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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

Histological analysis of triceps brachii muscle from 12- to 16-week-old miceH & E and Masson's trichrome staining of cross-sections from 12-week old mice.Masson's trichrome staining from 16-week-old mice. Pathological changes observed in dyW/dyW mice, such as fibrosis, variation of muscle fibre diameters, increase in small fibres and collagen-containing tissue (blue in Masson Trichrome staining), are reduced in dyW/mag and dyW/Bcl/mag mice but are similar or stronger in dyW/Bcl mice.Percentage of fibrotic area per triceps cross-section. Mini-agrin reduces (p = 0.007), whereas Bcl2 increases (p = 0.03) fibrosis in dyW/dyW muscle. Fibrosis is comparable in dyW/mag and dyW/Bcl/mag muscles at 12- (p = 0.22) and at 16- (p = 0.14) weeks of age. N ≥ 3 ≤ 5.Muscle fibre size distribution of 12-week-old mice. Values represent relative numbers of fibres in a given diameter class (5 µm/class). DyW/Bcl muscles contain many more small fibres than dyW/dyW muscles. The fibre size distribution of dyW/Bcl/mag is shifted to smaller fibres compared to dyW/mag or control (ctrl) muscles; N = 4.Change in fibre size distribution from 12- to 16-week-old dyW/Bcl/mag and dyW/mag mice. Percentage of fibres with larger diameters increases from 12 to 16 weeks of age in dyW/Bcl/mag (arrows) but not in dyW/mag mice; N = 4.Mean fibre diameter. The size of dyW/Bcl/mag fibres increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.02) and becomes similar to the mean fibre size in dyW/mag muscle (p = 0.22); N = 4.Muscle area (mm2) of mid-belly triceps cross-sections. Control muscle has nearly twice the size compared to all the other genotypes at 12 weeks of age. Although not significantly different over all time points (two-way ANOVA: p = 0.058), muscle area of dyW/Bcl/mag mice increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.026) and becomes significantly larger than in dyW/mag mice (p = 0.03) at 16 weeks of age; N = 4.Total number of fibres per triceps cross-section. At 12 weeks of age, dyW/dyW (p < 0.001) and dyW/mag (p = 0.001) muscles have a significantly reduced number of fibres, whereas, the fibre number is near normal in dyW/Bcl (p = 0.8) and dyW/Bcl/mag (p = 0.08) mice. In 16-week-old dyW/Bcl/mag mice, the number of fibres is similar to controls (p = 0.86). Two-way ANOVA analysis confirmed the normalization of the fibre number over all ages in dyW/Bcl/mag when compared to dyW/mag muscle (p < 0.0001). Bcl2 expression did not increase the number of fibres on WT background (Bcl); N ≥ 3 ≤ 5. 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 if indicated (°p ≤ 0.05). Size bars = 100 µm.
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fig01: Histological analysis of triceps brachii muscle from 12- to 16-week-old miceH & E and Masson's trichrome staining of cross-sections from 12-week old mice.Masson's trichrome staining from 16-week-old mice. Pathological changes observed in dyW/dyW mice, such as fibrosis, variation of muscle fibre diameters, increase in small fibres and collagen-containing tissue (blue in Masson Trichrome staining), are reduced in dyW/mag and dyW/Bcl/mag mice but are similar or stronger in dyW/Bcl mice.Percentage of fibrotic area per triceps cross-section. Mini-agrin reduces (p = 0.007), whereas Bcl2 increases (p = 0.03) fibrosis in dyW/dyW muscle. Fibrosis is comparable in dyW/mag and dyW/Bcl/mag muscles at 12- (p = 0.22) and at 16- (p = 0.14) weeks of age. N ≥ 3 ≤ 5.Muscle fibre size distribution of 12-week-old mice. Values represent relative numbers of fibres in a given diameter class (5 µm/class). DyW/Bcl muscles contain many more small fibres than dyW/dyW muscles. The fibre size distribution of dyW/Bcl/mag is shifted to smaller fibres compared to dyW/mag or control (ctrl) muscles; N = 4.Change in fibre size distribution from 12- to 16-week-old dyW/Bcl/mag and dyW/mag mice. Percentage of fibres with larger diameters increases from 12 to 16 weeks of age in dyW/Bcl/mag (arrows) but not in dyW/mag mice; N = 4.Mean fibre diameter. The size of dyW/Bcl/mag fibres increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.02) and becomes similar to the mean fibre size in dyW/mag muscle (p = 0.22); N = 4.Muscle area (mm2) of mid-belly triceps cross-sections. Control muscle has nearly twice the size compared to all the other genotypes at 12 weeks of age. Although not significantly different over all time points (two-way ANOVA: p = 0.058), muscle area of dyW/Bcl/mag mice increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.026) and becomes significantly larger than in dyW/mag mice (p = 0.03) at 16 weeks of age; N = 4.Total number of fibres per triceps cross-section. At 12 weeks of age, dyW/dyW (p < 0.001) and dyW/mag (p = 0.001) muscles have a significantly reduced number of fibres, whereas, the fibre number is near normal in dyW/Bcl (p = 0.8) and dyW/Bcl/mag (p = 0.08) mice. In 16-week-old dyW/Bcl/mag mice, the number of fibres is similar to controls (p = 0.86). Two-way ANOVA analysis confirmed the normalization of the fibre number over all ages in dyW/Bcl/mag when compared to dyW/mag muscle (p < 0.0001). Bcl2 expression did not increase the number of fibres on WT background (Bcl); N ≥ 3 ≤ 5. 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 if indicated (°p ≤ 0.05). Size bars = 100 µm.

Mentions: To determine the effect of the transgenes on the muscle pathology, we analysed the fast-twitch foreleg muscle triceps brachii (Fig 1) and the slow-twitch hindleg muscle soleus (Fig 2). The latter becomes progressively paralysed in dyW/dyW mice as a consequence of peripheral nerve demyelination (Kuang et al, 1998b). Haematoxylin & Eosin (H & E; Merck) and Masson's trichrome stainings of triceps muscle of 12- (Fig 1A) and 16-week-old animals (Fig 1B) are shown. They revealed extensive fibrosis and a high proportion of small, rounded fibres in dyW/dyW mice (Fig 1A). Expression of mini-agrin (dyW/mag) largely impeded the replacement of muscle with fibrotic tissue while Bcl2 expression (dyW/Bcl) seemed to increase fibrosis in dyW/dyW muscle (Fig 1A), a finding that was confirmed by quantification of the relative fibrotic area (Fig 1C) and by determining the amount of the collagen-specific amino acid hydroxyproline, which is a measure of fibrosis (Fig S2A of Supporting information). However, co-expression of mini-agrin (dyW/Bcl/mag) eliminated the fibrotic impact of Bcl2 (Fig 1A and C and Fig S2A of Supporting information). The high degree of fibrosis in Bcl2 transgenic dyW/dyW mice coincided with a strong increase in the number of macrophages in triceps of dyW/Bcl mice (Fig S2C and D of Supporting information). Like the fibrosis, inflammation was reduced by the co-expression of mini-agrin. The same histological results were also obtained in the soleus muscle of 12-week-old mice (Fig 2A and B and Fig S2B of Supporting information), indicating that the progressed paralysis of the hindlegs did not affect fibrosis.


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)

Histological analysis of triceps brachii muscle from 12- to 16-week-old miceH & E and Masson's trichrome staining of cross-sections from 12-week old mice.Masson's trichrome staining from 16-week-old mice. Pathological changes observed in dyW/dyW mice, such as fibrosis, variation of muscle fibre diameters, increase in small fibres and collagen-containing tissue (blue in Masson Trichrome staining), are reduced in dyW/mag and dyW/Bcl/mag mice but are similar or stronger in dyW/Bcl mice.Percentage of fibrotic area per triceps cross-section. Mini-agrin reduces (p = 0.007), whereas Bcl2 increases (p = 0.03) fibrosis in dyW/dyW muscle. Fibrosis is comparable in dyW/mag and dyW/Bcl/mag muscles at 12- (p = 0.22) and at 16- (p = 0.14) weeks of age. N ≥ 3 ≤ 5.Muscle fibre size distribution of 12-week-old mice. Values represent relative numbers of fibres in a given diameter class (5 µm/class). DyW/Bcl muscles contain many more small fibres than dyW/dyW muscles. The fibre size distribution of dyW/Bcl/mag is shifted to smaller fibres compared to dyW/mag or control (ctrl) muscles; N = 4.Change in fibre size distribution from 12- to 16-week-old dyW/Bcl/mag and dyW/mag mice. Percentage of fibres with larger diameters increases from 12 to 16 weeks of age in dyW/Bcl/mag (arrows) but not in dyW/mag mice; N = 4.Mean fibre diameter. The size of dyW/Bcl/mag fibres increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.02) and becomes similar to the mean fibre size in dyW/mag muscle (p = 0.22); N = 4.Muscle area (mm2) of mid-belly triceps cross-sections. Control muscle has nearly twice the size compared to all the other genotypes at 12 weeks of age. Although not significantly different over all time points (two-way ANOVA: p = 0.058), muscle area of dyW/Bcl/mag mice increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.026) and becomes significantly larger than in dyW/mag mice (p = 0.03) at 16 weeks of age; N = 4.Total number of fibres per triceps cross-section. At 12 weeks of age, dyW/dyW (p < 0.001) and dyW/mag (p = 0.001) muscles have a significantly reduced number of fibres, whereas, the fibre number is near normal in dyW/Bcl (p = 0.8) and dyW/Bcl/mag (p = 0.08) mice. In 16-week-old dyW/Bcl/mag mice, the number of fibres is similar to controls (p = 0.86). Two-way ANOVA analysis confirmed the normalization of the fibre number over all ages in dyW/Bcl/mag when compared to dyW/mag muscle (p < 0.0001). Bcl2 expression did not increase the number of fibres on WT background (Bcl); N ≥ 3 ≤ 5. 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 if indicated (°p ≤ 0.05). Size bars = 100 µm.
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fig01: Histological analysis of triceps brachii muscle from 12- to 16-week-old miceH & E and Masson's trichrome staining of cross-sections from 12-week old mice.Masson's trichrome staining from 16-week-old mice. Pathological changes observed in dyW/dyW mice, such as fibrosis, variation of muscle fibre diameters, increase in small fibres and collagen-containing tissue (blue in Masson Trichrome staining), are reduced in dyW/mag and dyW/Bcl/mag mice but are similar or stronger in dyW/Bcl mice.Percentage of fibrotic area per triceps cross-section. Mini-agrin reduces (p = 0.007), whereas Bcl2 increases (p = 0.03) fibrosis in dyW/dyW muscle. Fibrosis is comparable in dyW/mag and dyW/Bcl/mag muscles at 12- (p = 0.22) and at 16- (p = 0.14) weeks of age. N ≥ 3 ≤ 5.Muscle fibre size distribution of 12-week-old mice. Values represent relative numbers of fibres in a given diameter class (5 µm/class). DyW/Bcl muscles contain many more small fibres than dyW/dyW muscles. The fibre size distribution of dyW/Bcl/mag is shifted to smaller fibres compared to dyW/mag or control (ctrl) muscles; N = 4.Change in fibre size distribution from 12- to 16-week-old dyW/Bcl/mag and dyW/mag mice. Percentage of fibres with larger diameters increases from 12 to 16 weeks of age in dyW/Bcl/mag (arrows) but not in dyW/mag mice; N = 4.Mean fibre diameter. The size of dyW/Bcl/mag fibres increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.02) and becomes similar to the mean fibre size in dyW/mag muscle (p = 0.22); N = 4.Muscle area (mm2) of mid-belly triceps cross-sections. Control muscle has nearly twice the size compared to all the other genotypes at 12 weeks of age. Although not significantly different over all time points (two-way ANOVA: p = 0.058), muscle area of dyW/Bcl/mag mice increases from 12 to 16 weeks of age (two-way ANOVA: p = 0.026) and becomes significantly larger than in dyW/mag mice (p = 0.03) at 16 weeks of age; N = 4.Total number of fibres per triceps cross-section. At 12 weeks of age, dyW/dyW (p < 0.001) and dyW/mag (p = 0.001) muscles have a significantly reduced number of fibres, whereas, the fibre number is near normal in dyW/Bcl (p = 0.8) and dyW/Bcl/mag (p = 0.08) mice. In 16-week-old dyW/Bcl/mag mice, the number of fibres is similar to controls (p = 0.86). Two-way ANOVA analysis confirmed the normalization of the fibre number over all ages in dyW/Bcl/mag when compared to dyW/mag muscle (p < 0.0001). Bcl2 expression did not increase the number of fibres on WT background (Bcl); N ≥ 3 ≤ 5. 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 if indicated (°p ≤ 0.05). Size bars = 100 µm.
Mentions: To determine the effect of the transgenes on the muscle pathology, we analysed the fast-twitch foreleg muscle triceps brachii (Fig 1) and the slow-twitch hindleg muscle soleus (Fig 2). The latter becomes progressively paralysed in dyW/dyW mice as a consequence of peripheral nerve demyelination (Kuang et al, 1998b). Haematoxylin & Eosin (H & E; Merck) and Masson's trichrome stainings of triceps muscle of 12- (Fig 1A) and 16-week-old animals (Fig 1B) are shown. They revealed extensive fibrosis and a high proportion of small, rounded fibres in dyW/dyW mice (Fig 1A). Expression of mini-agrin (dyW/mag) largely impeded the replacement of muscle with fibrotic tissue while Bcl2 expression (dyW/Bcl) seemed to increase fibrosis in dyW/dyW muscle (Fig 1A), a finding that was confirmed by quantification of the relative fibrotic area (Fig 1C) and by determining the amount of the collagen-specific amino acid hydroxyproline, which is a measure of fibrosis (Fig S2A of Supporting information). However, co-expression of mini-agrin (dyW/Bcl/mag) eliminated the fibrotic impact of Bcl2 (Fig 1A and C and Fig S2A of Supporting information). The high degree of fibrosis in Bcl2 transgenic dyW/dyW mice coincided with a strong increase in the number of macrophages in triceps of dyW/Bcl mice (Fig S2C and D of Supporting information). Like the fibrosis, inflammation was reduced by the co-expression of mini-agrin. The same histological results were also obtained in the soleus muscle of 12-week-old mice (Fig 2A and B and Fig S2B of Supporting information), indicating that the progressed paralysis of the hindlegs did not affect fibrosis.

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