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Feb 24, 2012 - and Regeneration Pathways in Mouse Models for Muscular. Dystrophies. P. C. G. Onofre-Oliveira ... of X-linked Duchenne muscular dystrophy (DMD), and its more benign allelic ..... de Distrofia Muscular (ABDIM). References.
Neuromol Med (2012) 14:74–83 DOI 10.1007/s12017-012-8172-3

ORIGINAL PAPER

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies P. C. G. Onofre-Oliveira • A. L. F. Santos P. M. Martins • D. Ayub-Guerrieri • M. Vainzof



Received: 6 September 2011 / Accepted: 4 February 2012 / Published online: 24 February 2012  Springer Science+Business Media, LLC 2012

Abstract The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/ regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmdmdx, SJL/J, Largemyd and Lama2dy2J/J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-b1 and Pro-collagen 1a2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-b1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We

P. C. G. Onofre-Oliveira  A. L. F. Santos  P. M. Martins  D. Ayub-Guerrieri  M. Vainzof Human Research Genome Center, Bioscience Institute, University of Sa˜o Paulo, R. do Mata˜o, travessa 13, no. 106, Sa˜o Paulo, SP CEP 05508-090, Brazil M. Vainzof (&) Centro de Estudos do Genoma Humano, IB-USP, R. do Mata˜o, 277, sala 220, Sa˜o Paulo, SP CEP 05508-900, Brazil e-mail: [email protected]

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also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle. Keywords Muscular dystrophy  Gene expression  Mouse models  Muscular regeneration  Satellite cells Abbreviations a- and b-DG a, b-, c-, d- and e-SG CMD DGC DMD FKRP GAPDH LGMD MRF NMD PCOL TGF TGF-b1

Alpha and beta dystroglycan Alpha, beta, gamma, delta and epsilon sarcoglycans Congenital muscular dystrophy Dystroglycan complex Duchenne muscular dystrophy Fukutin-related protein Glyceraldehyde-3-phosphate dehydrogenase Limb-girdle muscular dystrophy Muscle regulatory factor Neuromuscular disorders Pro-collagen 1a2 Transforming growth factor Transforming growth factor beta 1

Introduction Muscular dystrophies are a heterogeneous group of genetic diseases, causing a progressive loss of the motor ability. More than 30 genetically defined forms are recognized, and in the last decade, mutations in many genes have been

Neuromol Med (2012) 14:74–83

reported, resulting in the deficiency or loss of function of different important muscle proteins. Biochemical and immunohistological analyses have localized these proteins in several compartments of the muscle fiber: dystrophin, sarcoglycans and dysferlin are sarcolemmal or peri-sarcolemmal proteins; a2-laminin and collagen VI are extracellular matrix proteins; telethonin and actin are sarcomeric proteins; calpain 3 and fukutin-related protein are cytosolic enzymes; and emerin and lamin A/C are nuclear proteins. Dystrophin is the intracellular link of the dystrophin– glycoprotein complex (DGC) in the muscle sarcolemma. The DGC is an oligomeric complex that consists of dystroglycan (a- and b-DG), sarcoglycan (a, b-, c-, d- and e-SG) and syntrophin/dystrobrevin subcomplexes and plays an important structural role in muscle fibers, connecting the subsarcolemmal cytoskeleton to the extracellular matrix (Yoshida and Ozawa 1990; Ervasti and Campbell 1993; Gouveia et al. 2007). Mutations in the dystrophin gene cause the most common and severe form of X-linked Duchenne muscular dystrophy (DMD), and its more benign allelic condition—Becker muscular dystrophy (BMD) (Hoffman et al. 1987). Mutations in the genes coding for a, b-, c- and d- SG proteins cause severe forms of limb-girdle muscular dystrophies type LGMD-2D, 2E, 2C and 2F, respectively (Guglieri et al. 2008). The peripheral membrane glycoprotein a-DG is highly glycosylated and a receptor for the heterotrimeric basement membrane protein laminin-2 (Straub and Campbell 1997). Some forms of muscular dystrophy have been recently associated with defects in the glycosylation of this protein. Among them, the allelic LGMD-2I and congenital muscular dystrophy type CMD-1C are caused by mutations in the putative glycosyltransferase FKRP protein, and CMD-1D is caused by mutations in the LARGE gene (Longman et al. 2003). Muscle protein analysis in these patients shows a hypoglycosylation of a-dystroglycan and a consequent reduction of numerous ligand components of the extracellular matrix, such as laminin-2 (Muntoni et al. 2004). Additionally, mutations in the LAMA2 gene, encoding the a2 chain of laminin-2, cause a severe form of congenital muscular dystrophy (CMD-1A) linked to chromosome 6q (Tome et al. 1994). Other milder forms of muscular dystrophy are caused by mutations in genes coding the enzyme calpain 3 (LGMD-2A), the sarcomeric protein telethonin (LGMD-2G), and the sarcolemmal protein dysferlin (LGMD-2B), which is involved in the fusion of membranes and may be responsible for its repair (Vainzof et al. 2001). Several mouse models for muscular dystrophies are recognized (Vainzof et al. 2008). Among them, the Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD) and carries a mutation in the dystrophin gene, resulting in the total deficiency of this

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protein in its muscle (Bulfield et al. 1984). The SJL/J mouse develops a spontaneous mild myopathy because of a mutation in the dysferlin gene, being a model for LGMD2B (Bittner et al. 1999). The Lama2dy2J/J mouse presents a significant reduction of a2-laminin in the muscle due to a mutation in the Lama2 gene (Anderson et al. 2005), and the myodystrophy mouse (Largemyd) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of a-DG (Browning et al. 2005). These mouse models present variable degrees of severity of the phenotype, observed by distinct patterns of weakness and disease progression, as well as different histopathological characteristics in muscles, reflecting the diversity of the mechanisms of muscle degeneration. However, similar to the phenotypes observed in the human dystrophies, all the dystrophic models present an active process of degeneration, usually accompanied by a tentative of regeneration to restore the function of muscles. Muscle regeneration is directed by the satellite cells, a subpopulation of partially undifferentiated myogenic precursor cells, located in the periphery of mature myotubes. In response to injury, and under the stimulus of several myogenic factors, these cells are activated, start to proliferate and differentiate, fusing to pre-existing fibers or generating new fibers, in a process that recapitulates muscle development (Hawke and Garry 2001). Myogenic determinants are the components of the family of transcription factors called muscle regulatory factors (MRF), including (a) Myf5 and MyoD, responsible for muscle-cell type determination and satellite-cell activation and (b) Myf6 (also called MRF4) and myogenin, responsible for muscle differentiation (Brand-Saberi and Christ 1999). The regenerative capacity of the satellite cells, however, is finite (Schultz and Lipton 1982), and the exhaustion of the pool of precursor cells is a significant factor that contributes to progressive muscle deterioration observed in human and murine muscular dystrophy. During the process of muscle degeneration, the transforming growth factor (TGF) superfamily acts as important cell growth regulatory cytokines (Charge and Rudnicki 2004). TGF-b1, a member of this family, is an inflammatory cytokine with a possible role in the fibrosis of dystrophic muscle (Gosselin et al. 2004), stimulating the synthesis of collagen and inhibiting its degradation (Ignotz and Massague 1986). Therefore, the replacement of muscle tissue by fat or connective tissue is the main cause of weakness in muscular dystrophies. The objective of the present study was to evaluate the effect of different mutations in the activation and expression of genes involved in the cascades of degeneration and regeneration, studying four mouse models for muscular dystrophies with diverse phenotype and muscle histopathological findings.

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Materials and Methods Eight-week-old mice from the five strains C57Black (N = 4 ? 6), Dmdmdx(N = 4), SJL/J (N = 4), Largemyd (N = 4 ? 8) and Lama2dy2J/J (N = 3) were obtained from our animal house, in the Human Genome Research Center at University of Sa˜o Paulo. The mice received routine required cares for good health, and all experiments were approved by the research ethics committee of the Biosciences Institute, University of Sa˜o Paulo. All the strains are available at Jackson Laboratory. In the initial experiments, about 3–4 animals from each strain were studied. After the first analysis, additional animals became available (C57Black: ?6; Largemyd: ?8) and were added in the study to confirm the significance of the observed results. Animals were euthanized using CO2 chamber, and the gastrocnemius and diaphragm muscles were dissected and collected for histological, DNA and RNA analyses. All tissues were frozen immediately in liquid nitrogen. The following mice strains were used: •





C57BL/10ScSn-Dmdmdx/J, also known as mdx, a model for DMD, carrying a point mutation in exon 23 of the dystrophin gene (Bulfield et al. 1984). The genetic background of this strain is C57Black, and this mouse has a very mild phenotype, retain a normal lifespan, and both males and females affected can reproduce (C57BL/ 10ScSn-Dmdmdx/J). SJL/J-Pde6brd, also known as SJL/J, which develops a spontaneous myopathy resulting from a splice-site mutation in the dysferlin gene (Weller et al. 1997). This Dysfim allele has been shown to result in decreased levels of dysferlin protein in SJL/J mice and makes this strain a good model for limb-girdle muscular dystrophy 2B (Bittner et al. 1999). This strain, which background is Swiss, is maintained only by affected animals, the phenotype is very mild, and the weakness is only noted after the age of 6 months (SJL/J). B6.WK-Lama2dy-2J/J, also a strain with C57Black background(Meier and Southard 1970). This mouse is homozygous for the dystrophia-muscularis spontaneous mutation, a G to A mutation in a splice-site consensus sequence of the Lama2 gene that causes abnormal splicing and expression of multiple mRNAs. One mRNA is translated into an alpha 2 polypeptide with a deletion in the domain VI. Affected animals are characterized by progressive weakness and paralysis beginning at about 3 1/2 weeks of age. The hind limbs are affected first, later the axial and forelimb musculature. Death usually occurs before 6 months of age, and mutant mice are usually sterile (B6.WK-Lama2dy-2 J/J). The affected mouse is created in a 25% proportion by crossing heterozygous and is used as a model to congenital muscular dystrophy 1A.

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B6C3Fe a/a-Largemyd/J, also known as Largemyd (Grewal and Hewitt 2002). The mutation underlying the myodystrophy phenotype has been determined to be an intragenic deletion in the glycosyltransferase gene Large, also in the C57Black background. The deletion of exons 5–7 causes a frame shift and a premature stop codon before the first two catalytic domains. The mouse shows abnormal skeletal muscle fiber morphology, decreased body weight, postnatal growth retardation, reduced fertility, and other signals of the effects of the hypoglycosylation of alpha dystroglycan (a/a-Largemyd/J). This animal is the model to congenital muscular dystrophy 1D. C57Black6—normal control mouse.

Histology and Histochemistry Animals were euthanized using CO2 chamber, and the gastrocnemius and diaphragm muscles were dissected and collected for histological, DNA and RNA analyses. All tissues were frozen immediately in liquid nitrogen. For histological analysis, one animal from each strain was evaluated, and frozen sections of 7 lm were prepared on slides using a cryostat (Zeiss, Jena, Germany). Cryosections were stained with hematoxylin and eosin for histopathological evaluation and stained with picrosirius for the quantification of connective tissues, using methodologies described in Dubowitz (Dubowitz et al. 2007). Histopathological parameters include the evaluation of the variation in fiber size, increase in connective tissue, atrophy, hypertrophy, the presence of necrotic and basophilic fibers and percentage of internally located nuclei. One experienced pathologist was responsible to examine and score sections of the different affected muscles, in blind test. The area of fibrotic tissue using picrosiriusstained sections was relatively measured using Image-Pro Plus software (Media Cybernetics) and using area of control mouse as 1. Relative Quantification of Gene Expression-Real-Time PCR (Q-PCR) Whole frozen muscles of the posterior legs of each animal were macerated and homogenized, and total RNA was extracted using Trizol (Invitrogen), according to the protocol. RNA was quantified and normalized for cDNA synthesis using oligo dT and random primers, and MMLV enzyme (Invitrogen), in a quick 3-step protocol of incubation in 65 or 37. For Q-PCR, we used the protocol described by Gosselin (Gosselin et al. 2004). Samples of cDNA of each animal were applied in triplicate in 96 wells

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plate. At each sample was added the pair of primers of the gene of interest and Sybr Green MasterMix (Applied Biosystems) in a total volume of 25lL. Each plate was run in the 7500 Applied Biosystems thermocycler for real-time PCR. The relative expression of the following genes was measured: • •

Pathway of regeneration: MyoD, Myf5, myogenin (Chen and Goldhamer 2003). Pathway of degeneration: TGF-b1 and pro-collagen 1a2 (PCOL) (Gosselin et al. 2004).

The used primers are described in Table 1: We used previously described primers for TGF-b1 and PCOL, with some corrections, and the others were selected from a realtime primer database with validation results (Primerbank), available online. The products of each reaction were also sequenced for the verification of the correct amplification. A dissociation curves for each reaction was obtained, as needed because of the use of SYBR Green.

directed) (RPII) and glucose-6-phosphate dehydrogenase 2 (G6PDH). The GAPDH gene was chosen and used as endogenous control for our subsequent experiments because it showed the lowest variation in the different studied strains. As the analysis uses one unique calibrator control, it was necessary to select one individual for this purpose. We decided to use the normal mouse sample with the lower expression of TGF-b1 in the diverse experiments carried out as the calibrator control. When the animal 5 was tested, all values of TGF-b1 in the remaining normal animals became positive, showing the normal variation. For the genes of regeneration, the same calibrator animal was used. Statistical Analysis The values obtained were compared for their statistical significance using the Minitab program. The test used was the nonparametric Mann–Whitney analysis, for small samples.

Standardization of the Quantification Results The low expression of these genes in normal adult animals hampered their analysis. Therefore, for the elaboration of the standard curve of each gene, we used a pool of cDNA samples, obtained by mixing samples of muscles from Dmdmdx and C57Black, including newborn and 2-monthold animals. These samples were chosen because they should express degeneration genes in older dystrophic animals and regeneration genes in dystrophic or not newborn animals, since the genes of regeneration are the same involved in normal muscle development (Wagers and Conboy 2005). Using this pool of samples, it was possible to obtain standard curves for each one of the studied genes. For the experiments, each one of the animals of the five tested groups was analyzed individually for the five genes. The choice of the endogenous gene was also a challenge, since the ideal gene is the one that would show the greater stability of expression in different strains, tissues and ages. Four genes were evaluated—glyceraldehyde-3phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), polypeptide A polymerase (RNA) II (DNA-

Table 1 Primers used in relative quantification of gene expression

Name

Analysis of the Expression of the 5 Genes, as Compared to Normal Controls The individual values of the relative expression obtained for gastrocnemius samples of the normal and dystrophic animals and then for each of the studied genes are shown in Fig. 1. The median of the expression of each gene was compared in the 4 affected versus the normal control groups (Table 2). Considering an increased or decreased expression due to the dystrophic process, statistically significant differences were obtained for the genes shown in Fig. 2. In the degeneration pathway, TGF-b1 expression was increased in the SJL/J and Dmdmdx models, while PCOL was increased in the SJL/J and Dmdmdx models and reduced in Lama2dy2J/J model. In the regeneration pathway, MyoD was increased in the Lama2dy2J/J and reduced in SJL/J, Myf5 was increased in the Dmdmdx and reduced in SJL/J and Largemyd, and myogenin was increased in the Dmdmdx, Lama2dy2J/J and Largemyd models (Fig. 2).

Forward

Reverse

TGF-b1

50 -CCC CAC TGA TAC GCC TGA GT-30

50 -AG CCC TGT ATT CCG TCT CCT T-30

PCOL

50 -GAT GGT CAC CCT GGA AAA CC-30

50 -CAC GAG CAC CCT GTG GTC C-30

MyoD Myf5 Myogenin GAPDH

0

0

0

0

5 -TAC AGT GGC GAC TCA GAT GC-3 5 -CTG TCT GGT CCC GAA AGA AC-3

50 -TAG TAG GCG GTG TCG TAG CC-30 50 -GAC GTG ATC CGA TCC ACA ATG-30

0

0

50 -GGA CCG AAC TCC AGT GCA T-30

0

0

50 -TGT AGA CCA TGT AGT TGA GGT CA-30

5 -CAG TAC ATT GAG CGC CTA CAG-3 5 -AGG TCG GTG TGA ACG GAT TTG-3

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Regeneration

Degeneration

MyoD

TGF-β1 Log10(2ΔΔCT)

4

Log10(2 ΔΔCT)

3.5 3 2.5 2 1.5 1 0.5

4 3.5 3 2.5 2 1.5 1 0.5 0 -0.5 1-1.5 -2 -2.5 -3

Normal

Largemyd

Myf5

0 SJL/J

DMDmdx Lama2dy2J/J

Largemyd

Log10(2ΔΔCT)

Normal

PCOL

4 3.5

Log10(2 ΔΔCT)

DMDmdx Lama2dy2J/J

SJL/J

3 2.5 2 1.5

4 3.5 3 2.5 2 1.5 1 0.5 0 -0.5 -1 -1.5 -2 -2.5 -3

Normal

DMDmdx Lama2dy2J/J

SJL/J

Largemyd

Myogenin

1

0 SJL/J DMDmdx Lama2dy2J/J

Normal

Largemyd

Log10(2ΔΔCT)

0.5

4 3.5 3 2.5 2 1.5 1 0.5 0 -0.5 -1 -1.5 -2 -2.5 -3

Normal

SJL/J

DMDmdx Lama2dy2J/J

Largemyd

Fig. 1 Normalized values of the expression of the genes TGF-b1, PCOL, MyoD, Myf5 and myogenin, in the gastrocnemius muscles from normal controls and the four dystrophic mice strains. In green, the median values for each group

Table 2 Medians ± SE of the relative expression of the genes TGF-b1, PCOL, MyoD, Myf5 and myogenin in the 5 tested groups of mouse strains Mouse strain

N

TGF-b1

PCOL

MyoD

Myf5

Myog -0.0186 ± 0.3515

Normal control

10

0.5302 ± 0.4203

0.4166 ± 0.3979

0.1276 ± 0.3006

-0.2881 ± 0.1971

SJL/J

4

1.5077 ± 0.6317

2.3791 ± 1.1733

-1.4815 ± 0.5440

-1.0706 ± 0.2972

-0.1733 ± 0.3663

p = 0.0403

p = 0.0133

p = 0.0142

p = 0.0346

p = 0,9326

DMDmdx

4

1.3830 ± 0.2662

1.7802 ± 0.3686

0.7815 ± 0.5354

0.2166 ± 0.4673

1.9670 ± 0.3590

p = 0.0089

p = 0.0089

p = 0.1039

p = 0.0284

p = 0.0058

Lama2dy2J/J

3

1.4881 ± 0.26334

-0.1831 ± 0.3293

1.3020 ± 0.4324

0.2730 ± 0.3641

1.6261 ± 0.2876

Largemyd

12

p = 0.1791 0.1662 ± 0.5304

p = 0.02250 0.5141 ± 0.4650

p = 0.0142 -0.2846 ± 0.6144

p = 0.1508 -0.7887 ± 0.3965

p = 0.0142 2.0187 ± 1.0506

p = 0.2766

p = 0.9212

p = 0.0518

p = 0.0041

p = 0.0011

Statistical analysis using Mann–Whitney test showing p value for each dystrophic group when compared to the respective normal control

Analysis of Expression in Gastrocnemius Versus Diaphragm

affected by the dystrophic process. This muscle also showed lower expression of the TGF-b1 and PCOL genes.

The comparison of the median expression of the gastrocnemius and the diaphragm muscle for each group showed that only the expression of the genes TGF-b1 and PCOL from the Dmdmdx model was significantly different, as shown in Fig. 3. Histological analysis allowed showing the difference in the pattern of degeneration in these two muscles, being the diaphragm muscle significantly more

Histopathological Analysis

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The analysis of histopathological alterations in each strain of the studied mice is described in Table 3, and illustrated in Fig. 4. The SJL/J and Dmdmdx models, which present more benign phenotype, showed also more discrete dystrophic signs in the muscle, while the more severely

Neuromol Med (2012) 14:74–83 Fig. 2 a Schematic representation of the degeneration and regeneration pathways, and the respective studied genes illustrated in different colors. b Median expression and standard error range of each gene in the normal control C57Black and in the four dystrophic strains. The asterisks show statistical significance level: *p \ 0.05; **p \ 0.01

79 Regeneration

Degeneration

A

Dystrophic process

TGF-b1

PCOL

B

myotube

* *

** **

4

*

3

3

2.5

2

Log10(2ΔΔCT)

Log10(2ΔΔCT)

Fibrosis

4 3.5

2 1.5 1 0.5 0 -1

* *

* ** *

*

** **

1 0 -1 -2

-0.5 Normal

SJL/J

DMDmdx

TGF-b1

Lama2dy2J/J

Largemyd

-3

Normal

SJL/J MyoD

PCOL

Dmd mdx

2.5

Gastrocnemius

myoblast

muscle fiber

inflammation

Diaphragm

DMDmdx Myf5

Lama2dy2J/J

Largemyd

Myog

while in the Lama2dy2J/J and Largemyd models, these fibers were more scattered and distributed throughout the muscle bundle. The quantification of connective tissue through the picrosirius methodology staining for collagen showed that again here, the two mouse models Largemyd and Lama2dy2J/J more severely affected, also showed the higher degree of connective tissue replacement, when compared to normal control (Fig. 4).

2.0 PCOL

Log10(2ΔΔCT)

Discussion 1.5

TGF-β1

1.0 PCOL

0.5 TGF-β1

0.0

-0.5

Fig. 3 Comparison of median expression of the TGF-b1 and PCOL gene in the gastrocnemius (left) versus the diaphragm (right) muscles in Dmdmdx mice of 2 months of age. Histopathological analysis in HE staining, showing the differences in the pattern of degeneration

affected models Largemyd and Lama2dy2J/J showed more severe degree of muscle degeneration concerning mainly variation in fiber size, necrosis and connective tissue replacement. Small basophilic fibers indicating regeneration were observed in the Dmdmdx mice, forming clusters,

Many factors are involved in the degeneration and regeneration of the dystrophic muscle, which is a complex process that requires a coordinated modulation between the satellite cells, growth factors, cytokines, inflammatory responses and vascular components of the extracellular matrix (Goetsch et al. 2003). Here, we standardized a quantitative method for the evaluation of the expression of five genes expressed in the degeneration and regeneration pathways of the dystrophic process. Normal Variability in Controls Normal control mice, with no muscle damage, are expected to express negligible levels of genes of degeneration and regeneration. We found that the expression of several genes occurred in different and variable levels in normal animals. We selected as an animal control calibrator the one with lower expression of TGF-b1, because it is expected that

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Table 3 Histopathological alteration in the 4 mouse models for muscular dystrophies Changes in fiber shape and size

Fibrosis and adipose tissue

Atrophy

Hypertrophy

Percentage of centronucleated nuclei

Necrosis

Basophilic fibers (regeneration)

C57Black

?

-

-

?

1%

-

-

SJL/J

?

-

?

?

3%

?

-

Dmdmdx

???

?

??

??

57%

?

???

Largemyd

???

??

??

???

64%

???

??

Lama2dy2j/j

???

??

??

??

34%

???

???

-, not present and/or not observed; ? to ???, present and with variable phenotype

this gene is not expressed in normal muscle, since it is closely connected with the emergence of tissue fibrosis (Gosselin et al. 2004; Cohn et al. 2007). However, the expression of TGF-b1 in the various controls was not totally negative, and low levels of expression could be identified in many normal animals, which may suggest basal levels of expression involved in other pathways of cell cycle control (Li et al. 2008a, b). On the other hand, the values of the regenerating genes oscillated between positive and negative values, within the variability of the group (Fig. 1). Evaluation of the Expression of Genes of Degeneration in Dystrophic Animals The analysis of the expression of the TGF-b1 gene showed a significantly higher expression in the four dystrophic animals when compared to controls, as already described for the Dmdmdx model (Li et al. 2008b; Yablonka-Reuveni et al. 2008). The expression of pro-collagen gene, however, was significantly higher only in Dmdmdx and SJL/J models. Interestingly, these two models are also the animals less affected, showing also milder dystrophic alteration pattern on muscle. Complementing these observations, the quantification of the staining of collagen picrosirius showed the greater amount of connective tissue in animals most affected Largemyd and Lama2dy-2J/J and less infiltration in animals with more benign dystrophies SJL/J and Dmdmdx (Fig. 4). These results support the hypothesis that at 2 months of age, the degenerative process in animals with more severe phenotype is already in advanced stage, with greater loss of muscle fibers and replacement of degenerated muscle by connective tissue. Therefore, considering the data of gene expression, and the dystrophic pattern, an inverse relation between the pattern of degeneration and the expression of the PCOL gene was observed for the gastrocnemius muscle of dystrophic animals. This inverse relation was also observed when comparing two different muscles from of the Dmdmdx mice. The mildly affected gastrocnemius muscle showed a significantly higher expression of TGF-b1 gene and PCOL

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while the diaphragm, which is much more severely affected (Fig. 3) (Ghedini et al. 2008), showed a reduction in the expression of these genes. These results show that in general, degeneration genes are more expressed in the gastrocnemius than in the diaphragm, which confirms the hypothesis of an elevation of expression of these genes in muscle more preserved or in initial stages of the process of degeneration. Together, these results suggest that while the TGFb-1 gene is activated by the dystrophic process in any degree of degeneration, the activation of pro-collagen gene expression occurs in the early stages of this process. The dystrophic animals’ degeneration path exhibits increased expression of TGFb-1, cytokine that signals to replace tissue and activation of inflammatory cascade (Gosselin et al. 2004), which induces the expression of pro-collagen (Li et al. 2004), though the pro-collagen expression may be reduced with the disease’s progression. Evaluation of the Expression of Genes of Regeneration in Dystrophic Animals The analysis of the genes of the regeneration cascade showed great variability in the levels of expression in different murine models. Considering that these genes are expressed by satellite cells, responsible for regeneration of muscle tissue, the expression profile observed may reflect how these cells are acting in each type of primary mutation, leading to the process of muscle degeneration (Wagers and Conboy 2005; Yablonka-Reuveni et al. 2008). High expression of MyoD and Myf5 genes reflects the commitment and proliferation of satellite cells, when activated to start the process of regeneration, while myogenin is an important inducer of muscle differentiation, leading to fusion of myoblasts to form differentiated myotubes. The low expression of MyoD and Myf5 observed in SJL/J animals suggests that the satellite cells in these muscles are quiescent. The lack of expression of myogenin confirms this result, because there was no stimulus for the differentiation of these cells. This result is consistent with the histopathological pattern observed in this model at the

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Fig. 4 Histological findings in the different mouse models for NMD, showing the areas of degeneration and regeneration. At the right column the staining for picrosirius, showing connective tissue replacement and the relative quantification, as compared to normal control

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age of 2 months, where the muscle is very preserved without significant evidence of degeneration and subsequent activation of regeneration. On the other hand, we observed an increase in the expression of genes MyoD and Myf5 in Dmdmdx and Lama2dy-2J/J animals, reflecting the activation of satellite cells for their proliferation. We also observed increase in the expression of myogenin, compatible with the stimulus for the differentiation of these cells. This pattern of expression was compatible with the presence of groups of small fibers with regenerative characteristics through histological analysis of these models. In both models, therefore, the degenerative process has, as expected, stimulated the regenerative cascade. A very active regeneration is well described in the Dmdmdx model. The muscle of these animals displays a very large increase in the number of centronucleated fibers after the 3rd or 4th weeks of life, and these fibers are considered newly regenerated (DiMario et al. 1991). Our results in Lama2dy-2J/J model show that this pattern of degeneration followed by a process of regeneration also occurs in these mice. On the other hand, in Largemyd mice, which shows a very similar histopathological pattern to the Lama2dy-2J/J mouse, we observed a low expression of genes MyoD and Myf5, suggesting low-proliferative activity of satellite cells in these animals (Yablonka-Reuveni et al. 2008) (Fig. 2). Indeed, studies of the satellite cells in conditional knockout mice for the expression of the protein a-dystroglycan (aDG) led the authors to the suggestion that a functional mechanism for glycosylation of a-DG is an important requirement for the proper functioning and renewal of these cells (Cohn et al. 2002). Therefore, considering the fact that the Largemyd model presents a defect in the glycosylation of a-DG, this could contribute to a failure in the activation and/or proliferation capacity of the satellite cells in this mouse model.

Conclusions In conclusion, our results suggest that there are differences in the expression of genes of the pathway of degeneration depending on the degree of disorder of muscle, because the TGFb-1 gene was expressed in dystrophic muscle irrespective of the degree of the tissue, while the PCOL gene appears to be activated in initial stages of this process. Additionally, considering that satellite cells play a key role in the balance between the degenerative and regenerative capacity of muscle, the cascade of degeneration of each pathophysiological pathway differently activated the regeneration, with distinctions in the induction of proliferation of satellite cells, but without changes in the differentiation stimulus. Defects in dystrophin-associated

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glycoprotein complex (DGC) can, therefore, also lead to specific changes in the ability of proliferation of satellite cells, which can result in poor regeneration of the models involved. Thus, the dysfunction in the population of the satellite cells may represent an important mechanism in the pathogenesis of muscular dystrophy. Acknowledgments The authors would like to thank the following researchers for scientific and technical support: Dra. Mayana Zatz, Dra. Helga C. Silva, Lydia Yamamoto and Marta Canovas. We would also like to thank Dr. Paulo Otto for the statistical analyses, Dr. Peter Pearson for the English revision, and Ms. Claudia Mori and IPEN for taking care of the mice. This work was supported by Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo—Centro de Pesquisa, Inovac¸a˜o e Difusa˜o (FAPESP-CEPID), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Instituto Nacional de Cieˆncia e Tecnologia (INCT), FINEP, and Associac¸a˜o Brasileira de Distrofia Muscular (ABDIM).

References a/a-Largemyd/J, B. C. F. ‘‘http://jaxmice.jax.org/strain/000226.html’’. Anderson, J. L., Head, S. I., et al. (2005). Synaptic plasticity in the dy2J mouse model of laminin alpha2-deficient congenital muscular dystrophy. Brain Research, 1042(1), 23–28. B6.WK-Lama2dy-2J/J ‘‘http://jaxmice.jax.org/strain/000524.html’’. Bittner, R. E., Anderson, L. V., et al. (1999). Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B. Nature Genetics, 23(2), 141–142. Brand-Saberi, B., & Christ, B. (1999). Genetic and epigenetic control of muscle development in vertebrates. Cell and Tissue Research, 296(1), 199–212. Browning, C. A., Grewal, P. K., et al. (2005). A rapid PCR method for genotyping the Large(myd) mouse, a model of glycosylationdeficient congenital muscular dystrophy. Neuromuscular Disorders, 15(5), 331–335. Bulfield, G., Siller, W. G., et al. (1984). X chromosome-linked muscular dystrophy (mdx) in the mouse. Proceedings of the National Academy of Sciences of the USA, 81(4), 1189–1192. C57BL/10ScSn-Dmdmdx/J. ‘‘http://jaxmice.jax.org/strain/001801. html.’’ Retrieved August-13th-2010. Charge, S. B., & Rudnicki, M. A. (2004). Cellular and molecular regulation of muscle regeneration. Physiological Reviews, 84(1), 209–238. Chen, J. C., & Goldhamer, D. J. (2003). Skeletal muscle stem cells. Reproductive Biology and Endocrinology, 1, 101. Cohn, R. D., Henry, M. D., et al. (2002). Disruption of DAG1 in differentiated skeletal muscle reveals a role for dystroglycan in muscle regeneration. Cell, 110(5), 639–648. Cohn, R. D., van Erp, C., et al. (2007). Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states. Nature Medicine, 13(2), 204–210. DiMario, J. X., Uzman, A., et al. (1991). Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle. Developmental Biology, 148(1), 314–321. Dubowitz, V., Sewry, C. A., et al. (2007). Muscle biopsy : A practical approach. Philadelphia: Saunders Elsevier. Ervasti, J. M., & Campbell, K. P. (1993). A role for the dystrophinglycoprotein complex as a transmembrane linker between laminin and actin. Journal of Cell Biology, 122(4), 809–823.

Neuromol Med (2012) 14:74–83 Ghedini, P. C., Viel, T. A., et al. (2008). Increased expression of acetylcholine receptors in the diaphragm muscle of MDX mice. Muscle and Nerve, 38(6), 1585–1594. Goetsch, S. C., Hawke, T. J., et al. (2003). Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration. Physiological Genomics, 14(3), 261–271. Gosselin, L. E., Williams, J. E., et al. (2004). Localization and early time course of TGF-beta 1 mRNA expression in dystrophic muscle. Muscle and Nerve, 30(5), 645–653. Gouveia, T. L., Kossugue, P. M., et al. (2007). A new evidence for the maintenance of the sarcoglycan complex in muscle sarcolemma in spite of the primary absence of delta-SG protein. Journal of Molecular Medicine, 85(4), 415–420. Grewal, P. K., & Hewitt, J. E. (2002). Mutation of Large, which encodes a putative glycosyltransferase, in an animal model of muscular dystrophy. Biochimica et Biophysica Acta, 1573(3), 216–224. Guglieri, M., Straub, V., et al. (2008). Limb-girdle muscular dystrophies. Current Opinion in Neurology, 21(5), 576–584. Hawke, T. J., & Garry, D. J. (2001). Myogenic satellite cells: Physiology to molecular biology. Journal of Applied Physiology, 91(2), 534–551. Hoffman, E. P., Brown, R. H., Jr, et al. (1987). Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell, 51(6), 919–928. Ignotz, R. A., & Massague, J. (1986). Transforming growth factorbeta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. Journal of Biological Chemistry, 261(9), 4337–4345. Li, Y., Foster, W., et al. (2004). Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: A key event in muscle fibrogenesis. American Journal of Pathology, 164(3), 1007–1019. Li, X., McFarland, D. C., et al. (2008a). Effect of Smad3-mediated transforming growth factor-beta1 signaling on satellite cell proliferation and differentiation in chickens. Poultry Science, 87(9), 1823–1833. Li, X., McFarland, D. C., et al. (2008b). Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells. Domestic Animal Endocrinology, 35(3), 263–273.

83 Longman, C., Brockington, M., et al. (2003). Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Human Molecular Genetics, 12(21), 2853–2861. Meier, H., & Southard, J. L. (1970). Muscular dystrophy in the mouse caused by an allele at the dy-locus. Life Science, 9(3), 137–144. Muntoni, F., Brockington, M., et al. (2004). Defective glycosylation in congenital muscular dystrophies. Current Opinion in Neurology, 17(2), 205–209. Primerbank. ‘‘http://pga.mgh.harvard.edu/primerbank/index.html’’. Retrieved August-13th-2010. Schultz, E., & Lipton, B. H. (1982). Skeletal muscle satellite cells: Changes in proliferation potential as a function of age. Mechanisms of Ageing and Development, 20(4), 377–383. SJL/J. ‘‘http://jaxmice.jax.org/strain/000686.html’’. Straub, V., & Campbell, K. P. (1997). Muscular dystrophies and the dystrophin-glycoprotein complex. Current Opinion in Neurology, 10(2), 168–175. Tome, F. M., Evangelista, T., et al. (1994). Congenital muscular dystrophy with merosin deficiency. C R Academic Science III, 317(4), 351–357. Vainzof, M., Anderson, L. V., et al. (2001). Dysferlin protein analysis in limb-girdle muscular dystrophies. Journal of Molecular Neuroscience, 17(1), 71–80. Vainzof, M., Ayub-Guerrieri, D., Onofre, P. C., Martins, P. C., Lopes, V. F., Zilberztajn, D., et al. (2008). Animal models for genetic neuromuscular diseases. Journal of Molecular Neuroscience, 34(3), 241–248. Wagers, A. J., & Conboy, I. M. (2005). Cellular and molecular signatures of muscle regeneration: Current concepts and controversies in adult myogenesis. Cell, 122(5), 659–667. Weller, A. H., Magliato, S. A., et al. (1997). Spontaneous myopathy in the SJL/J mouse: Pathology and strength loss. Muscle and Nerve, 20(1), 72–82. Yablonka-Reuveni, Z., Day, K., et al. (2008). Defining the transcriptional signature of skeletal muscle stem cells. Journal of Animal Science, 86(14 Suppl), E207–E216. Yoshida, M., & Ozawa, E. (1990). Glycoprotein complex anchoring dystrophin to sarcolemma. Journal of Biochemistry, 108(5), 748–752.

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