Limits...
Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction.

Sassoli C, Formigli L, Bini F, Tani A, Squecco R, Battistini C, Zecchi-Orlandini S, Francini F, Meacci E - J. Cell. Mol. Med. (2011)

Bottom Line: In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells.Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration.In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

Show MeSH

Related in: MedlinePlus

Effects of S1P treatment on EC-induced apoptosis in EDL muscle. (A) Western blotting analysis of pro-apoptotic protein, Bax and Cytochrome c (Cytc) expression. Protein aliquots (10–25 μg) corresponding to the 15,000 ×g fraction (mitochondria) and the cytosol obtained from control (C), S1P-treated, EC- and EC + S1P-injured muscle fibres were immunodetected by specific antibodies and revealed by enhanced chemiluminescence. The relative percentage of band intensity to control set as 100 (mean ± S.E.M.) and normalized to gel staining or β-actin is shown in the graphs. Student’s t-test, *P < 0.05 versus specific control; n= 3; §P < 0.05 versus EC, n= 3. (B) Determination of caspase 3/7 activity. The caspase 3/7 assay was performed in lysates obtained from the muscle samples as indicated in (A). RFU resulting from the release of free fluorescent caspase 3/7 substrate (Ac-DEVD-AMC) are reported as mean ± S.E.M. (Student’s t-test, *P < 0.05 versus specific control). (C) TUNEL assay. Cryostat sections from control, EC- and EC + S1P- injured muscles. Sparse apoptotic nuclei showing positive TUNEL labelling (green) are detected within the muscle fibres and the surrounding cells in EC injured muscles. Only scanty TUNEL+ cells can be seen in EC + S1P-injured muscles, whereas no apoptotic cells are detected in control muscles.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3822960&req=5

fig02: Effects of S1P treatment on EC-induced apoptosis in EDL muscle. (A) Western blotting analysis of pro-apoptotic protein, Bax and Cytochrome c (Cytc) expression. Protein aliquots (10–25 μg) corresponding to the 15,000 ×g fraction (mitochondria) and the cytosol obtained from control (C), S1P-treated, EC- and EC + S1P-injured muscle fibres were immunodetected by specific antibodies and revealed by enhanced chemiluminescence. The relative percentage of band intensity to control set as 100 (mean ± S.E.M.) and normalized to gel staining or β-actin is shown in the graphs. Student’s t-test, *P < 0.05 versus specific control; n= 3; §P < 0.05 versus EC, n= 3. (B) Determination of caspase 3/7 activity. The caspase 3/7 assay was performed in lysates obtained from the muscle samples as indicated in (A). RFU resulting from the release of free fluorescent caspase 3/7 substrate (Ac-DEVD-AMC) are reported as mean ± S.E.M. (Student’s t-test, *P < 0.05 versus specific control). (C) TUNEL assay. Cryostat sections from control, EC- and EC + S1P- injured muscles. Sparse apoptotic nuclei showing positive TUNEL labelling (green) are detected within the muscle fibres and the surrounding cells in EC injured muscles. Only scanty TUNEL+ cells can be seen in EC + S1P-injured muscles, whereas no apoptotic cells are detected in control muscles.

Mentions: We first evaluated the effects of EC on EDL skeletal muscle by morphological, biochemical and electrophysiological analyses. At light microscopic examination, skeletal muscle fibres subjected to EC showed extensive and severe morphological alterations (histological score of 2.7 ± .0.1, Table 1; Fig. 1A and B). At the ultrastructural level, the majority of the injured fibres displayed variable degree of myofibrillar disarrangement and Z-disk disruption, dilated cistaerne and tubules of sarcoplasmic reticulum, and swollen mitochondria with shortening and disappearance of cristae (Fig. 1C and D). Biochemical analysis revealed that LDH activity was significantly decreased of approximately 35% in EC-injured muscle compared to control (8.9 ± 0.9 versus 13.7 ± 1.2; mean ± S.E.M.; P < 0.05, n= 3). Moreover, we demonstrated a significant increase in the association of the pro-apoptotic protein, Bax, with mitochondrial fractions, and in the release of respiratory chain protein, Cytc, into the cytosolic fractions obtained from EC-damaged muscle compared to control (Fig. 2A). However, EC damage provoked only a slightly increase in caspase 3/7 activity (Fig. 2B), and a certain positivity to the TUNEL reaction. In particular, positive nuclei were found both in the myofibres and in cells located in the close vicinity, and represented an approximately 2% of the total nuclei (Fig. 2C). The electrophysiological assessment of the effects of EC performed on single muscle fibres showed the occurrence of significant changes in the sarcolemnic functionality (reduced plasma membrane resistance (RmCm) and resting membrane depolarization, Table 2), and in the myofibre excitability (reduced Na+ current amplitude (INa) and altered kinetic, Fig. 3A and C; Table 2). EC also affected the excitation-contraction coupling, reducing L-type-mediated Ca2+ current (ICa) and altering the channel kinetics (Fig. 3B and D; Table 2).


Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction.

Sassoli C, Formigli L, Bini F, Tani A, Squecco R, Battistini C, Zecchi-Orlandini S, Francini F, Meacci E - J. Cell. Mol. Med. (2011)

Effects of S1P treatment on EC-induced apoptosis in EDL muscle. (A) Western blotting analysis of pro-apoptotic protein, Bax and Cytochrome c (Cytc) expression. Protein aliquots (10–25 μg) corresponding to the 15,000 ×g fraction (mitochondria) and the cytosol obtained from control (C), S1P-treated, EC- and EC + S1P-injured muscle fibres were immunodetected by specific antibodies and revealed by enhanced chemiluminescence. The relative percentage of band intensity to control set as 100 (mean ± S.E.M.) and normalized to gel staining or β-actin is shown in the graphs. Student’s t-test, *P < 0.05 versus specific control; n= 3; §P < 0.05 versus EC, n= 3. (B) Determination of caspase 3/7 activity. The caspase 3/7 assay was performed in lysates obtained from the muscle samples as indicated in (A). RFU resulting from the release of free fluorescent caspase 3/7 substrate (Ac-DEVD-AMC) are reported as mean ± S.E.M. (Student’s t-test, *P < 0.05 versus specific control). (C) TUNEL assay. Cryostat sections from control, EC- and EC + S1P- injured muscles. Sparse apoptotic nuclei showing positive TUNEL labelling (green) are detected within the muscle fibres and the surrounding cells in EC injured muscles. Only scanty TUNEL+ cells can be seen in EC + S1P-injured muscles, whereas no apoptotic cells are detected in control muscles.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822960&req=5

fig02: Effects of S1P treatment on EC-induced apoptosis in EDL muscle. (A) Western blotting analysis of pro-apoptotic protein, Bax and Cytochrome c (Cytc) expression. Protein aliquots (10–25 μg) corresponding to the 15,000 ×g fraction (mitochondria) and the cytosol obtained from control (C), S1P-treated, EC- and EC + S1P-injured muscle fibres were immunodetected by specific antibodies and revealed by enhanced chemiluminescence. The relative percentage of band intensity to control set as 100 (mean ± S.E.M.) and normalized to gel staining or β-actin is shown in the graphs. Student’s t-test, *P < 0.05 versus specific control; n= 3; §P < 0.05 versus EC, n= 3. (B) Determination of caspase 3/7 activity. The caspase 3/7 assay was performed in lysates obtained from the muscle samples as indicated in (A). RFU resulting from the release of free fluorescent caspase 3/7 substrate (Ac-DEVD-AMC) are reported as mean ± S.E.M. (Student’s t-test, *P < 0.05 versus specific control). (C) TUNEL assay. Cryostat sections from control, EC- and EC + S1P- injured muscles. Sparse apoptotic nuclei showing positive TUNEL labelling (green) are detected within the muscle fibres and the surrounding cells in EC injured muscles. Only scanty TUNEL+ cells can be seen in EC + S1P-injured muscles, whereas no apoptotic cells are detected in control muscles.
Mentions: We first evaluated the effects of EC on EDL skeletal muscle by morphological, biochemical and electrophysiological analyses. At light microscopic examination, skeletal muscle fibres subjected to EC showed extensive and severe morphological alterations (histological score of 2.7 ± .0.1, Table 1; Fig. 1A and B). At the ultrastructural level, the majority of the injured fibres displayed variable degree of myofibrillar disarrangement and Z-disk disruption, dilated cistaerne and tubules of sarcoplasmic reticulum, and swollen mitochondria with shortening and disappearance of cristae (Fig. 1C and D). Biochemical analysis revealed that LDH activity was significantly decreased of approximately 35% in EC-injured muscle compared to control (8.9 ± 0.9 versus 13.7 ± 1.2; mean ± S.E.M.; P < 0.05, n= 3). Moreover, we demonstrated a significant increase in the association of the pro-apoptotic protein, Bax, with mitochondrial fractions, and in the release of respiratory chain protein, Cytc, into the cytosolic fractions obtained from EC-damaged muscle compared to control (Fig. 2A). However, EC damage provoked only a slightly increase in caspase 3/7 activity (Fig. 2B), and a certain positivity to the TUNEL reaction. In particular, positive nuclei were found both in the myofibres and in cells located in the close vicinity, and represented an approximately 2% of the total nuclei (Fig. 2C). The electrophysiological assessment of the effects of EC performed on single muscle fibres showed the occurrence of significant changes in the sarcolemnic functionality (reduced plasma membrane resistance (RmCm) and resting membrane depolarization, Table 2), and in the myofibre excitability (reduced Na+ current amplitude (INa) and altered kinetic, Fig. 3A and C; Table 2). EC also affected the excitation-contraction coupling, reducing L-type-mediated Ca2+ current (ICa) and altering the channel kinetics (Fig. 3B and D; Table 2).

Bottom Line: In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells.Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration.In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

Show MeSH
Related in: MedlinePlus