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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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Related in: MedlinePlus

Apoptosis in triceps brachiiApoptotic nuclei recognized by TUNEL staining (green, separately shown in the bottom row) of triceps brachii cross-sections from 12-week-old mice. Muscles were counterstained with WGA (red) to visualize membranes and fibrotic areas and with DAPI (blue) to stain nuclei. Muscle fibres containing apoptotic nuclei are found in dyW/dyW and to a lower extent in dyW/mag muscle (arrows). Apoptotic nuclei recognized in dyW/Bcl and dyW/Bcl/mag muscle reside outside of the muscle fibres (arrowheads).Relative number of the TUNEL-positive muscle fibres. Bcl2 expression inhibits apoptosis in muscle fibres of dyW/dyW (p = 0.004). Similarly, Bcl2 prevents apoptosis in dyW/mag mice at 12 weeks (p ≤ 0.001), at 16 weeks (p = 0.002) as well as over both ages (two-way ANOVA: p < 0.0001); N ≥ 3 ≤ 4.Percentage of centrally localized nuclei that undergo apoptosis. In dyW/dyW muscles almost 7% and in dyW/mag muscles 3.5% of the regenerating fibres are TUNEL-positive. Apoptosis of regenerating fibres is strongly decreased by expression of Bcl2 in muscles of dyW/dyW (p = 0.02) and dyW/mag mice (12 weeks: p = 0.03; 16 weeks: p = 0.04; two-way ANOVA over both ages: p = 0.0027); N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per 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 above. Size bars = 50 µm.
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fig03: Apoptosis in triceps brachiiApoptotic nuclei recognized by TUNEL staining (green, separately shown in the bottom row) of triceps brachii cross-sections from 12-week-old mice. Muscles were counterstained with WGA (red) to visualize membranes and fibrotic areas and with DAPI (blue) to stain nuclei. Muscle fibres containing apoptotic nuclei are found in dyW/dyW and to a lower extent in dyW/mag muscle (arrows). Apoptotic nuclei recognized in dyW/Bcl and dyW/Bcl/mag muscle reside outside of the muscle fibres (arrowheads).Relative number of the TUNEL-positive muscle fibres. Bcl2 expression inhibits apoptosis in muscle fibres of dyW/dyW (p = 0.004). Similarly, Bcl2 prevents apoptosis in dyW/mag mice at 12 weeks (p ≤ 0.001), at 16 weeks (p = 0.002) as well as over both ages (two-way ANOVA: p < 0.0001); N ≥ 3 ≤ 4.Percentage of centrally localized nuclei that undergo apoptosis. In dyW/dyW muscles almost 7% and in dyW/mag muscles 3.5% of the regenerating fibres are TUNEL-positive. Apoptosis of regenerating fibres is strongly decreased by expression of Bcl2 in muscles of dyW/dyW (p = 0.02) and dyW/mag mice (12 weeks: p = 0.03; 16 weeks: p = 0.04; two-way ANOVA over both ages: p = 0.0027); N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per 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 above. Size bars = 50 µm.

Mentions: To verify the role of apoptosis in the loss of fibres in dyW/dyW mice, we used TdT-mediated dUTP nick end labelling (TUNEL) which recognizes nuclear in situ DNA-strand breaks that occur in nuclei undergoing apoptosis. In dyW/dyW and dyW/mag muscles, apoptotic TUNEL-positive muscle fibres could be identified (Fig 3A, arrows) and represented 1.7 and 1% of all fibres, respectively (Fig 3B). In contrast, no TUNEL-positive myonuclei were found upon Bcl2 expression. The TUNEL-positive nuclei detected in dyW/Bcl and dyW/Bcl/mag mice all seemed to belong to non-muscle cells (Fig 3A, arrowheads). Apoptosis has been suggested to increase during abortive muscle regeneration in MDC1A patients (Hayashi et al, 2001) and mouse models thereof (Bentzinger et al, 2005; Dominov et al, 2005; Girgenrath et al, 2004; Kuang et al, 1999). To account for this, we also counted the number of TUNEL-positive, centrally localized myonuclei. Central nucleation has been shown to be indicative of regenerative processes. As shown in Fig 3C, triceps muscle from dyW/dyW and dyW/mag contained more regenerating fibres that were TUNEL-positive than those expressing Bcl2. These results indicate that Bcl2 largely prevents the death of fibres both in normal and regenerating muscle, which may maintain the number of fibres to the levels of control mice.


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)

Apoptosis in triceps brachiiApoptotic nuclei recognized by TUNEL staining (green, separately shown in the bottom row) of triceps brachii cross-sections from 12-week-old mice. Muscles were counterstained with WGA (red) to visualize membranes and fibrotic areas and with DAPI (blue) to stain nuclei. Muscle fibres containing apoptotic nuclei are found in dyW/dyW and to a lower extent in dyW/mag muscle (arrows). Apoptotic nuclei recognized in dyW/Bcl and dyW/Bcl/mag muscle reside outside of the muscle fibres (arrowheads).Relative number of the TUNEL-positive muscle fibres. Bcl2 expression inhibits apoptosis in muscle fibres of dyW/dyW (p = 0.004). Similarly, Bcl2 prevents apoptosis in dyW/mag mice at 12 weeks (p ≤ 0.001), at 16 weeks (p = 0.002) as well as over both ages (two-way ANOVA: p < 0.0001); N ≥ 3 ≤ 4.Percentage of centrally localized nuclei that undergo apoptosis. In dyW/dyW muscles almost 7% and in dyW/mag muscles 3.5% of the regenerating fibres are TUNEL-positive. Apoptosis of regenerating fibres is strongly decreased by expression of Bcl2 in muscles of dyW/dyW (p = 0.02) and dyW/mag mice (12 weeks: p = 0.03; 16 weeks: p = 0.04; two-way ANOVA over both ages: p = 0.0027); N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per 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 above. Size bars = 50 µm.
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fig03: Apoptosis in triceps brachiiApoptotic nuclei recognized by TUNEL staining (green, separately shown in the bottom row) of triceps brachii cross-sections from 12-week-old mice. Muscles were counterstained with WGA (red) to visualize membranes and fibrotic areas and with DAPI (blue) to stain nuclei. Muscle fibres containing apoptotic nuclei are found in dyW/dyW and to a lower extent in dyW/mag muscle (arrows). Apoptotic nuclei recognized in dyW/Bcl and dyW/Bcl/mag muscle reside outside of the muscle fibres (arrowheads).Relative number of the TUNEL-positive muscle fibres. Bcl2 expression inhibits apoptosis in muscle fibres of dyW/dyW (p = 0.004). Similarly, Bcl2 prevents apoptosis in dyW/mag mice at 12 weeks (p ≤ 0.001), at 16 weeks (p = 0.002) as well as over both ages (two-way ANOVA: p < 0.0001); N ≥ 3 ≤ 4.Percentage of centrally localized nuclei that undergo apoptosis. In dyW/dyW muscles almost 7% and in dyW/mag muscles 3.5% of the regenerating fibres are TUNEL-positive. Apoptosis of regenerating fibres is strongly decreased by expression of Bcl2 in muscles of dyW/dyW (p = 0.02) and dyW/mag mice (12 weeks: p = 0.03; 16 weeks: p = 0.04; two-way ANOVA over both ages: p = 0.0027); N ≥ 3 ≤ 4. All values represent the mean ± SEM; N indicates the animal number per 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 above. Size bars = 50 µm.
Mentions: To verify the role of apoptosis in the loss of fibres in dyW/dyW mice, we used TdT-mediated dUTP nick end labelling (TUNEL) which recognizes nuclear in situ DNA-strand breaks that occur in nuclei undergoing apoptosis. In dyW/dyW and dyW/mag muscles, apoptotic TUNEL-positive muscle fibres could be identified (Fig 3A, arrows) and represented 1.7 and 1% of all fibres, respectively (Fig 3B). In contrast, no TUNEL-positive myonuclei were found upon Bcl2 expression. The TUNEL-positive nuclei detected in dyW/Bcl and dyW/Bcl/mag mice all seemed to belong to non-muscle cells (Fig 3A, arrowheads). Apoptosis has been suggested to increase during abortive muscle regeneration in MDC1A patients (Hayashi et al, 2001) and mouse models thereof (Bentzinger et al, 2005; Dominov et al, 2005; Girgenrath et al, 2004; Kuang et al, 1999). To account for this, we also counted the number of TUNEL-positive, centrally localized myonuclei. Central nucleation has been shown to be indicative of regenerative processes. As shown in Fig 3C, triceps muscle from dyW/dyW and dyW/mag contained more regenerating fibres that were TUNEL-positive than those expressing Bcl2. These results indicate that Bcl2 largely prevents the death of fibres both in normal and regenerating muscle, which may maintain the number of fibres to the levels of control mice.

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