice as they progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the initial days in culture the myofibers isolated from both the MyoD-/- and the wildtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a very small number of cells transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of MyoD-/- satellite cells, we identified numerous cells whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD-/- cultures were often positive for desmin, similar to the MyoD+ cells in the wildtype cultures. Myogenin+ cells were identified in the MyoD-/- primary cultures, but their appearance was delayed compared to the wildtype cells. These "delayed" myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precursor pool in the adult muscle.     

Satellite cells of striated muscle after compression injury so slight as not to cause degeneration of the muscle fibres

Summary Electron microscopy of musculus bulbi rectus superior in the rat reveales increase in the number of. muscle satellite cells during the first 10–12 hours following compression injury so slight as not to cause degeneration of the muscle fibre. These satellite cells are situated close to the muscle nuclei and, in contrast to those in the intact rectus superior, they have a pale nucleus with a conspicuous nucleolus, sparse, pale cytoplasm containing a few small mitochondria, a mainly agranular endoplasmic reticulum and numbers of pinocytotic vesicles along their cell membrane. Later on, between 24 and 48 hours, the cytoplasm increases greatly in amount and extendes in long processess over a considerable length of the muscle fibre. An increase in the number of mitochondria and free ribosomes, the appearance of polysomes and great numbers of Golgi complexes and a decrease in the number of pinocytotic vesicles is noted. On the 4th day, some of the satellite cells have still further extended their cytoplasm beneath the muscle basement membrane, while others are apposed to the muscle fibre only with a minor part of their membrane. On the 9th day, only satellite cells comparable in number and structure with those in intact muscles are found.The possibility is discussed that the increase and subsequent decrease in the number of these satellite cells as well as the changes in their morphology at different periods after the injury reflect morphogenesis of satellite cells originating from the nuclei of the muscle fibre by mitotic division even after a trauma, too slight to cause any visible muscle injury. The observations are interpreted as giving new support to the hypothesis that muscle satellite cells may be of importance in posttraumatic muscle regeneration.     

Regenerative potential of human skeletal muscle during aging

In this study, we have investigated the consequences of aging on the regenerative capacity of human skeletal muscle by evaluating two parameters: (i) variation in telomere length which was used to evaluate the in vivo turn-over and (ii) the proportion of satellite cells calculated as compared to the total number of nuclei in a muscle fibre. Two skeletal muscles which have different types of innervation were analysed: the biceps brachii, a limb muscle, and the masseter, a masticatory muscle. The biopsies were obtained from two groups: young adults (23 +/- 1.15 years old) and aged adults (74 +/- 4.25 years old). Our results showed that during adult life, minimum telomere lengths and mean telomere lengths remained stable in the two muscles. The mean number of myonuclei per fibre was lower in the biceps brachii than in the masseter but no significant change was observed in either muscle with increasing age. However, the number of satellite cells, expressed as a proportion of myonuclei, decreased with age in both muscles. Therefore, normal aging of skeletal muscle in vivo is reflected by the number of satellite cells available for regeneration, but not by the mean number of myonuclei per fibre or by telomere lengths. We conclude that a decrease in regenerative capacity with age may be partially explained by a reduced availability of satellite cells.     

Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.     

The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of ³HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.     

Subcellular localization of muscle FBPase in carp (Cyprinus carpio) tissues

Subcellular localization of muscle FBPase-a regulatory enzyme of glyconeogenesis-was investigated in carp using immunohistochemistry and protein exchange method. Results of the experiments revealed that, in striated muscles, FBPase associates with alpha-actinin of the Z-line and co-localizes with aldolase. Additionally, in cardiac and smooth muscle cells FBPase is present inside the nuclei. In the light of findings on mammalian muscle FBPase, the data presented here indicates that interaction of the enzyme with specific cellular partners and nuclear presence of FBPase is a general phenomenon in contemporary vertebrates.     

Survey of fructose 1,6-bisphosphatase isoenzyme in rat organs and ontogenic expression of the enzyme in rat fetus

1. Among eleven tissues of rat, the liver type of fructose 1,6-bisphosphatase (FBPase) subunit was detected in the liver, kidney, testis, pancreas and lung by Western blot analysis using anti-(liver FBPase) or anti-(muscle FBPase) serum. 2. The muscle type of the enzyme subunit was detected only in the pancreas other than skeletal muscle. Both types of the enzyme subunit were found in the pancreas. 3. Neither anti-(liver FBPase) nor anti-(muscle FBPase) serum detected the band of enzyme subunit on the blots of the extracts of brain, heart, small intestinal mucosa, spleen and placenta. 4. FBPase is present in fetal rat liver at least as early as the 14th day of gestation. 5. In agreement with the increase in immunological staining density, the level of the enzyme activity in fetal liver increased exponentially during fetal development. 6. The muscle enzyme was not detected until the fetus reached the 19th day of gestation.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro

Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9–12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.     

Frequency of M-cadherin-stained satellite cells declines in human muscles during aging.

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30- microm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 +/- 2.3 years) and six old (70.8 +/- 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p < 0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.     

A satellite cell mitogen from crushed adult muscle

Single fiber-satellite cell units from skeletal muscle of adult rats were used to study the regulation of satellite cell proliferation. The satellite cells remained quiescent during culture in serum-containing medium but could be induced to enter the cell cycle by exposure to a saline extract of crushed adult muscle. The activity in the extract has a molecular weight greater than 30K and is heat and trypsin sensitive. The mitogenic activity does not result from transferrin. Little or no activity was obtained from crushed extracts of heterologous tissues. Proliferation of myogenic cells from rat embryos was also stimulated by the muscle mitogen but growth of muscle fibroblasts was not enhanced. The time response of satellite cell proliferation after exposure to the muscle mitogen showed that the cells enter DNA synthesis after a lag period of 18 hr and proliferate with a generation time of 12 hr. This confirms that satellite cells in adult muscle are in G0, or an extended G1. The mitogen is also effective in stimulating muscle growth and myoblast fusion in vivo when injected into 1-week-old rat pups. These experiments suggest that muscle regeneration is initiated by the release of an endogenous mitogen from traumatized muscle.     

Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats

Dividing cardiomyocytes are observed in autopsied human hearts following recent myocardial infarction, however there is a lack of information in the literature on the division of these cells. In this study we used a rat model to investigate how and when adult mammalian cardiomyocytes proliferate by cell division after myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. The rats were sacrificed periodically up to 28 days following induced myocardial infarction, and the hearts subjected to microscopic investigation. Cardiomyocytes entering the cell cycle were assayed by observation of nuclear morphology and measuring expression of Ki-67, a proliferating cell marker. Ki-67 positive cardiomyocytes and dividing nuclei were observed initially after 1 day. After 2 days dividing cells gradually increased in number at the ischemic border zone, reaching a peak increase of 1.12% after 3 days, then gradually decreasing in number. Dividing nuclei increased at the ischemic border zone after 3 days, peaked by 0.14% at day 5, and then decreased. In contrast, Ki-67 positive cells and dividing nuclei were limited in number in the non-ischemic area throughout all experiments. In conclusion, mitogenic cardiomyocytes are present in the adult rat heart following myocardial infarction, but were spatially and temporally restricted.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

Nuclear Localization of Fructose 1,6-bisphosphatase in Smooth Muscle Cells

Summary Fructose 1,6-bisphosphatase (FBPase) – a key enzyme of gluconeogenesis – for a long time was regarded to be soluble, and freely diffused in the cytoplasm. Our recent investigation revealed however, that in skeletal muscles of mammals, FBPase is located on both sides of the Z-line and, in cardiomyocytes, it is also present inside the cells’ nuclei. In the current paper we demonstrate that, in smooth muscle cells, FBPase is located in the cytoplasm and the nucleus, and that the presence of the enzyme in the nucleus is almost completely restricted to the heterochromatin area. In search for additional evidence for the nuclear localization of FBPase and for a possible explanation of its role in the nucleus, we have analyzed the primary structures of muscle FBPases, finding on their molecular surface a number of domains specific for proteins transported into the nucleus.     

3H-thymidine and 3H-uridine incorporation into the heart cells of the freshwater crayfish Astacus astacus and the swimming crab Portunus holsatus

DNA and RNA syntheses in the heart cells of two decapod species were investigated with the aid of electron microscopic autoradiography. Isotopes were injected in the cavity of adult animals 4 hours before fixation. 3H-thymidine labeling was found in several satellite cell nuclei and in some particular epicardial cell nuclei. None of myonuclei was labeled. 3H-uridine incorporated in all the nuclei of muscle fibers. Satellite cells were labeled with 3H-uridine very slightly, if at all. Such a peculiarity of biosynthetic processes in the decapod heart satellite cell suggests their myoblastic nature similar to that of satellite cells of somatic muscles. The active 3H-thymidine uptake by the heart satellite cells of adult animals may be accounted for by the permanent growth of the decapods through their whole life span.     

Localization of chicken muscle FBPase in cardiomyocyte nuclei

Fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) localization in cardiomyocyte nuclei has recently been investigated in mammals [FEBS Lett. 539 (2003) 51]. In this study, nuclear localization of FBPase in the cardiac muscle of the chicken was studied by immunohistochemistry and other methods. A result of the electron microscopic investigation was confirmed by immunoblotting analysis. Using MALDI Q-TOF mass spectrometry and Mascot program, the nuclear FBPase was identified as muscle chicken FBPase. FBPase activity in isolated cardiomyocyte nuclei was 5.9 mU/g. Nuclear FBPase was strongly inhibited by allosteric inhibitor AMP. I(0.5) for AMP was 0.16 microM and was the same as for the purified chicken muscle enzyme.     

Membrane currents of rat satellite cells attached to intact skeletal muscle fibers

Muscle satellite cells play an important role in the postnatal growth of skeletal muscle and in the regeneration of damaged muscle during adult life. Little is known about the physiological properties of satellite cells in their dormant state as they lie adjacent to the intact muscle fibers, underneath the basement membrane. Our recent experiments, using patch clamp techniques, indicate that no tight electrical coupling is present between satellite cells and the muscle fiber dissociated from rat flexor digitorum brevis. Satellite cells possess sodium channels with low sensitivity to tetrodotoxin and at a much lower density than muscle. In addition, satellite cells are insensitive to acetylcholine (ACh) for at least 24 hr after having been removed from the animal, even when detached from their muscle fiber. However, we could measure ACh-evoked currents from satellite cells 48-72 hr in culture, indicating that ACh sensitivity develops with time.