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Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes

      Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder of children. The mdx (C57BL/10 background, C57BL/10-mdx) mouse is a widely used model of DMD, but the histopathological hallmarks of DMD, such as the smaller number of myofibers, accumulation of fat and fibrosis, and insufficient regeneration of myofibers, are not observed in adult C57BL/10-mdx except for in the diaphragm. In this study, we showed that DBA/2 mice exhibited decreased muscle weight, as well as lower myofiber numbers after repeated degeneration–regeneration cycles. Furthermore, the self-renewal efficiency of satellite cells of DBA/2 is lower than that of C57BL/6. Therefore, we produced a DBA/2-mdx strain by crossing DBA/2 and C57BL/10-mdx. The hind limb muscles of DBA/2-mdx mice exhibited lower muscle weight, fewer myofibers, and increased fat and fibrosis, in comparison with C57BL/10-mdx. Moreover, remarkable muscle weakness was observed in DBA/2-mdx. These results indicate that the DBA/2-mdx mouse is a more suitable model for DMD studies, and the efficient satellite cell self-renewal ability of C57BL/10-mdx might explain the difference in pathologies between humans and mice.
      Duchenne muscular dystrophy (DMD) is a progressive and lethal X-linked muscular disorder caused by mutations in the dystrophin gene.
      • Koenig M
      • Monaco AP
      • Kunkel LM
      The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein.
      The dystrophin gene encodes a 427-kDa cytoskeletal protein that forms the dystrophin/glycoprotein complex at the sarcolemma with α- and β-dystroglycans, α-, β-, γ-, and δ-sarcoglycans, and other molecules, and links the cytoskeleton of myofibers to the extracellular matrix in skeletal muscle.
      • Suzuki A
      • Yoshida M
      • Hayashi K
      • Mizuno Y
      • Hagiwara Y
      • Ozawa E
      Molecular organization at the glycoprotein-complex-binding site of dystrophin. Three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin.
      • Ervasti JM
      • Campbell KP
      A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin.
      The lack of dystrophin in the sarcolemma disturbs the assembly of the dystrophin/glycoprotein complex and causes instability of the muscle membrane, leading to muscle degeneration and myofiber loss. The histopathological hallmarks of DMD include degeneration, necrosis, accumulation of fat and fibrosis, and insufficient regeneration of myofibers accompanied by a loss of myofibers.
      • Carpenter S
      • Karpati G
      Disease of skeletal muscle.
      Therefore, the manifestations of DMD are considered to result from an imbalance between degeneration and regeneration.
      The function and structure of dystrophin has been elucidated by studies of a variety of dystrophin-deficient animals. Among these animal models, the mdx mouse (the correct nomenclature is C57BL/10-Dmdmdx), first described in1984, is the most prolific. A spontaneous mutation (mdx) arose in an inbred colony of C57BL/10 mice, which have a high level of serum pyruvate kinase.
      • Bulfield G
      • Siller WG
      • Wight PA
      • Moore KJ
      X chromosome-linked muscular dystrophy (mdx) in the mouse.
      The muscle pathology of the mice includes active fiber necrosis, cellular infiltration, a wide range of fiber sizes, and numerous centrally nucleated regenerating fibers. However, in contrast to DMD, replacement of muscle with fat and fibrosis is not prominent, and no losses of muscle fiber and muscle weight are observed in the skeletal muscle of mdx mice except in the diaphragm.
      • Stedman HH
      • Sweeney HL
      • Shrager JB
      • Maguire HC
      • Panettieri RA
      • Petrof B
      • Narusawa M
      • Leferovich JM
      • Sladky JT
      • Kelly AM
      The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.
      • Tanabe Y
      • Esaki K
      • Nomura T
      Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse.
      In contrast, most of the limb muscles of the mdx mouse maintain hypertrophy and increased skeletal muscle mass throughout much of their life span.
      • Pastoret C
      • Sebille A
      mdx mice show progressive weakness and muscle deterioration with age.
      One reason for the difference between DMD and mdx is explained by the up-regulation of expression of utrophin, a homolog of dystrophin.
      • Deconinck AE
      • Rafael JA
      • Skinner JA
      • Brown SC
      • Potter AC
      • Metzinger L
      • Watt DJ
      • Dickson JG
      • Tinsley JM
      • Davies KE
      Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.
      • Grady RM
      • Teng H
      • Nichol MC
      • Cunningham JC
      • Wilkinson RS
      • Sanes JR
      Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy.
      Another reason has been supposed to be the excellent regeneration capacity of mdx compared with DMD. However, this hypothesis has not been verified.
      Regeneration of skeletal muscle depends on the competence of muscle satellite cells. Muscle satellite cells, which account for 2 to 5% of the total nuclei in adult skeletal muscle, play a major role in muscle regeneration.
      • Bischoff R
      Analysis of muscle regeneration using single myofibers in culture.
      Under normal conditions, satellite cells are found external to the myofiber plasma membrane and beneath the muscle basal lamina,
      • Mauro A
      Satellite cell of skeletal muscle fibers.
      and they are mitotically quiescent in adult skeletal muscle.
      • Schultz E
      • Gibson MC
      • Champion T
      Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study.
      When activated by muscle damage, satellite cells proliferate, differentiate, fuse with each other or injured myofibers, and eventually regenerate mature myofibers. During the regenerative processes, satellite cells not only produce large amounts of muscle, but also renew themselves to maintain their own population.
      • Collins CA
      • Olsen I
      • Zammit PS
      • Heslop L
      • Petrie A
      • Partridge TA
      • Morgan JE
      Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.
      In fact, it is reported that the satellite cell pool of C57BL/10 continues to respond efficiently even when the skeletal muscle is subjected to as many as 50 cycles of severe damage.
      • Luz MA
      • Marques MJ
      • Santo Neto H
      Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.
      Therefore, it is thought that maintenance of the satellite cell pool is indispensable to retain the long-term regenerative potential for skeletal muscle injury, including in muscular dystrophies.
      To investigate genetic differences in long-term regeneration potential, we first induced repeated degeneration–regeneration cycles in four inbred strains of mice. Among these strains, C57BL/6, a widely used strain akin to C57BL/10, was tolerant of repeated injury. This is consistent with the results of C57BL/10 previously described.
      • Luz MA
      • Marques MJ
      • Santo Neto H
      Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.
      In contrast, among four inbred strains, DBA/2 mice exhibited the most remarkable skeletal muscle loss and impaired regeneration after repeated injury. Importantly, the self-renewal potential of DBA/2 satellite cells was significantly lower than that of C57BL/6. In addition, in vitro colony formation and proliferation assays indicated that intrinsic difference between C57BL/6 and DBA/2 satellite cells exist. Finally, we crossed the mdx genotype with the DBA/2 for more than five generations. At the fifth backcross, the mice are not yet fully congenic (D2.B10-DMDmdx), and thus we refer to them as DBA/2-mdx hereafter. We investigated their phenotypes. Intriguingly, severe loss of skeletal muscle weight, decreased myofiber number, increased fat and fibrosis volume, and apparent muscle weakness were observed in the DBA/2-mdx mice. These results indicate that the intrinsic genetic program affects the properties of satellite cells, and DBA/2-mdx will be a more useful model of DMD than C57BL/10-mdx. It is also speculated that the high self-renewal potential of C57BL/10 satellite cells might explain the difference in pathologies between humans and mice.

      Materials and Methods

      Mice

      Six-week-old, specific pathogen-free, BALB/c, C3H/HeN, C57BL/6, and DBA/2 mice were purchased from Charles River Japan (Yokohama, Japan). Six-week-old, specific pathogen-free C57BL/10 mice were purchased from Shimizu Laboratory Supplies Co., Ltd (Kyoto, Japan). Specific pathogen-free mdx mice (of C57BL/10 background) were provided by Central Laboratories of Experimental Animals (Kanagawa, Japan) and maintained in our animal facility by brother-sister matings. Mdx of C57BL/10 background were backcrossed into DBA/2 genetic background. Mice backcrossed more than five generations were used in this study. Genotyping was performed according to previous reports.
      • Amalfitano A
      • Chamberlain JS
      The mdx-amplification-resistant mutation system assay, a simple and rapid polymerase chain reaction-based detection of the mdx allele.
      All procedures for experimental animals were approved by the Experimental Animal Care and Use Committee at Osaka University.

      Muscle Injury

      Muscle injury was induced by injecting cardiotoxin (10 μmol/L in saline, Wako Pure Chemical Industries, Tokyo, Japan) into tibialis anterior (50 μl), gastrocnemius (150 μl), and quadriceps femoris (100 μl) muscles as described.
      • Fukada S
      • Yamamoto Y
      • Segawa M
      • Sakamoto K
      • Nakajima M
      • Sato M
      • Morikawa D
      • Uezumi A
      • Miyagoe-Suzuki Y
      • Takeda S
      • Tsujikawa K
      • Yamamoto H
      CD90-positive cells, an additional cell population, produce laminin alpha2 upon transplantation to dy(3k)/dy(3k) mice.
      All injections were first done when mice were 8 to 10 weeks of age.

      Histological Analysis

      Tibialis anterior, gastrocnemius, and quadriceps femoris muscles were isolated and frozen in liquid nitrogen-cooled isopentane (Wako Pure Chemical Industries). Cryosections (10 μm) were stained with H&E, Oil red-O (Sigma-Aldrich, St. Louis, MO), or Sirius Red (Sigma-Aldrich).

      Immunohistochemistry

      For immunohistochemical examinations, transverse cryosections (6 μm) were stained with various antibodies. Monoclonal rat anti-laminin α2 (1:200; clone: 4H8-2) and mouse anti-Pax7 antibodies were purchased from Alexis Biochemical (Lausen, Switzerland) and Developmental Studies Hybridoma Bank (Iowa, IA), respectively. For Pax7 staining, a M.O.M. kit (Vector Laboratories, Burlingame, CA) was used to block endogenous mouse IgG. After the first staining at 4°C overnight, sections were reacted with secondary antibodies conjugated with Alexa 488 or Alexa 568 (Molecular Probes, Eugene, OR). Sections were shielded using Vectashield (Vector Laboratories, Inc). The signals were recorded photographically using an Axiophot microscope (Carl Zeiss, Oberkochen, Germany).

      Preparation of Muscle Satellite Cells and Culture

      Satellite cells were isolated from uninjured adult skeletal muscle using biotinylated-SM/C-2.6
      • Fukada S
      • Higuchi S
      • Segawa M
      • Koda K
      • Yamamoto Y
      • Tsujikawa K
      • Kohama Y
      • Uezumi A
      • Imamura M
      • Miyagoe-Suzuki Y
      • Takeda S
      • Yamamoto H
      Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody.
      and IMag methods (BD Immunocytometry Systems, Mountain View, CA) as described in a previous report.
      • Fukada S
      • Yamamoto Y
      • Segawa M
      • Sakamoto K
      • Nakajima M
      • Sato M
      • Morikawa D
      • Uezumi A
      • Miyagoe-Suzuki Y
      • Takeda S
      • Tsujikawa K
      • Yamamoto H
      CD90-positive cells, an additional cell population, produce laminin alpha2 upon transplantation to dy(3k)/dy(3k) mice.
      Satellite cells were cultured in a growth medium of high-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing 20% fetal calf serum (Trace Biosciences, N.S.W., Australia), 2.5 ng/ml basic fibroblast growth factor (PeproTech, London, UK), leukemia inhibitory factor (Alexis Biochemical), and penicillin (100 U/ml)-streptomycin (100 μg/ml) (Gibco BRL, Gaithersburg, MD) on culture dishes coated with Matrigel (BD Bioscience, San Diego, CA).

      Colony Forming Assay

      Clonal cultures of freshly isolated satellite cells were performed in 96-well plates coated with type I collagen (Sumilon, Tokyo, Japan) in growth medium for a week. The frequency of colony formation and number of cells in each well were counted under a phase-contrast microscope.

      Cell Proliferation Assay

      Isolated satellite cells were cultured in growth medium for 3 to 4 days, and expanded primary myoblasts were harvested and additional culture was performed in 96-well dishes for 1 day. Eight hours later, bromodeoxyuridine (BrdU) uptake was quantified using the Cell Proliferation ELISA, BrdU Kit (Roche Diagnostics, Basel, Switzerland) and a microplate reader (Model 680, Bio-Rad, Hercules, CA).

      Measurement of Sizes of Myofibers and Oil Red-O-Positive and Fibrotic Areas

      Image J software was used to measure myofiber sizes and Oil red-O- and Sirius Red-positive areas.

      Evans Blue Dye Injection

      Evans blue (Wako Pure Chemical Industries) was dissolved in PBS and injected intraperitoneally into mice (1 mg/100 μl/10g body weight).
      • Matsuda R
      • Nishikawa A
      • Tanaka H
      Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle.
      Sixteen to 18 hours later, muscle tissues were removed, and frozen in liquid nitrogen-cooled isopentane. The muscle fibers with Evans Blue incorporated were then counted as injured muscles.

      Muscle Endurance and Grip Strength Test

      The muscle endurance test was referred to the studies by Handschin et al.
      • Handschin C
      • Chin S
      • Li P
      • Liu F
      • Maratos-Flier E
      • Lebrasseur NK
      • Yan Z
      • Spiegelman BM
      Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1alpha muscle-specific knock-out animals.
      In brief, we used a MK-680S treadmill (Muromachi Kikai Co., Ltd., Tokyo, Japan). For 3 days, animals were acclimated to treadmill running for 5 minutes at a speed of 10 m/min on a 0% grade. After the acclimation, animals ran on a treadmill with a 10% uphill grade starting at a speed of 10 m/min for 5 minutes. Every subsequent 2 minutes, the speed was increased by 2 m/min until the mice were exhausted. Exhaustion was defined as the inability of the animal to remain on the treadmill despite mechanical prodding. Running time and speed were measured, and the distance was calculated. Grip strength was measured using a MK-380M grip strength meter (Muromachi Kikai Co., Ltd). The grip strength of each individual mouse was measured 10 times, the same measurements were repeated on the next day, and the highest value of each experiment was used.

      Statistics

      Values were expressed as means ± SD. Statistical significance was assessed by Student's t-test. In comparisons of more than two groups, nonrepeated measures analysis of variance (analysis of variance) followed by the Student-Newman-Keuls test were used. A probability of less than 5% (P < 0.05) or 1% (P < 0.01) was considered statistically significant.

      Results

      Genetic Differences in Skeletal Muscle Regeneration

      To examine the long-term regeneration ability of four inbred strains of mice, repeated cycles of degeneration–regeneration were induced by injection of cardiotoxin (CTX). CTX was injected into one side of the tibialis anterior (TA), gastrocnemius (GC), and quadriceps (Qu) muscle every 2 weeks. At the last (sixth) CTX injection, another intact TA muscle received CTX once to examine the regenerative potential in one cycle of each mouse at this age. Four weeks later, the muscles were removed and analyzed. As shown in Figure 1, A and B, none of the strains displayed a striking difference in either skeletal muscle weight or histochemistry after one CTX injection (CTX-1), except for the appearance of adipocytes in BALB/c. However, the DBA/2 mice that received six CTX injections (CTX-6) exhibited remarkably impaired regeneration (Figure 1A) and loss of TA muscle weight (Figure 1B). A similar loss of muscle weight was also observed in GC and Qu of DBA/2 (CTX-6 in Figure 1C). In contrast, none of the other strains showed a significant difference in uninjured muscle weight at this age (uninjured in Figure 1C). Fat was observed in DBA/2, BALB/c, and C3H/HeN after six injections, but the sclerosis and loss of muscle weight was remarkable in DBA/2. Therefore, the following experiments were performed on C57BL/6 and DBA/2.
      Figure thumbnail gr1
      Figure 1Impaired regeneration and loss of muscle weight in DBA/2 mice after injured six times. A: TA (tibialis anterior) muscles were examined histologically in four inbred strains of mice after one (CTX-1) or six (CTX-6) cardiotoxin (CTX) injections. The cross sections were stained with H&E. Scale bar =100 μm. B: The TA muscle weight (mg) per body weight (g) in each inbred strain after one or six CTX injections. B6, D2, Bc, and C3 indicate C57BL/6, DBA/2, BALB/c, and C3H/HeN mice, respectively. C: The GC (gastrocnemius) and Qu (quadriceps) muscle weights (mg) per body weight (g) in each inbred strain from uninjured or muscle injured six times. The number in the each graph indicates the number of mice used in these experiments. *P < 0.05, **P < 0.01 (analysis of variance, SNK-test).

      Regeneration Impairment in DBA/2 Is Inherited Recessively

      To assess the inheritance of the lower regeneration ability of DBA/2, we injected CTX into C57BL/6, DBA/2, and their F1 mice (B6D2F1). To allow more sufficient regeneration time, the interval between CTX injections was changed to 4 weeks. As shown in Figure 2A, we found marked muscle weight loss in DBA/2 after three CTX injections (4 weeks × 3). The results of B6D2F1 mice were similar to those of C57BL/6 (Figure 2, A and B).
      Figure thumbnail gr2
      Figure 2Impaired regeneration of DBA/2 phenotype is recessively inherited. A: The TA, GC, and Qu muscle weights (mg) per body weight (g) in C57BL/6 (closed circles), DBA/2 (closed squares), and B6D2F1 (closed triangles) mice after one (4 weeks × 1) or three CTX injections (4 weeks × 3). The cross sections were stained with H&E (B), Oil red-O (C), or Sirius Red (D). Scale bars: 100 μm (B); 500 μm (C and D). E: The y axis shows the mean percentage of Oil red-O or Sirius Red-positive areas per section. White and black columns indicate the results of C57BL/6 and DBA/2, respectively. The number in the each graph indicates the number of mice used in this analysis. *P < 0.05, **P < 0.01 (analysis of variance, SNK-test).
      As shown in Figure 1A, DBA/2 mice exhibited impaired regeneration accompanied by accumulation of fat and fibrosis after three CTX injections (4 weeks × 3), but not in the 4 weeks × 1 experiment (Figure 2B). Oil red-O (Figure 2C) and Sirius Red (Figure 2D) stainings were performed to determine the amount of fat and fibrosis, respectively. As shown in Figure 2E, increments in fat and fibrotic areas were observed in DBA/2 mice receiving three injections (4 weeks × 3). In 4 weeks × 1 DBA/2, the fat accumulation of one mouse was a slightly higher volume (2.02%), but three mice showed little fat accumulation (less than 0.6%). In contrast to DBA/2, C57BL/6, and B6D2F1 mice did not show any sign of impaired regeneration. These results indicate that the impaired regeneration ability of the DBA/2 strain after repeated injury is recessive heredity.

      Loss of Muscle Mass Results from Decreased Number and Size of Myofibers

      To assess the cause of muscle weight loss in DBA/2, the numbers and sizes of myofibers were quantified. In uninjured muscle, no significant difference between the numbers of myofibers was observed in C57BL/6 and DBA/2 (Figure 3A). However, as shown in Figure 3B, decreased numbers of myofibers were observed in DBA/2 after three CTX injections (4 weeks × 3), as compared with 4 weeks × 1 or uninjured muscle. C57BL/6 showed more myofibers than uninjured muscle after one or three injections (Figure 3B).
      Figure thumbnail gr3
      Figure 3Decreased numbers of myofibers and satellite cells in DBA/2 mice after three repeated injuries. A: The number of myofibers in uninjured TA muscle at 10 weeks old. The y axis shows the mean number of myofibers per section. B6 and D2 indicate C57BL/6 and DBA/2, respectively. B: The mean numbers of myofiber in TA muscles after one or three injuries. *P < 0.05 (analysis of variance, SNK-test). The sizes of myofibers in TA muscle after one or three CTX injections. C: Cross sections were stained with anti-laminin α2 antibody (green). D: The size of each myofiber in TA muscle was measured after one or three injections. Closed circles or squares show the results of C57BL/6 or DBA/2, respectively. E: Arrowhead indicates Pax7 expressing cells lying beneath the basal lamina. F: The number of satellite cells in noninjured TA muscle (uninjured) or TA muscle injured three times (4 weeks × 3). The y axis shows the mean number of satellite cells per section. The number in each graph indicates the number of mice used in these analyses. **P < 0.01 (Student's t-test).
      The sizes of myofibers were also measured. Four weeks after one CTX injection (4 weeks × 1), the size of myofibers in DBA/2 was similar to that in C57BL/6 (Figure 3, C and D). However, the regenerated myofibers of DBA/2 (4 weeks × 3) were slightly smaller than those of C57BL/6 (Figure 3, C and D). These data indicate that the loss of muscle weight in DBA/2 results from the decreased number and size of myofibers.

      Decreased Number of Self-Renewed Satellite Cells in DBA/2

      We hypothesized that a decreased number of satellite cells leads to the loss of myofibers, because myofibers are mainly made by satellite cells. To elucidate this hypothesis, we examined the number of satellite cells. As shown in Figure 3E, cells positive for Pax7, a specific marker of satellite cells,
      • Seale P
      • Sabourin LA
      • Girgis-Gabardo A
      • Mansouri A
      • Gruss P
      • Rudnicki MA
      Pax7 is required for the specification of myogenic satellite cells.
      lying beneath the basal lamina were counted. There was no significant difference between the uninjured TA muscles of C57BL/6 and DBA/2 mice. However, a remarkable decrease in the number of satellite cells was observed in DBA/2 after three CTX injections (Figure 3F). These results imply that the functions (including self-renewal potential) of satellite cells include responsibility for most of the regeneration of impaired muscle in DBA/2.

      Colony Formation and Proliferation of Satellite Cells from DBA/2

      To examine whether there is an intrinsic difference between the satellite cells of C57BL/6 and DBA/2, satellite cells were isolated and cultured in vitro. As shown in Figure 4A, the BrdU uptake of primary myoblasts of DBA/2 was inferior to that of C57BL/6 myoblasts. Next, we performed a colony-forming assay of single satellite cells. As shown in Figure 4C, single DBA/2 satellite cell did not produce large colonies similar to those of C57BL/6. The frequencies of colony forming cells did not differ in C57BL/6 and DBA/2 (Figure 4B). These results indicate that intrinsic factors affect the properties of satellite cells.
      Figure thumbnail gr4
      Figure 4Satellite cells of DBA/2 strain show inferior BrdU uptake and colony-forming potential. A: BrdU uptake of primary myoblasts derived from C57BL/6 or DBA/2 satellite cells. The y axis shows the mean with SD of three independent experiments. *P < 0.05 (Student's t-test). Frequency of colony formation by a single satellite cell derived from C57BL/6 or DBA/2 (B) and the size of single cell-derived colonies (C). The picture shows representative colonies of each strain. Colonies were categorized into three groups: >50 cells/well, 10 to 49 cells/well, and 2 to 9 cells/well. The y axis indicates the frequency (B) or percentage of each category (C) from three independent experiments. Scale bar = 100 μm.

      Loss of Muscle Weight in DBA/2-mdx

      To assess whether the low regenerative potential of mice with the dystrophin mutation exhibit DMD-like features, we crossed C57BL/10-mdx (B10-mdx) into DBA2. It was reported that body weight of B10-mdx is heavier than that of the control wild-type.
      • Connolly AM
      • Keeling RM
      • Mehta S
      • Pestronk A
      • Sanes JR
      Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin alpha2-deficient mice.
      In contrast to B10-mdx, DBA/2-mdx (D2-mdx) mice showed the decreased body weight regardless of gender (Figure 5A). A more remarkable phenotype of D2-mdx was the loss of skeletal muscle mass (Figure 5B). As previously reported, the muscle weight of B10-mdx was heavier than that of controls,
      • Pastoret C
      • Sebille A
      mdx mice show progressive weakness and muscle deterioration with age.
      but the TA, GC, and Qu muscle weights of D2-mdx males were 71%, 59%, and 54% of those of controls, respectively (Figure 5C). Female muscles were 85% (TA), 61% (GC), and 52% (Qu) of each control muscle, respectively. The loss of muscle weight did not simply reflect the decreased body weight because there is also a significant difference in muscle weight (mg) per body weight (g) between D2-mdx and control littermates (Figure 5C). Control littermates of D2-mdx and normal DBA/2 exhibited similar results in muscle weight per body weight ratios (data not shown).
      Figure thumbnail gr5
      Figure 5DBA/2-mdx mice show decreased body weight and remarkable muscle weight loss. A: Body weight of D2-mdx (closed squares) and their wild or heterozygous littermates (open circles) related to age. *P < 0.05, **P < 0.01 (Student's t-test) B: Photographs of hind limb muscles of male B10-mdx (15 months) and D2-mdx (12 months). Scale bar = 1 cm. C: TA, GC, and Qu muscle weights (mg) or per body weight (g) of 6-month-old mice. x axis shows the mean with SD. The numbers of muscles used in each study are shown in each graph. *P < 0.05, **P < 0.01.

      Histology of DBA/2-mdx

      In contrast to the histology of DMD, it is widely accepted that fibrosis and fat replacement are minimal in B10-mdx.
      • Tanabe Y
      • Esaki K
      • Nomura T
      Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse.
      In addition, there was no apparent fiber loss. To examine the accumulation of fibrosis and fat tissue in D2-mdx, cross sections were stained with Sirius Red or Oil red-O. As shown in Figure 6, A and B, there was no sign of fibrosis or adipogenesis in B10-mdx. However, D2-mdx mice exhibited increased fibrosis and fat accumulation in comparison with B10-mdx. In contrast to B10-mdx, a decreased number of total myofibers was also observed in D2-mdx(Figure 6C).
      Figure thumbnail gr6
      Figure 6Histological analyses of DBA/2-mdx mice. Sirius red staining (A) and Oil red-O staining (B) of Qu muscle of 8-month-old B10-mdx and D2-mdx mice. The y axis indicates the mean percentage of Sirius Red- (A) or Oil red-O (B) -positive areas per section. The x axis indicates the age of mice. Black and white columns show the results for B10-mdx and D2-mdx, respectively. The numbers of mice used in each study are shown in each graph. C: The y axis indicates the mean number of Evans blue-positive or total myofibers of B10-mdx and D2-mdx at 8 months of age. *P < 0.05, **P < 0.01.
      To enumerate the number of necrotic fibers, the mice were injected with Evans blue dye to visualize necrotic fibers. As shown in Figure 6C, fewer total necrotic fibers were observed in D2-mdx. This result suggests that the D2-mdx phenotype does not result from acceleration of degeneration.

      Decreased Skeletal Muscle Function in DBA/2-mdx

      Skeletal muscle endurance was assessed by treadmill running to exhaustion as an indicator of maximal muscle capacity. After acclimatization, mice were run on a 10% slope at increasing speed until the animals were unable to remain on the treadmill despite prodding. We then recorded the end time and speed to calculate the distance run. As shown in Figure 7A, male and female D2-mdx ran 45% and 56% shorter distances than control littermates. The maximum speed of D2-mdx was also lower than that of their littermate. The distance run showed the most significantly difference because of the protocol of increasing speed (Figure 7A). The average distance run by male controls was 544 meters, but that of D2-mdx males was 205 meters (38% of the control). A similar result was shown by female D2-mdx (25% of the control). B10-mdx also showed lower values compared with normal C57BL/10 mice (data not shown), but the decreased ratio of each parameter in D2-mdx was more remarkable than that in B10-mdx(Figure 7B).
      Figure thumbnail gr7
      Figure 7Comparison of muscle strength in DBA/2-mdx and B10-mdx. A: Treadmill running test of mice at 24 weeks old. Final time, speed, and distance were recorded and calculated for the individual performance score. The averages are shown with SD. Control indicates heterozygous or wild-type littermates of D2-mdx. The numbers of mice used in each study are shown in each graph. *P < 0.05, **P < 0.01. B: Comparison of C57BL/10-mdx and DBA/2-mdx in treadmill running test. The y axis indicates the percentage of mdx per control value. The numbers of male C57BL/10, male C57BL/10-mdx, female C57BL/10, and female C57BL/10-mdx are 4, 4, 4, and 8, respectively. Grip strength test of D2-mdx (C) or B10-mdx (D). Black and white columns indicate the results for mdx or control mice, respectively. The y axis indicates the average score of each mouse with SD. The x axis shows the ages of mice. The number in the each graph indicates the number of mice taking this test. *P < 0.05, **P < 0.01.
      A grip strength test was also performed as an indicator of motor function and whether D2-mdx exhibited muscle weakness compared with controls. As shown in Figure 7C, D2-mdx earned a lower score than control mice regardless of gender. However, there was no significant difference between B10-mdx and control mice.

      Discussion

      Repeated Injury Models

      Muscle satellite cells play central roles in skeletal muscle regeneration.
      • Charge SB
      • Rudnicki MA
      Cellular and molecular regulation of muscle regeneration.
      Satellite cells produce a vast number of progenitor cells (myoblasts) that finally become myofibers. During this process, at least some of the satellite cells have self-renewal potential,
      • Collins CA
      • Olsen I
      • Zammit PS
      • Heslop L
      • Petrie A
      • Partridge TA
      • Morgan JE
      Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.
      • Sacco A
      • Doyonnas R
      • Kraft P
      • Vitorovic S
      • Blau HM
      Self-renewal and expansion of single transplanted muscle stem cells.
      but are quiescent and will respond efficiently to the next damages. In fact, Luz et al
      • Luz MA
      • Marques MJ
      • Santo Neto H
      Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.
      indicated that C57BL/10 could regenerate after 50 bupivacaine injections without the loss of myofibers or gain of fibrotic areas in the TA muscle. Importantly, C57BL/10-mdx mice exhibited decreased numbers of myofibers after 50 bupivacaine injections
      • Luz MA
      • Marques MJ
      • Santo Neto H
      Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.
      because C57BL10-mdx mice already have dystrophic degeneration–regeneration cycles. Sadeh et al
      • Sadeh M
      • Czyewski K
      • Stern LZ
      Chronic myopathy induced by repeated bupivacaine injections.
      also showed active regeneration cycles in rats that received weekly injections of bupivacaine for 6 months. They reported that there was lack of evidence for reduction or exhaustion of muscle fiber capacity to regenerate despite ongoing degeneration–regeneration over a period approximating one fourth of the rat life expectancy. These results indicate that the satellite cell pool was efficiently maintained for multiple degeneration–regeneration cycles in these animals, and that dystrophic mice exhibit less regeneration ability. However, DBA/2 showed significantly decreased numbers of myofibers and self-renewed satellite cells after only three injections of CTX.
      The number of DBA/2 satellite cells in uninjured TA muscle is similar to that of C57BL/6. Although, the myofibers in DBA/2 were smaller than those in C57BL/6 2 weeks after one CTX injection (data not shown), the myofiber size and histological characteristics showed few significant differences between and DBA/2 and C57BL/6 4 weeks after a single CTX injection. These results suggest that the self-renewal ability of DBA/2 satellite cells is incomplete and that the exhaustion of muscle satellite cells leads to a decreased number of myofiber and loss of skeletal muscle weight. Nonmyogenic cells, for example, macrophages, also play important roles in skeletal muscle regeneration. However, dysfunction of macrophages leads to impaired regeneration after one CTX injection.
      • Arnold L
      • Henry A
      • Poron F
      • Baba-Am Y
      • van Rooijen N
      • Plonquet A
      • Gherardi RK
      • Chazaud B
      Inflammatory monocytes recruited after skeletal muscle injury switch into anti-inflammatory macrophages to support myogenesis.
      • Segawa M
      • Fukada S
      • Yamamoto Y
      • Yahagi H
      • Kanematsu M
      • Sato M
      • Ito T
      • Uezumi A
      • Hayashi S
      • Miyagoe-Suzuki Y
      • Takeda S
      • Tsujikawa K
      • Yamamoto H
      Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis.
      Furthermore, the remarkable regeneration deficit was not observed in DBA/2 4 weeks after one CTX injection in TA muscle. These results suggest that repeated injury is a suitable model to assess the long-term regeneration potential of skeletal muscle, and that the self-renewal ability of satellite cells is responsible at least in part for the result of repeatedly injured muscle in DBA/2.

      Strain Differences of Muscle Regeneration Ability

      C57BL/6, a strain akin to C57BL/10, is the most widely used strain for skeletal muscle regeneration studies. As shown in Figure 1, C57BL/6 has the best ability to regenerate skeletal muscle among the four inbred strains examined. An early study by Grounds and McGeachie
      • Grounds MD
      • McGeachie JK
      A comparison of muscle precursor replication in crush-injured skeletal muscle of Swiss and BALBc mice.
      indicated a strain difference in skeletal muscle regeneration between BALB/c and Swiss SJL/J. They showed that superior and faster regeneration was observed in the Swiss SJL/J strain. The most outstanding phenotype of DBA/2 is the remarkable decrease of muscle weight compared with the three other inbred strains, including BALB/c. Intriguingly, DBA/2 mice have a shorter life span than C57BL/6.
      • Gelman R
      • Watson A
      • Bronson R
      • Yunis E
      Murine chromosomal regions correlated with longevity.
      In addition, it is reported that muscle weight loss is increased during aging (sarcopenia) in DBA/2 mice compared with C57BL/6.
      • Lionikas A
      • Blizard DA
      • Vandenbergh DJ
      • Stout JT
      • Vogler GP
      • McClearn GE
      • Larsson L
      Genetic determinants of weight of fast- and slow-twitch skeletal muscles in old mice.
      The reason why the DBA/2 strain exhibits the loss of muscle weight is unknown, but our results imply a relationship between the impaired function of satellite cells and sarcopenia in DBA/2.
      Heydemann et al
      • Heydemann A
      • Huber JM
      • Demonbreun A
      • Hadhazy M
      • McNally EM
      Genetic background influences muscular dystrophy.
      reported that γ-sarcoglycan-null mice with DBA/2 background showed decreased skeletal muscle weight, increased Evans Blue uptake, and a higher hydroxyproline concentration than C57BL/6, CD1, and 129 background null mice. Although they ruled out the voluntary activities of DBA/2, they did not discuss the cause of these results. Our results suggest that the low regeneration potential of DBA/2 leads to a severe skeletal muscle phenotype in various dystrophic mouse models.
      The DBA/2J strain has been used in sarcopenia and γ-sarcoglycan-null mouse studies.
      • Lionikas A
      • Blizard DA
      • Vandenbergh DJ
      • Stout JT
      • Vogler GP
      • McClearn GE
      • Larsson L
      Genetic determinants of weight of fast- and slow-twitch skeletal muscles in old mice.
      • Heydemann A
      • Huber JM
      • Demonbreun A
      • Hadhazy M
      • McNally EM
      Genetic background influences muscular dystrophy.
      To exclude the possibility that DBA/2 substrain differences exist, we compared the BrdU uptake of primary myoblasts in DBA/2N (used in this study) and DBA/2J. Because we observed similar low BrdU uptakes by primary myoblasts in both DBA/2N and DBA/2J (data not shown), these results suggest that lower muscle regeneration is common to the DBA/2 strain.

      Stem (Satellite) Cell Function and Mouse Strains

      As mentioned above, some previous reports indicated different responses in skeletal muscle regeneration among inbred strains of mice. However, to our knowledge, this is the first evidence that there is an intrinsic difference in satellite cells among inbred mice. The exact relationship between in vitro and in vivo results of satellite cells is not clear. However, low or slow proliferation of satellite cells might explain the decreased muscle weight and slow regeneration after a single injury in DBA/2 in comparison with C57BL/6 and B6D2F1, which showed increased muscle weight in their TA muscle (Figure 2A). It is unlikely that telomere erosion contributes to the in vitro and in vivo results of DBA/2 satellite cells because DBA/2 mice have longer telomeres than C57BL/6 mice.
      • Manning EL
      • Crossland J
      • Dewey MJ
      • Van Zant G
      Influences of inbreeding and genetics on telomere length in mice.
      Recently Kuang et al
      • Kuang S
      • Kuroda K
      • Le Grand F
      • Rudnicki MA
      Asymmetric self-renewal and commitment of satellite stem cells in muscle.
      reported that satellite cells are a heterogeneous population of stem cells (satellite stem cells) and committed progenitor cells, and that they can be distinguished from others by Myf5 expression. They showed that Myf5-negative (satellite stem) cells self-renewed three times more frequently than Myf-5-positive (progenitor) cells in vivo. Schultz and Lipton
      • Schultz E
      • Lipton BH
      Skeletal muscle satellite cells: changes in proliferation potential as a function of age.
      first described the heterogeneity of satellite cells by the different colony sizes of each satellite cell and found decreased colony sizes in aging muscle in the rat. Although it was not determined whether satellite stem cells form a large-colonies or not in vitro, our results showed that mice having low self-renewing satellite cells (DBA/2) exhibit smaller colony formations than mice having high self-renewing satellite cells (C57BL/6). These results suggest that satellite stem cells may form larger colonies in vitro.
      In contrast to satellite cells, a highly strain-dependent function of hematopoietic stem cells was reported.
      • Dykstra B
      • de Haan G
      Hematopoietic stem cell aging and self-renewal.
      Chen et al
      • Chen J
      • Astle CM
      • Harrison DE
      Genetic regulation of primitive hematopoietic stem cell senescence.
      reported that DBA/2 showed a decline in primitive hematopoietic stem cell function with age, but that it increased with age in C57BL/6 in a vivo transplantation study. Recombinant inbred mice, named BXD strains, are available. Using BXD, Liang et al
      • Liang Y
      • Jansen M
      • Aronow B
      • Geiger H
      • Van Zant G
      The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice.
      identified latexin as affecting the size of the hematopoietic stem cell population in mice. A similar approach might lead to the discovery of key genes that affect the properties of satellite cells.

      DBA/2-mdx as Model for DMD

      Mdx was discovered a quarter of a century ago.
      • Bulfield G
      • Siller WG
      • Wight PA
      • Moore KJ
      X chromosome-linked muscular dystrophy (mdx) in the mouse.
      In 1989, the mdx mutation, a C to T transition within exon 23, was identified in the dystrophin gene on the X chromosome.
      • Sicinski P
      • Geng Y
      • Ryder-Cook AS
      • Barnard EA
      • Darlison MG
      • Barnard PJ
      The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.
      Nearly all mdx colonies are maintained as homozygous inbred lines; in addition, the difficulty of point mutation typing might impede the effect of genetic background on mdx phenotype. However, Amalfitano and Chamberlain
      • Amalfitano A
      • Chamberlain JS
      The mdx-amplification-resistant mutation system assay, a simple and rapid polymerase chain reaction-based detection of the mdx allele.
      reported a rapid and simple typing strategy, and we established DBA/2-mdx following their protocol. C57BL/10-mdx mice have played central roles in a vast array of pathological, clinical, and physiological studies as a model for DMD. However, they do not reflect human pathology in some aspects, including little fat and fibrosis accumulation, no loss of myofiber numbers, and muscle weight. Recently, Gargioli et al
      • Gargioli C
      • Coletta M
      • De Grandis F
      • Cannata SM
      • Cossu G
      PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle.
      showed that the advanced stage of dystrophy including sclerosis precluded treatment by stem cell therapy. Therefore, assessment of therapeutic effect in more severe disease conditions is needed.
      In marked contrast to the severe phenotype observed in DMD, early studies using C57BL/10-mdx concluded that they do not show obvious functional disability.
      • Bulfield G
      • Siller WG
      • Wight PA
      • Moore KJ
      X chromosome-linked muscular dystrophy (mdx) in the mouse.
      • Tanabe Y
      • Esaki K
      • Nomura T
      Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse.
      However, some later reports indicated functional differences between C57BL/10-mdx and control mice.
      • Muntoni F
      • Mateddu A
      • Marchei F
      • Clerk A
      • Serra G
      Muscular weakness in the mdx mouse.
      • Hara H
      • Nolan PM
      • Scott MO
      • Bucan M
      • Wakayama Y
      • Fischbeck KH
      Running endurance abnormality in mdx mice.
      • Carter GT
      • Wineinger MA
      • Walsh SA
      • Horasek SJ
      • Abresch RT
      • Fowler Jr, WM
      Effect of voluntary wheel-running exercise on muscles of the mdx mouse.
      • Lynch GS
      • Hinkle RT
      • Chamberlain JS
      • Brooks SV
      • Faulkner JA
      Force and power output of fast and slow skeletal muscles from mdx mice 6–28 months old.
      As shown in Figure 7, C57BL/6-mdx also showed muscle weakness in the treadmill test. However, the muscle weakness of DBA/2-mdx is more remarkable than that of C57BL/10-mdx. Therefore, DBA/2-mdx is a more appropriate model to assess skeletal muscle function after therapeutic treatment.
      Chamberlain et al
      • Chamberlain JS
      • Metzger J
      • Reyes M
      • Townsend D
      • Faulkner JA
      Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.
      reported that the average life spans of female and male C57BL/10-mdx mice were 22.5 and 21.5 months, respectively. Pastoret et al
      • Pastoret C
      • Sebille A
      mdx mice show progressive weakness and muscle deterioration with age.
      also reported that C57BL/10-mdx mice have short life spans and that C57BL/10-mdx older than 78 weeks exhibit progressive weakness. We have not determined the life span of DBA/2-mdx, but it will be clarified in the future. Intriguingly, Chamberlain et al
      • Chamberlain JS
      • Metzger J
      • Reyes M
      • Townsend D
      • Faulkner JA
      Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.
      observed the appearance of rhabdomyosarcoma-like tumors in C57BL/10-mdx. They speculate that the lifelong continuous myofiber degeneration and regeneration that characterize this animal model are associated with continuous and massive activation and proliferation of satellite cells, which greatly increase the chance of developing random and spontaneous mutations. To date, we have observed tumors in C57BL/10-mdx but not in DBA/2-mdx. This observation supports their speculations.
      The reasons why mdx mice do not show the human-like pathology have been investigated. One reason for the difference between DMD and mdx is explained by the presence of utrophin, a homolog of dystrophin. Utrophin is located in the neuromuscular junction in normal muscle. In dystrophic muscle, utrophin is up-regulated in the sarcolemma and compensates for dystrophin function. As shown in Figure 6, the results of Evans blue uptake in DBA/2-mdx indicated that the degeneration of myofiber was not accelerated, but that the regeneration potential was inferior. These results clearly indicate that not only utrophin expression but also regeneration potential, perhaps a satellite cell function, directly leads to the pathological condition. The identification of genes that determine the DBA/2 phenotype will provide new therapeutic strategies for the treatment of muscular dystrophy.

      Acknowledgements

      We thank Katherine Ono for reading this manuscript.

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