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Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice.

Meinen S, Lin S, Thurnherr R, Erb M, Meier T, Rüegg MA - EMBO Mol Med (2011)

Bottom Line: By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres.Treatment of mice with both agents results in improved muscle regeneration and increased force.Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Switzerland. markus-a.ruegg@unibas.ch

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Spontaneous and injury-induced muscle regenerationA. Percentage of CNF. In dyW/dyW mice, only 9% of the muscle fibres are centrally nucleated, indicative of impaired regeneration. In dyW/mag muscle the number of CNF is increased and is further elevated by co-expression of Bcl2 in both 12- and 16-week-old dyW/Bcl/mag mice (two-way ANOVA over all ages: p < 0.0001); N = 4.B. Spontaneous muscle regeneration in 12-week-old muscles. Antibodies to dMyHC (green) and laminin-γ1 (red) were used. DAPI (blue) visualizes nuclei. Only few dMyHC-positive fibres are found in dyW/dyW mice. Some more dMyHC-positive fibres are present in dyW/mag (arrows) muscle, whereas, many more are detected upon Bcl2 expression in dyW/Bcl and dyW/Bcl/mag (encircled areas).C. Quantification of dMyHC-positive fibres. The number of dMyHC-positive fibres is highest in Bcl2 transgenic mice (dyW/Bcl and dyW/Bcl/mag) followed by dyW/mag and dyW/dyW mice. Although not significant at 12 or 16 weeks of age (t-test), the increase of dMyHC-positive fibres in dyW/Bcl/mag compared to dyW/mag muscles becomes significant when measured over all ages (two-way ANOVA: p = 0.022). No spontaneous regeneration was detected in control muscles (ctrl); N ≥ 4 ≤ 6.D.–F. Regenerative response of tibialis anterior muscle of 6-week-old mice after notexin injection.D. Muscle cross-sections were stained with antibodies to dMyHC (green) and laminin-γ1 (red), and with the nuclear marker DAPI (blue) 1 week after notexin injection. Only very few dMyHC-positive fibres are detected in dyW/dyW mice, while many muscle fibres are dMyHC-positive in dyW/Bcl, dyW/mag, dyW/Bcl/mag and control (ctrl) mice. Note that muscle fibres in dyW/Bcl mice are small; N ≥ 4 ≤ 5.E. Regeneration status of tibialis anterior 2 weeks after notexin injection. Antibodies to dMyHC (green), laminin-γ1 (red) and the nuclear marker DAPI (blue) were used. Absence of dMyHC-positive fibres and large areas devoid of laminin-γ1 staining (asterisks) are indicators of the failed regeneration in dyW/dyW and dyW/Bcl muscles. The presence of few dMyHC-positive fibres and the non-disrupted laminin-γ1 staining indicate successful muscle regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4.F. H & E staining of tibialis anterior 2 weeks after notexin injection shows the failure in regeneration leading to fibrosis in dyW/dyW and dyW/Bcl muscles (asterisks) and the successful regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per each experimental group. p-Values are Student's t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s. p > 0.05) or two-way ANOVA as noted in the text. Size bars = 50 µm.
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fig04: Spontaneous and injury-induced muscle regenerationA. Percentage of CNF. In dyW/dyW mice, only 9% of the muscle fibres are centrally nucleated, indicative of impaired regeneration. In dyW/mag muscle the number of CNF is increased and is further elevated by co-expression of Bcl2 in both 12- and 16-week-old dyW/Bcl/mag mice (two-way ANOVA over all ages: p < 0.0001); N = 4.B. Spontaneous muscle regeneration in 12-week-old muscles. Antibodies to dMyHC (green) and laminin-γ1 (red) were used. DAPI (blue) visualizes nuclei. Only few dMyHC-positive fibres are found in dyW/dyW mice. Some more dMyHC-positive fibres are present in dyW/mag (arrows) muscle, whereas, many more are detected upon Bcl2 expression in dyW/Bcl and dyW/Bcl/mag (encircled areas).C. Quantification of dMyHC-positive fibres. The number of dMyHC-positive fibres is highest in Bcl2 transgenic mice (dyW/Bcl and dyW/Bcl/mag) followed by dyW/mag and dyW/dyW mice. Although not significant at 12 or 16 weeks of age (t-test), the increase of dMyHC-positive fibres in dyW/Bcl/mag compared to dyW/mag muscles becomes significant when measured over all ages (two-way ANOVA: p = 0.022). No spontaneous regeneration was detected in control muscles (ctrl); N ≥ 4 ≤ 6.D.–F. Regenerative response of tibialis anterior muscle of 6-week-old mice after notexin injection.D. Muscle cross-sections were stained with antibodies to dMyHC (green) and laminin-γ1 (red), and with the nuclear marker DAPI (blue) 1 week after notexin injection. Only very few dMyHC-positive fibres are detected in dyW/dyW mice, while many muscle fibres are dMyHC-positive in dyW/Bcl, dyW/mag, dyW/Bcl/mag and control (ctrl) mice. Note that muscle fibres in dyW/Bcl mice are small; N ≥ 4 ≤ 5.E. Regeneration status of tibialis anterior 2 weeks after notexin injection. Antibodies to dMyHC (green), laminin-γ1 (red) and the nuclear marker DAPI (blue) were used. Absence of dMyHC-positive fibres and large areas devoid of laminin-γ1 staining (asterisks) are indicators of the failed regeneration in dyW/dyW and dyW/Bcl muscles. The presence of few dMyHC-positive fibres and the non-disrupted laminin-γ1 staining indicate successful muscle regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4.F. H & E staining of tibialis anterior 2 weeks after notexin injection shows the failure in regeneration leading to fibrosis in dyW/dyW and dyW/Bcl muscles (asterisks) and the successful regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per each experimental group. p-Values are Student's t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s. p > 0.05) or two-way ANOVA as noted in the text. Size bars = 50 µm.

Mentions: Besides its function in stabilizing muscle fibres during contraction, LM-211 has also been shown to be important for the successful regeneration of muscle (Bentzinger et al, 2005; Kuang et al, 1999). In addition, regeneration is improved in dyW/mag mice (Meinen et al, 2007). Regenerating muscle fibres transiently express the developmental form of myosin heavy chain (dMyHC; Novocastra, NCL-MHCd) and newly regenerated fibres have their nuclei in the centre and not in the periphery. Thus, the number of dMyHC-positive and centrally nucleated fibres (CNF) is a measure of ongoing and successful regeneration. To assess the role of apoptosis inhibition in the regeneration process, we first counted the number of CNF in the different mouse models. As shown earlier, the number of CNF was increased in dyW/dyW mice due to ongoing muscle degeneration when compared to WT controls (Fig 4A). Expression of Bcl2 resulted in a further threefold increase in the number of CNF, suggesting that regeneration of muscle fibres is more effective. Similarly, the number of CNF was increased in dyW/mag mice compared to dyW/dyW but was lower than in dyW/Bcl mice. This finding is consistent with the previous interpretation that mini-agrin enables successful regeneration but primarily prevents muscle degeneration by its linking of the basement membrane to α-dystroglycan (Moll et al, 2001). Interestingly, expression of both Bcl2 and mini-agrin resulted in a further increase of the number of CNF in 12- and 16-week-old mice compared to dyW/mag mice. To test for ongoing regeneration, we also stained for dMyHC. As with CNF, the number of dMyHC-positive fibres in triceps muscle was several fold higher in dyW/Bcl than in dyW/dyW mice and the effect of Bcl2 was also seen when combined with mini-agrin (Fig 4B and C).


Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice.

Meinen S, Lin S, Thurnherr R, Erb M, Meier T, Rüegg MA - EMBO Mol Med (2011)

Spontaneous and injury-induced muscle regenerationA. Percentage of CNF. In dyW/dyW mice, only 9% of the muscle fibres are centrally nucleated, indicative of impaired regeneration. In dyW/mag muscle the number of CNF is increased and is further elevated by co-expression of Bcl2 in both 12- and 16-week-old dyW/Bcl/mag mice (two-way ANOVA over all ages: p < 0.0001); N = 4.B. Spontaneous muscle regeneration in 12-week-old muscles. Antibodies to dMyHC (green) and laminin-γ1 (red) were used. DAPI (blue) visualizes nuclei. Only few dMyHC-positive fibres are found in dyW/dyW mice. Some more dMyHC-positive fibres are present in dyW/mag (arrows) muscle, whereas, many more are detected upon Bcl2 expression in dyW/Bcl and dyW/Bcl/mag (encircled areas).C. Quantification of dMyHC-positive fibres. The number of dMyHC-positive fibres is highest in Bcl2 transgenic mice (dyW/Bcl and dyW/Bcl/mag) followed by dyW/mag and dyW/dyW mice. Although not significant at 12 or 16 weeks of age (t-test), the increase of dMyHC-positive fibres in dyW/Bcl/mag compared to dyW/mag muscles becomes significant when measured over all ages (two-way ANOVA: p = 0.022). No spontaneous regeneration was detected in control muscles (ctrl); N ≥ 4 ≤ 6.D.–F. Regenerative response of tibialis anterior muscle of 6-week-old mice after notexin injection.D. Muscle cross-sections were stained with antibodies to dMyHC (green) and laminin-γ1 (red), and with the nuclear marker DAPI (blue) 1 week after notexin injection. Only very few dMyHC-positive fibres are detected in dyW/dyW mice, while many muscle fibres are dMyHC-positive in dyW/Bcl, dyW/mag, dyW/Bcl/mag and control (ctrl) mice. Note that muscle fibres in dyW/Bcl mice are small; N ≥ 4 ≤ 5.E. Regeneration status of tibialis anterior 2 weeks after notexin injection. Antibodies to dMyHC (green), laminin-γ1 (red) and the nuclear marker DAPI (blue) were used. Absence of dMyHC-positive fibres and large areas devoid of laminin-γ1 staining (asterisks) are indicators of the failed regeneration in dyW/dyW and dyW/Bcl muscles. The presence of few dMyHC-positive fibres and the non-disrupted laminin-γ1 staining indicate successful muscle regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4.F. H & E staining of tibialis anterior 2 weeks after notexin injection shows the failure in regeneration leading to fibrosis in dyW/dyW and dyW/Bcl muscles (asterisks) and the successful regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per each experimental group. p-Values are Student's t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s. p > 0.05) or two-way ANOVA as noted in the text. Size bars = 50 µm.
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fig04: Spontaneous and injury-induced muscle regenerationA. Percentage of CNF. In dyW/dyW mice, only 9% of the muscle fibres are centrally nucleated, indicative of impaired regeneration. In dyW/mag muscle the number of CNF is increased and is further elevated by co-expression of Bcl2 in both 12- and 16-week-old dyW/Bcl/mag mice (two-way ANOVA over all ages: p < 0.0001); N = 4.B. Spontaneous muscle regeneration in 12-week-old muscles. Antibodies to dMyHC (green) and laminin-γ1 (red) were used. DAPI (blue) visualizes nuclei. Only few dMyHC-positive fibres are found in dyW/dyW mice. Some more dMyHC-positive fibres are present in dyW/mag (arrows) muscle, whereas, many more are detected upon Bcl2 expression in dyW/Bcl and dyW/Bcl/mag (encircled areas).C. Quantification of dMyHC-positive fibres. The number of dMyHC-positive fibres is highest in Bcl2 transgenic mice (dyW/Bcl and dyW/Bcl/mag) followed by dyW/mag and dyW/dyW mice. Although not significant at 12 or 16 weeks of age (t-test), the increase of dMyHC-positive fibres in dyW/Bcl/mag compared to dyW/mag muscles becomes significant when measured over all ages (two-way ANOVA: p = 0.022). No spontaneous regeneration was detected in control muscles (ctrl); N ≥ 4 ≤ 6.D.–F. Regenerative response of tibialis anterior muscle of 6-week-old mice after notexin injection.D. Muscle cross-sections were stained with antibodies to dMyHC (green) and laminin-γ1 (red), and with the nuclear marker DAPI (blue) 1 week after notexin injection. Only very few dMyHC-positive fibres are detected in dyW/dyW mice, while many muscle fibres are dMyHC-positive in dyW/Bcl, dyW/mag, dyW/Bcl/mag and control (ctrl) mice. Note that muscle fibres in dyW/Bcl mice are small; N ≥ 4 ≤ 5.E. Regeneration status of tibialis anterior 2 weeks after notexin injection. Antibodies to dMyHC (green), laminin-γ1 (red) and the nuclear marker DAPI (blue) were used. Absence of dMyHC-positive fibres and large areas devoid of laminin-γ1 staining (asterisks) are indicators of the failed regeneration in dyW/dyW and dyW/Bcl muscles. The presence of few dMyHC-positive fibres and the non-disrupted laminin-γ1 staining indicate successful muscle regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4.F. H & E staining of tibialis anterior 2 weeks after notexin injection shows the failure in regeneration leading to fibrosis in dyW/dyW and dyW/Bcl muscles (asterisks) and the successful regeneration in dyW/mag, dyW/Bcl/mag and control mice; N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per each experimental group. p-Values are Student's t-test (***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s. p > 0.05) or two-way ANOVA as noted in the text. Size bars = 50 µm.
Mentions: Besides its function in stabilizing muscle fibres during contraction, LM-211 has also been shown to be important for the successful regeneration of muscle (Bentzinger et al, 2005; Kuang et al, 1999). In addition, regeneration is improved in dyW/mag mice (Meinen et al, 2007). Regenerating muscle fibres transiently express the developmental form of myosin heavy chain (dMyHC; Novocastra, NCL-MHCd) and newly regenerated fibres have their nuclei in the centre and not in the periphery. Thus, the number of dMyHC-positive and centrally nucleated fibres (CNF) is a measure of ongoing and successful regeneration. To assess the role of apoptosis inhibition in the regeneration process, we first counted the number of CNF in the different mouse models. As shown earlier, the number of CNF was increased in dyW/dyW mice due to ongoing muscle degeneration when compared to WT controls (Fig 4A). Expression of Bcl2 resulted in a further threefold increase in the number of CNF, suggesting that regeneration of muscle fibres is more effective. Similarly, the number of CNF was increased in dyW/mag mice compared to dyW/dyW but was lower than in dyW/Bcl mice. This finding is consistent with the previous interpretation that mini-agrin enables successful regeneration but primarily prevents muscle degeneration by its linking of the basement membrane to α-dystroglycan (Moll et al, 2001). Interestingly, expression of both Bcl2 and mini-agrin resulted in a further increase of the number of CNF in 12- and 16-week-old mice compared to dyW/mag mice. To test for ongoing regeneration, we also stained for dMyHC. As with CNF, the number of dMyHC-positive fibres in triceps muscle was several fold higher in dyW/Bcl than in dyW/dyW mice and the effect of Bcl2 was also seen when combined with mini-agrin (Fig 4B and C).

Bottom Line: By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres.Treatment of mice with both agents results in improved muscle regeneration and increased force.Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Switzerland. markus-a.ruegg@unibas.ch

Show MeSH
Related in: MedlinePlus