Elevated collagen levels in skeletal muscles in SGCA- and SGCD-null mice and in the heart of SGCD-null mice.

<p><b>(a)</b> Immunofluorescent images of skeletal muscles stained with collagen type I (fibrotic marker, green) and laminin (extracellular matrix of muscle fibres, red) Scale bar: 100 μm. <b>(b)</b> Quantification of collagen type I positive area in skeletal muscles relative to wild type muscles. A significant increase in the percentage of collagen type I positive area was found in diaphragm and quadriceps muscles of SGCA- and SGCD-null mice, while no difference was found in the gastrocnemius. Tibialis anterior muscles of female SGCD-null mice showed a significant increase in collagen type I positive area compared to wild type. <b>(c)</b> Fibrotic and inflammatory gene expression measured by qPCR, normalized to <i>Gapdh</i> (n = 5 mice per group) in skeletal muscles of LGMD and wild type mice. A significant increase in <i>Col1a1</i> and <i>Cd68</i> expression was found in SGCA- and SGCD-null muscles when compared to wild type muscles. <b>(d)</b> Immunofluorescence images of the heart stained with collagen type I (fibrotic marker, green). Scale bar: 1000 μm. <b>(e)</b> The percentage of collagen type I positive area, measured with Image J was significantly increased in SGCD-null mice compared to wild type mice; males showed a significantly higher increase in collagen than did female SGCD-null mice. Q, quadriceps; GC, gastrocnemius; TA, tibialis anterior; Dia, diaphragm. * Indicates a significant difference from WT controls. # Indicates a significant difference from female SGCD-null mice. Error bars represent ± SD.</p

Experimental timeline.

<p>Mice were subjected to the functional test regime every other week. Time points are indicated by the black ellipses. Black stars donate time points at which creatine kinase (CK) levels were measured. Grey rhombuses indicate time points at which the respiratory function was assessed. The grey triangle denotes time point at which mice were sacrificed and terminal analyses were performed.</p

Respiratory function and creatine kinase levels in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice.

<p><b>(a)</b> Respiration rate was significantly lower (<i>P</i><0.001) in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice at 15 and 34 weeks of age, as compared to wild type mice (WT). <i>Dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice aged 34 weeks showed a significantly lower (<i>P</i><0.05) respiration rate than 15 weeks old mice, while no differences were found with age in wild type mice. <b>(b)</b> Respiration amplitude normalized to body weight was significantly higher (<i>P</i><0.001) in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice at 15 and 34 weeks of age, as compared to wild type mice. No differences in normalized respiration amplitude were found with age and between genders in wild type and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice. Males are indicated by squares and females by circles. <b>(c)</b> Creatine kinase levels were significantly higher (<i>P</i><0.01) in females <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> and wild type mice at four and eight weeks of age. * Indicates a significant difference. Error bars represent ± SEM, n = 6 per genotype per gender.</p

Functional test regime did not interfere with muscle pathology in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice.

<p><b>(a)</b> Body weight did not differ between functionally challenged and sedentary mice. <b>(b)</b> Respiration rate and amplitude were comparable between functionally challenged and sedentary mice. <b>(c)</b> CK levels did not differ between functionally challenged and sedentary mice <b>(d)</b> Gene expression analysis of pathogenic markers measured by qPCR, normalized to <i>Gapdh</i> in gastrocnemius and triceps muscle of functionally challenged and sedentary mice. No differences were found in <i>Gadd45a</i>, <i>Ctgf</i> and <i>Lgals3</i> mRNA levels between functionally challenged and sedentary groups. Func, functionally challenged mice; sed, sedentary mice. Error bars represent in a and c ± SEM and in d ± SD. Sample sizes of body weight, respiratory function and CK analysis n = 6 males and 6 females group, while n = 5 males per group were used for gene expression analysis.</p

Impaired muscle function and integrity in SGCA- and SGCD-null mice.

<p><b>(a)</b> Normalized four limb grip strength was significantly lower in SGCD-null mice than in wild type and SGCA-null mice <b>(b)</b> Maximum hanging time with two limbs was significantly shorter in SGCA- and SGCD-null mice when compared to wild type mice. SGCD-null mice outperformed SGCA-null mice. Maximum hanging time was significantly shorter in male SGCD-null mice than in female SGCD-null mice. <b>(c)</b> Maximum hanging time with four limbs was significantly shorter in SGCA- and SGCD-null mice when compared to wild type mice. SGCD-null mice outperformed SGCA-null mice. Maximum hanging time was significantly shorter in male SGCD-null mice than in female SGCD-null mice. <b>(d)</b> Creatine kinase levels were significantly elevated in both LGMD strains. <b>(e)</b> No significant differences were detected in respiration amplitude between the mouse models. <b>(f)</b> Respiration rate was significantly decreased in SGCA- and SGCD-null mice at 15 and 34 weeks of age compared to wild type mice. At 15 and 34 weeks of age, SGCA-null mice showed significantly lower respiration rate than SGCD-null mice. No differences were found between males (in blue) and females (in pink). <b>(g)</b> Force frequency relationship of the tibialis anterior muscle. Each data point represents the force measured at each frequency. Muscles of both SGCA- and SGCD-null mice showed a significantly lower specific force than those of wild type mice. SGCD-null muscles generated a significantly lower specific force than those of SGCA-null mice. <b>(h)</b> Relative changes in tetanic force during eleven cycles of eccentric contraction in tibialis anterior muscle. The tetanic tension developed during the first cycle was taken as 100%. The isometric force significantly dropped by 10–15% in SGCA- and SGCD-null mice, while it remained unchanged in wild type mice. For e and f * Indicates a significant difference from wild type (WT) controls. # Indicates a significant difference from SGCA-null mice. Error bars represent standard error of the mean (± SEM).</p

Increased muscle fibrosis and inflammation in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice.

<p><b>(a)</b> Representative H&E-stained gastrocnemius and triceps from 34 weeks old male wild type and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice. Scale bar: 100 μm <b>(b)</b> Percentage of pathological area quantified on H&E stained sections. A significant increase (<i>P</i><0.0001) in pathological tissue area was found in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> gastrocnemius and triceps relative to wild type muscles. Pathology was more pronounced (<i>P</i><0.0001) in gastrocnemius than in triceps of <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice. <b>(c)</b> Representative Sirius red-stained gastrocnemius and triceps from 34 weeks old male wild type and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice. Scale bar: 100 μm. (<b>d</b>) Percentage of collagen quantified on Sirius red stained sections. A significant increase in Sirius red positive area was found in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> gastrocnemius and triceps relative to wild type muscles. <b>(e)</b> Gene expression of fibrotic markers measured by qPCR. Gastrocnemius of <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice expressed significantly higher levels of <i>Ctgf</i>, <i>Col1a1</i> and <i>Col3a1</i> (<i>P</i><0.01) when compared to <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> triceps, diaphragm and wild type muscles. <i>Col1a1</i>and <i>Col3a1</i> levels (<i>P</i><0.05) were significantly higher in triceps muscle of <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice than in wild type and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> diaphragm. <b>(f)</b> Gene expression of inflammatory markers measured by qPCR. A significant upregulation of <i>Cd68</i> transcript (<i>P</i><0.01) was found in gastrocnemius of <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice when compared to wild type mice and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> diaphragm. <i>Lgals3</i> levels were significantly higher (<i>P</i><0.01) in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> gastrocnemius and triceps when compared to wild type and <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> diaphragm. <i>Lgals3</i> levels of gastrocnemius exceeded (<i>P</i><0.01) those of the triceps in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice. Data were normalized to <i>Gapdh</i>. Gas, gastrocnemius; Tri, triceps and Dia, diaphragm. * Indicates a significant difference from muscle type-matched wild type (WT) controls. # Indicates a significant difference from <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> gastrocnemius muscle. $ Indicates a significant difference from <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> triceps. Error bars represent ± SD, n = 5 (functionally challenged males per group). For Sirius red analysis: n = 5 functionally challenged wild type males and n = 4 functionally challenged <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> males.</p

MSTN/TGF-β signalling contributes to LGMD disease pathology.

<p><b>(a)</b> Gene expression analysis of three <i>Tgf-β</i> isoforms and <i>Mstn</i>. <i>Tgf-β1</i>, <i>Tgf-β2</i> and <i>Tgf-β3</i> levels were significantly higher in diaphragm muscles of LGMD compared to wild type, while they did not differ in gastrocnemius muscles. <i>Mstn</i> levels were significantly lower in gastrocnemius and diaphragm muscles of LGMD mice. <b>(b)</b> Gene expression analysis of type II receptors <i>Acvr2a</i>, <i>Acvr2b</i> and <i>Tgfbr2</i>. <i>Acvr2a</i> levels were upregulated in male SGCD-null compared to wild type and female SGCD-null diaphragm muscles. <i>Acvr2b</i> levels were significantly higher in diaphragm muscles of SGCD-null males compared to females. <i>Tgfbr2</i> expression was significantly increased in gastrocnemius and diaphragm muscles of LGMD compared to wild type mice. <b>(c)</b> qPCR analysis of type I receptors: <i>Alk1</i>, <i>Alk4</i> and <i>Alk5</i>. <i>Alk1</i> levels were upregulated in LGMD gastrocnemius and SGCA- and male SGCD-null diaphragm muscles compared to wild type muscles. <i>Alk4</i> was significantly increased in diaphragm muscles of LGMD mice relative to wild type. In addition, <i>Alk4</i> levels were significantly lower in SGCD-null females than males. <i>Alk5</i> was significantly higher in diaphragm muscles of SGCA- and female SGCD-null mice relative to wild type and male SGCD-null mice. Data were normalized to <i>Gapdh</i> (all n = 5 mice per group). GC, gastrocnemius; Dia, diaphragm. * Indicates a significant difference from WT controls. # Indicates a significant difference from male SGCD-null mice. Error bars represent ± SD.</p

Functional test regime did not interfere with muscle pathology in both LGMD strains.

<p><b>(a)</b> CK levels were similar in functionally challenged and sedentary mice. <b>(b)</b> Tibialis anterior physiology of the functional challenged and sedentary groups. The functional test regime did not have an effect on muscle physiology <b>(c)</b> Gene expression analysis of pathogenic markers in gastrocnemius muscle of functionally challenged and sedentary LGMD mice. <i>Myh3</i>, <i>Col1a1</i> and <i>Cd68</i> mRNA levels did not differ between functionally challenged and sedentary groups. <i>Mstn</i> levels were lower in functionally challenged compared to sedentary SGCA-null mice. # Indicates a significant difference from non-functional SGCA-null mice. Error bars represent in a, b ± SEM and in c ± SD.</p

Impaired muscle function in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice.

<p><b>(a)</b> A lower body weight (<i>P</i><0.01) was detected in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice when compared to wild type mice. <b>(b)</b> Normalized four limb grip strength was lower (<i>P</i><0.0001) and decreased with age in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> compared to wild type mice. <b>(c)</b> <i>Dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice ran for shorter time (<i>P</i><0.001) than wild type mice. <b>(d)</b> <i>Dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice hung for shorter time (<i>P</i><0.0001) compared to wild type mice. <b>(e)</b> Maximum hanging time with four limbs was shorter (<i>P</i><0.0001) in <i>dy</i>^<i>2J</i><i>/dy</i>^<i>2J</i> mice when compared to wild type mice. * Indicates a significant difference from wild type (WT) controls. Error bars represent ± SEM and n = 6 per genotype per gender.</p

Smaller fiber sizes and increased muscle regeneration in SGCA- and SGCD-null mice.

<p><b>(a)</b> Fiber size distribution of skeletal muscles of 34-week-old SGCA-null, SGCD-null and wild type mice. Values represent relative number of fibers in a given diameter class (10 μm/class). The fiber size distribution was shifted to smaller fibers in SGCA- and SGCD-null mice when compared to wild type mice in gastrocnemius, tibialis anterior and diaphragm muscles. Fiber size distribution did not differ between male and female SGCD-null mice. <b>(b)</b> Immunofluorescent images of diaphragm muscles stained with embryonic myosin heavy chain (eMHC) (regenerative marker, green) and laminin (extracellular matrix of muscle fibres, red) Scale bar: 100 μm. <b>(c)</b> Increase in myogenic gene expression (<i>Myh3</i>, <i>Myog</i>, <i>Stat3</i> and <i>Myod</i>) measured by qPCR and normalized to <i>Gapdh</i> in skeletal muscles of 34-week-old SGCA-null, female and male SGCD-null mice when compared to wild type mice (all n = 5 mice per group). Q, quadriceps; GC, gastrocnemius; TA, tibialis anterior; Dia, diaphragm. * Indicates a significant difference from WT controls. # Indicates a significant difference from female SGCD-null mice Error bars represent standard deviation (±SD).</p