Abstract
Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.
| Original language | English |
|---|---|
| Pages (from-to) | 21-32 |
| Number of pages | 12 |
| Journal | Histology and Histopathology |
| Volume | 25 |
| Issue number | 1 |
| Publication status | Published - Jan 2010 |
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Keywords
- Apoptosis
- C2C12 cells
- Electron microscopy
- TUNEL
- UVB
ASJC Scopus subject areas
- Histology
- Pathology and Forensic Medicine
Cite this
Morphological and biochemical patterns in skeletal muscle apoptosis. / D'Emilio, A.; Biagiotti, L.; Burattini, S.; Battistelli, M.; Canonico, B.; Evangelisti, C.; Ferri, P.; Papa, S.; Martelli, A. M.; Falcieri, Elisabetta.
In: Histology and Histopathology, Vol. 25, No. 1, 01.2010, p. 21-32.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Morphological and biochemical patterns in skeletal muscle apoptosis
AU - D'Emilio, A.
AU - Biagiotti, L.
AU - Burattini, S.
AU - Battistelli, M.
AU - Canonico, B.
AU - Evangelisti, C.
AU - Ferri, P.
AU - Papa, S.
AU - Martelli, A. M.
AU - Falcieri, Elisabetta
PY - 2010/1
Y1 - 2010/1
N2 - Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.
AB - Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.
KW - Apoptosis
KW - C2C12 cells
KW - Electron microscopy
KW - TUNEL
KW - UVB
UR - http://www.scopus.com/inward/record.url?scp=74749093858&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=74749093858&partnerID=8YFLogxK
M3 - Article
C2 - 19924638
AN - SCOPUS:74749093858
VL - 25
SP - 21
EP - 32
JO - Histology and Histopathology
JF - Histology and Histopathology
SN - 0213-3911
IS - 1
ER -