Evidences of apoptosis during the early phases of soleus muscle atrophy in hindlimb suspended mice (original) (raw)
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European Journal of Applied Physiology, 2007
The purpose of the present study was to investigate the mechanisms of cell death (apoptosis vs. necrosis) during muscle atrophy induced by 1 week of hindlimb suspension. Biochemical and morphological parameters were examined in murine soleus and gastrocnemius muscles. A total of 70 male Charles River CD1 mice were randomly assigned to seven groups (n = 10/group): Cont (loading control conditions) and 6HS, 12HS, 24HS, 48HS, 72HS and 1wkHS with respect to the period of hindlimb suspension (HS). Compared to the Cont, skeletal muscle atrophy was confirmed by a significant decrease of 44 and of 17% in fiber cross-sectional areas in the gastrocnemius and soleus, respectively. A significant increase in caspase-3 activity was noticed in 6HS (196%, P < 0.05) and in 12HS (201%, P < 0.05), as well as the amount of cytosolic mono- and oligonucleosomes at 12HS (142%, P < 0.05) and 24HS (203%, P < 0.05) in the gastrocnemius and soleus, respectively. The profile of necrotic markers showed a peak of myeloperoxidase activity at 24HS (170%, P < 0.05) and at 72HS (114%, P < 0.05) in the gastrocnemius and soleus, respectively. The analysis of N-acetylglucosaminidase activity evidenced more increment in the soleus at 72HS (60%, P < 0.05). The analysis of the basal values of these parameters suggested that apoptosis prevailed in the slow-twitch muscle analyzed, whereas lysosomic activity seemed to be more pronounced in the gastrocnemius. The morphological data supported the biochemical results pointing towards a shift from apoptosis to necrosis, which seems to corroborate the aponecrosis theory.
2004
The contributions of potential autophagic or inflammatory reactions to the induction of skeletal muscle atrophy during immobilization are not yet completely understood. The aim of this study was to comparably investigate them over a short period of immobilization, with special emphasis on biochemical data and their probable morphological correlates. Twenty-four Charles River mice were divided into four experimental groups (each n=6) with one hindlimb immobilized for different periods of time (24h, 48h, 72h, and 7days, respectively). After cervical dislocation, both soleus muscles were completely removed and their wet weight was determined. The β-glycuronidase activity (βGlu), for muscle fiber autophagic response, and myeloperoxidase activity (MPO), for lysosomic activity of leukocytes were determined, always related to muscle protein content. All data were compared to the contralateral control muscles (=100%). A part of each muscle was examined morphologically in LM and EM. Muscle wet weight decreased progressively, most pronounced at 24h (15%), but slowing down already after 48h. βGlu activity increased significantly at 24h, returning to the basal levels at 48h group. At 48h and 72h, MPO showed a significant increase. The morphologic examination revealed the presence of lysosomes, mitochondrial swelling, and edema especially at 24h and 48h. Leucocytes infiltration was largely observed after 48 hours of immobilization. These data suggest that during immobilization atrophy, muscle wasting is initiated by an autophagic reaction followed by an inflammatory response. It therefore appears that necrosis is more important than apoptosis in immobilization atrophy.
European Journal of Applied Physiology, 2006
The comprehension of the cellular mechanisms underlying skeletal muscle atrophy has been the aim of several experimental studies. However, the majority of them focused on alterations of the myocytes induced by different experimental conditions yet disregarding the contribution of other cells such as endothelial cells and fibroblasts. In this sense, 70 Charles River CD1 male mice were randomly assigned to seven groups (n = 10 per group): control and 6, 12, 24, 48, 72 h and 1 week with respect to the period of hindlimb suspension. Forty-eight hours before sacrifice, the animals were injected with bromodeoxyuridine (BrdU) in order to identify proliferating cells. Immunohistochemistry and south-western blotting techniques were used to evaluate across the whole gastrocnemius muscle BrdU incorporation into the different proliferating cells. The contribution of the apoptotic response was also measured in order to ascertain whether the balance between cell survival and death was preserved. The results observed during 1 week of unloading-induced atrophy evidenced an intense peak of proliferating activity only after 6 h, mainly due to the duplication of satellite cells. Consequently to this unexpected activation of satellite cells, the addition of nuclei to the fibre syncytium was recognized at 12 h of unloading. After 48 h of weightlessness, the proliferating activity observed was largely due to an interstitial fibrosis. According to the apoptotic index profile observed during the analysed unloading period, this general proliferative activity was balanced by apoptosis, which strongly suggests the existence of a regulatory feedback response between anabolic and catabolic events in unloading-induced skeletal muscle atrophy.
Gravitational unloading inhibits the regenerative potential of atrophied soleus muscle in mice
Acta Physiologica, 2009
The present study was performed to investigate the influence of unloading on the regeneration of atrophied and injured skeletal muscle. Methods: Male mice (C57BL/6J), aged 8 weeks, were used. Cardiotoxin (CTX) was injected into soleus muscles bilaterally. Gravitational unloading on soleus muscle was performed by hind limb suspension for 2 weeks before and additionally 6 weeks after CTX injection in one group. Soleus muscles in the remaining groups were loaded keeping the mice in the cages and were dissected 14, 28 and 42 days after the injection. Results: Recovery of the wet weight and protein content of soleus in the CTX-injected group was inhibited by unloading. Increase in satellite cell number, induced by CTX injection and loading, was also inhibited by unloading. Disappearance of infiltration of mononucleated cells into the necrotic area was also delayed. This phenomenon suggests that regeneration, which is indicated by the appearance of fibres with central nuclei, was inhibited by unloading. Conclusion: Results suggested that loading plays an important role in the activation of the regenerating potential of injured skeletal muscle.
Apoptosis in Denervated Skeletal Muscle
International Journal of Morphology, 2007
The apoptosis phenomenon happens in normal tissues and in many pathological conditions as described by several researchers. Most studied in the 70s, this phenomenon remained almost forgotten in the 90s, when, with the advent of more advanced techniques, returned to laboratories. Thus, electronic microscopy, Comet Assays and histochemistry techniques, among others, were utilized in an effort to elucidate in a thorough way, the mechanisms that lead the cell to "suicide". No one can deny the importance of the knowledge of such phenomenon, so as to control its induction or blocking in the treatment of pathologies such as cancer. In this work, the apoptosis study was aimed at denervated muscles in different periods. Furthermore, two types of skeletal striated muscles were compared: the one called red muscle (soleo) which is resistant to fatigue, having a slow contraction, and the one named white muscle (EDL) of precocious fatigue and fast contraction. Previoulsy carried out in the techniques mentioned above, this work has been accomplished using the TUNEL technique, which is most accepted in the identification of apoptosis. It was demonstrated that this phenomenon takes place in a more marked way, in the beginning of the denervation process. In addition, it was observed that the red muscle responds more intensely to denervation than the white muscle. One can infer that the removal of the main cell function, the contraction, in the case of the skeletal striated muscle, triggers the chain reactions that culminate in apoptosis. Other projects are in process using mutant animals aiming at studying degenerative muscular illnesses and correlating them with denervation.
Postmortem muscle cells die through apoptosis
European Food Research and Technology, 2010
Several reports suggested the activation of caspases in postmortem muscle implicating the onset of a caspase-dependent cell death process after animal bleeding. It has been further well established that apoptosis and necrosis are the two major cell death pathways. The questions addressed in the present work were as follows: (a) in postmortem muscle, do cells die as in vivo? and (b) if so, by which dying process this goal is achieved? Selected hallmarks of apoptosis (phosphatidylserine externalization (PS), cell shrinkage, actin degradation) were analyzed in postmortem rat muscles and compared to usual cell behavior in apoptotic and necrotic processes. Results presented clearly demonstrate a rapid PS externalization and cell shrinkage extending during the first 24 h postexsanguination together with a progressive degradation of cytoskeletal and thin filaments of actin. It was therefore concluded that, in postmortem muscle, cells commit suicide soon after animal bleeding through apoptosis.
Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals
American Journal of …, 2000
Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals. Am J Physiol Endocrinol Metab 279: E1114-E1121, 2000.-Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4-12 h with peak changes occurring at 3-7 days. Maximal apoptosis was also evident at distant limb muscles at 3-7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting. muscle wasting; mitochondria; caspase; thermal injury ANY FORM OF CRITICAL ILLNESS, including burn injury, with or without sepsis, is associated with many functional, metabolic, and pharmacological aberrations (2, 3, 29, 32). The important functional change in skeletal muscle during and after critical illness is weakness or decreased tension-generating capacity, resulting in hypoventilation, difficulty in weaning off respirators, decreased mobilization, and/or muscle contractures (3, 13, 17). The characteristic morphological alteration occurring in muscle is the loss of muscle mass. The muscle wasting that occurs in catabolic states such as burns, sepsis, lipopolysaccharide injection (8), and/or denervation syndromes results largely from an accelerated breakdown of muscle protein (5, 31, 32).
Susceptibility to apoptosis in different murine muscle cell lines
Journal of Muscle Research and Cell Motility, 2000
Objective of the study was to investigate growth characteristics and susceptibility to apoptosis in different murine muscle cell lines (selected for high body weight, DU-6; randomly mated control, DU-Ks; immortalized myoblast cell line, C 2 C 12 ). Apoptosis was induced by serum deprivation. At days 4, 5, and 6 of cultivation, protein, DNA and the frequency of apoptotic cells were determined. Until day 4, C 2 C 12 accumulated more DNA and protein compared with DU-Ks and DU-6, while exhibiting a lower percentage of apoptotic cells. Serum deprivation impaired the growth of each cell line. C 2 C 12 continued to accumulate DNA and protein after serum deprivation, whereas reductions, indicative of cell death, were apparent in DU-Ks and DU-6. Serum deprivation did not enhance apoptosis in C 2 C 12 . Higher percentages of apoptosis were observed in DU-Ks and DU-6 after 2 days of serum deprivation with greater responsiveness of DU-6 to serum deprivation. The results suggest that cell loss in response to serum deprivation is in part due to induction of apoptosis. C 2 C 12 are less sensitive to sub-optimal culture conditions compared with DU-Ks and DU-6 which are at a closer distance to the in vivo status. Moreover, longterm selection for growth decreases the basic frequency of apoptosis of muscle satellite cells, but increases their susceptibility to apoptosis induction.
Opposing responses of apoptosis and autophagy to moderate compression in skeletal muscle
Acta Physiologica, 2011
The molecular mechanism that contributes to the pathogenesis of deep pressure ulcer remains to be elucidated. This study tested the hypotheses that: (1) apoptosis and autophagy are activated in compression-induced muscle pathology and (2) apoptotic and autophagic changes precede pathohistological changes in skeletal muscle in response to prolonged moderate compression. Methods: Adult Sprague-Dawley rats were subjected to an experimental model of pressure-induced deep tissue injury. Static pressure of 100 mmHg was applied to an area of 1.5 cm 2 over the mid-tibialis region of right limb of rats for one single session of 6-h compression (1D) or two sessions of 6-h compression over two consecutive days with rats sacrificed one day (2D) or immediately after (2D-IM) the compression. The left uncompressed limb served as the intra-animal control. Muscle tissues underneath compression region were collected for analysis. Results: Our histological analysis indicated that pathohistological characteristics including rounding contour of myofibres and massive nuclei accumulation were apparently demonstrated in muscles of 2D and 2D-IM. In contrast, these pathohistological changes were generally not found in muscle following 1D. Apoptotic DNA fragmentation, terminal dUTP nick-end labelling index and caspase-3 protease activity were significantly elevated in compressed muscles of all groups. Caspase-9 enzymatic activity was found to be significantly increased in compressed muscles of 2D and 2D-IM whereas increase in caspase-8 activity was exclusively found in compressed muscle of 1D. According to our immunoblot analysis, FoxO3 was significantly reduced in compressed muscles of all groups whereas Beclin-1 was decreased only in 2D. LC3-I was significantly reduced in compressed muscles of all groups while LC3-II was decreased in 2D and 1D. No significant differences were found in the protein abundance of Akt and phospho-Akt in muscles among all groups. Conclusion: These data demonstrate the opposing responses of apoptosis and autophagy to moderate compression in muscle. Moreover, our findings suggest that cellular changes in apoptosis and autophagy have already taken place in the very early stage in which apparent histopathology has yet to develop in the process of compression-induced muscle pathology. Keywords bedsore, caspase, deep pressure ulcer, programmed cell death.