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Human Molecular Genetics, 2004, Vol. 13, No. 2 213-221
DOI: 10.1093/hmg/ddh018

Characterization of ARC, apoptosis repressor interacting with CARD, in normal and dystrophin-deficient skeletal muscle

S. Abmayr, R.W. Crawford and J.S. Chamberlain*

Department of Neurology, University of Washington School of Medicine, HSB Room K233, Box 357720, Seattle, WA 98195-7720, USA

Received September 8, 2003; Accepted November 11, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Duchenne muscular dystrophy is an X-linked recessive disorder, primarily characterized by progressive muscle weakness and wasting. The disease results from the absence of dystrophin, however the precise molecular mechanisms leading to muscle pathology are poorly understood. Dystrophic muscles undergo increased oxidative stress and altered calcium homeostasis, which may contribute to myofiber loss by triggering both necrosis and apoptosis. Recent studies have identified ARC (apoptosis repressor with caspase recruitment domain) as an abundant protein in human muscle that can inhibit both hypoxia and caspase-8-induced apoptosis as well as protect cells from oxidative stress. To explore a potential role for ARC in protecting muscle fibers from dystrophic breakdown, we have cloned and characterized murine ARC and studied its expression in normal and dystrophic mouse muscle. ARC is expressed at high levels in striated muscle and displays fiber-type restricted expression patterns. ARC expression levels are normal in dystrophic mdx mice, although the intracellular localization pattern of ARC is slightly altered compared with normal muscles. Overexpression of ARC in transgenic mdx mice failed to alleviate the dystrophic pathology in skeletal muscles, suggesting that misregulation of the molecular pathways regulated by ARC does not significantly contribute to myofiber death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Duchenne muscular dystrophy (DMD) is an X-linked recessive, lethal disorder, caused by mutations in the dystrophin gene (1). DMD affects one in 3500 newborn males and is characterized by a progressive muscle weakness and wasting, resulting in a wheelchair dependency by the age of 11 and death in the second or third decade from cardiac or respiratory failure (2). The relationship between the absence of dystrophin and the pathological mechanism of the disease are poorly understood. Dystrophin is thought to play a structural role in providing a link between intracellular actin and extracellular laminin via its interaction with a complex of peripheral and integral membrane proteins, called ‘the dystrophin–glycoprotein complex’ (DGC) (3). Disruption of this linkage results in membrane instability and dystrophic muscle fibers are highly susceptible to contraction-induced injury (4–10). Besides providing mechanical stability, several proteins of the DGC play a role in cell signaling (11–16). Altered cell signaling is thought to increase the susceptibility of muscle fibers to secondary triggers, such as functional ischemia and oxidative stress (17–21). One signaling component of the DGC is the neuronal isoform of nitric oxide synthase (nNOS), which is absent from the sarcolemma in DMD (22). Muscles from mice deficient in nNOS, as well as dystrophin-deficient mdx muscles, show insufficient production of NO, resulting in impaired metabolic modulation of {alpha}-adrenergic vasoconstriction and functional ischemia (17,18,23). Dystrophin-deficient mdx muscles demonstrate oxidative injury prior to muscle pathology, and muscle cells display an increased susceptibility to oxidative stress compared with normal muscles (19,21). Reduced NO-mediated cell protection and increased oxidative damage might therefore contribute significantly to the pathology of muscular dystrophy. However, it remains unclear to what extent abnormal DGC-mediated signaling and/or loss of mechanical stability are responsible for the onset and the progression of the disease.

It also remains unclear if muscle fiber breakdown occurs primarily through apoptotic or necrotic processes. Recent studies suggest that cell death in mdx muscle may be initiated by apoptosis and followed by necrotic processes (24). Tissue sections of dystrophic muscle demonstrate apoptotic myonuclei in degenerating muscle fibers (25–28). Several groups have proposed that the intensity of the signal, such as intracellular ATP levels, hypoxia and/or reactive oxygen species can dictate whether a cell dies by a primarily necrotic, or an apoptotic, pathway (29–31).

The potential contribution of ischemia, oxidative stress and inducers of apoptosis to the dystrophic process are of interest in view of the recent identification of ARC [apoptosis repressor interacting with CARD (caspase recruitment domain)]. ARC was identified in the GenBank database using a screen for proteins with homology to the CARD of caspase-9, a key initiator of apoptosis in many cell types (32). The CARD domain is conserved in numerous proteins and mediates binding to, and regulation of, various caspases (33–35). ARC was shown to interact selectively with caspase-2 and caspase-8 via its CARD and to inhibit caspase-8 induced apoptosis (32). ARC has also been shown to inhibit both hypoxia-induced and hydrogen peroxidase-mediated cell death in H9C2 cells and to protect the heart from postischemic cardiomyopathy (36–40). Intriguingly, ARC expression in humans is restricted primarily to striated muscles, tissues that do not normally undergo rapid cell turnover or apoptosis (32). This high-level expression of an apoptosis inhibitor in long-lived cell types raises the possibility that ARC could help protect muscle fibers from apoptotic death resulting from mechanical stress or oxidative damage. Since myofiber death in dystrophic muscles has been linked to increased oxidative stress, ischemia and mechanical injury, we sought to study the expression of ARC in a mouse model for DMD, the mdx mouse. We have cloned and characterized murine ARC and shown that it is highly expressed in skeletal and cardiac muscle, and at lower levels in brain and testis. ARC displays a slightly abnormal intracellular localization pattern in dystrophic muscle. However, overexpression of ARC in mdx mice failed to alleviate the dystrophic process in skeletal muscle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of mouse ARC cDNA
The full-length mouse ARC cDNA was isolated by direct PCR amplification from a muscle cDNA library (41) using primers derived from an EST clone (accession number AA596110) and vector specific primers. Direct sequencing of PCR products revealed that the longest open reading frame encoded a protein of 220 amino acids with a predicted molecular mass of 24.5 kDa (GenBank accession no. AY459322). The protein sequence displayed 80% identity to the previously identified human ARC (32) and 91% identity to the rat ortholog (42) (data not shown). Alignment analysis confirmed that mouse ARC contains a N-terminal CARD and a C-terminal proline/glutamic acid-rich (P/E) domain. The CARD domain of ARC has significant amino acid similarity to the CARD domains from caspase-2, caspase-9, RAIDD and APAF-1 (32) and is structurally related to the death effector domain (DED) shared by effector caspases (35).

The human ARC gene has been reported to encode two proteins that differ by alternative RNA splicing. The alternative product has been named nucleolar protein Nop30 and has been shown to interact with the splicing factor SRp30c (43). Nop30 contains a 10 bp deletion that leads to a frame shift between amino acids 95 and 96, resulting in a different C-terminal domain consisting of 124 amino acids (43). To determine whether the mouse ARC gene also encoded a Nop30 like protein we amplified ARC from a mouse muscle cDNA library and subcloned the PCR product. Ten independent clones were sequenced and all encoded the normal, full-length ARC. Furthermore, we digested the PCR product with PmlI, which cuts within the 10 bp deletion of the potential Nop30 cDNA and would distinguish between ARC and Nop30. We were able to detect the ARC cDNA, but less than 5% of the product remained undigested (data not shown). Analysis of the amino acid sequence demonstrated that the 10 bp deletion observed in the human alternative transcript would only yield a 56 amino acid C-terminal domain, compared with the 124 amino acids in human Nop30. These data indicate that in mouse skeletal muscle, the ARC gene does not encode a Nop30-related protein.

Chromosomal localization of mouse ARC
The mouse ARC gene was mapped to chromosome 8 by typing a backcross panel (kindly provided by the Jackson Laboratories). The loci was named Nol3, nucleolar protein 3, in correspondence with the previously mapped human ARC gene product Nop30 (43). The mouse loci on chromosome 8 corresponds to human chromosome 16q22.1 and agrees with the mapping data of human ARC. ARC cosegregates with Psmb10 and Sntb2 (Fig. 1A).



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  		Figure 1. Characterization of mouse ARC. (A) Chromosomal localization of the mouse ARC gene. Figures from the TJL BSS backcross showing part of chromosome 8 with loci linked to Nol3. The map is depicted with the centromere toward the top. A 3 cM scale bar is shown to the right of the figure. Loci mapping to the same position are listed in alphabetical order. In the haplotype figure loci are listed in order with the most proximal at the top. The black boxes represent the C57BL6/JEi allele and the white boxes the SPRET/Ei allele. The number of animals with each haplotype is given at the bottom of each column of boxes. The percentage recombination (R) between adjacent loci is given to the right of the figure, with the standard error (SE) for each R. Missing typings were inferred from surrounding data where assignment was unambiguous. Raw data from the Jackson Laboratory were obtained from www.jax.org/resources/documents/cmdata. (B) Northern blot analysis of mouse ARC expression. mRNAs from various mouse tissues were hybridized with a cDNA for the full-length coding region of mouse ARC. The arrow indicates the 1.0 kb transcript characterized in this study, which is expressed in skeletal muscle and heart and at a lower level in brain. The nature of the larger transcripts observed in heart and testis (asterix) is unclear.

 
ARC expression in mice
To examine ARC gene expression in mice we initially probed a multiple tissue northern blot. Mouse ARC was highly expressed in heart, and at a slightly lower level in skeletal muscle (Fig. 1B). Some ARC expression was detected in brain and testis, with very low levels in kidney and lung. No ARC expression was detected in liver or spleen. Interestingly, in testis ARC mRNA was expressed as a longer transcript than in other tissues possibly due to an alternative polyadenylation site as seen in human tissue (43). The nature of the larger transcript in heart is unclear and could be the result of alternative splicing or polyadenylation site usage. This expression agrees with the reported ARC mRNA expression pattern in human and rat tissues (32,42). As the mdx mouse represents a good model for myofiber breakdown and turnover, we asked if ARC displayed a different expression pattern in dystrophic muscle. We compared ARC protein levels in wild-type C57BL/10J and dystrophic mdx mouse skeletal muscles at 2 and 6 months of age via western blotting. ARC was found to be expressed at essentially the same levels in C57BL/10J and mdx muscles at both ages (Fig. 2A).



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  		Figure 2. Expression and localization of endogenous mouse ARC in muscle tissue. (A) Immunoblot staining of quadriceps muscle extracts from 2- and 6-month-old C57Bl/10J and mdx mice using an anti-ARC antibody. The positive control represents 293T cells transfected with a human ARC FLAG expression cassette. (B) Immunofluorescent staining of quadriceps muscle sections for endogenous mouse ARC and for the mitochondrial marker COX (cytochrome oxidase subunit V). ARC shows a fiber-type specific expression pattern and co-localizes with COX. Scale bar: 50 µm.

 
Co-localization of ARC with mitochondria
To examine the localization of ARC in muscle fibers, we immuno-stained serial frozen sections of C57BL/10J and mdx mice for ARC (Fig. 2B). ARC staining showed a non-uniform expression pattern across the muscle section, suggesting that ARC is expressed in a fiber-type specific manner. This pattern was observed in diaphragm, quadriceps and tibialis anterior muscles, while the expression in heart showed a uniform expression pattern (Figs 2B and 4C and data not shown). ARC was expressed at similar levels in wild-type and mdx animals in these muscle types and appeared to be predominantly cytoplasmic. Hypercontracted myofibers in mdx muscle demonstrated a shift of ARC to the sarcolemma (data not shown). ARC expression in wild-type muscle showed a distinct regular pattern of ARC-positive and -negative fibers, while ARC expression in the mdx background appeared as a less distinct and more irregular pattern. To determine if ARC expression is fiber type-specific, we co-stained for ARC and for the mitochondrial-specific protein cytochrome oxidase. Cytochrome oxidase is mainly restricted to oxidative fibers, which display an oxidative metabolism and contain numerous mitochondria (44). ARC co-localized with cytochrome oxidase, showing that ARC is expressed mainly in oxidative fibers and that it is co-localized with mitochondria within the muscle fiber (Fig. 2B).



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  		Figure 4. Morphological analysis of muscle tissue from transgenic mice. (A) Hematoxylin and eosin (H&E) staining of quadriceps and diaphragm muscle sections of 6-week-old transgenic ARC/mdx, transgenic ARC/C57BL/10J, mdx and C57BL/10J mice. Transgenic ARC/mdx and mdx sections display a characteristic mdx pathology including centrally located nuclei, variation in fiber size, infiltrating immune cells (<-) and fibrosis. Transgenic ARC/C57BL/10J sections were not different from C57BL/10J sections. (B) Morphology of quadriceps and diaphragm muscle of age-matched 2-year-old transgenic ARC/mdx and mdx mice demonstrates a dystrophic pathology despite ARC overexpression (bottom). (C) Immunofluorescent staining of age-matched 18-month-old transgenic ARC/mdx and mdx soleus sections display a mosaic overexpression of ARC in the transgenic animals. H&E staining of transgenic ARC/mdx muscle sections show no morphological difference with mdx muscle sections. Expression of the transgene in heart was observed only in a few isolated myocytes (arrow). Scale bar: 50 µm.

 
Transgenic ARC expression and localization
We generated transgenic mice in order to test the hypothesis that forced overexpression of ARC might maintain myofiber survival and alleviate the dystrophic muscle pathology of mdx mice (Fig. 3A). Transgenic ARC expression was analyzed in C57BL/10J and mdx mice and expression levels were compared with endogenous ARC levels (Fig. 3B).



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  		Figure 3. Overexpression of human ARC in mouse muscle tissue. (A) Schematic illustration of the expression cassette used to generate transgenic mice. A human ARC cDNA with a FLAG tag epitope was driven by the human skeletal {alpha}-actin promoter (HSA). In addition, the expression cassette included the SV40 VP1 intron and the SV40 polyA adenylation site. (B) Western analysis of transgenic and endogenous ARC expression. Quadriceps and diaphragm muscle extracts from 6-week-old transgenic ARC/mdx, transgenic ARC/C57BL/10J, mdx and C57BL/10J mice were probed with an anti-ARC and an anti-FLAG antibody. (C) Immunofluorescence analysis of ARC and FLAG expression in serial quadriceps muscle sections from 6-week-old transgenic ARC/mdx, transgenic ARC/C57BL/10J and C57BL/10J mice. Muscle sections demonstrate a uniform expression pattern of the transgene. (D) Immunofluorescent staining of transgenic ARC/mdx quadriceps muscle section for ARC and for the mitochondrial marker COX. Transgenic ARC co-localized with COX in oxidative fibers. Scale bar: 50 µm.

 
Western analysis showed that the transgene was highly expressed in quadriceps and diaphragm muscle on both the C57Bl/10J and the mdx background. Immunohistochemical analysis revealed that the transgene was uniformly expressed in quadriceps and diaphragm muscle and that it localized predominantly to the sarcoplasm in wild-type muscle and shifted towards the sarcolemma in the mutant mdx background (Fig. 3C). Immunohistochemical analysis of soleus and heart muscle showed a mosaic overexpression pattern in soleus, while expression in heart muscle was observed only in rare myocytes (Fig. 4C). This latter observation is consistent with our previous results showing that the HSA expression cassette used in this study is generally not active in cardiac muscle (10,45,46).

To examine whether transgenic ARC co-localized with mitochondria, we co-stained transgenic quadriceps muscle sections for ARC and for the mitochondrial marker cytochrome oxidase (Fig. 3D). Transgenic ARC was expressed much more uniformly in all fiber types from the {alpha}-skeletal actin promoter in both normal and mdx muscle, compared with the endogenous ARC gene. However, transgenic ARC co-localized with cytochrome oxidase in oxidative fibers.

Morphological analysis of transgenic ARC/mdx mice
Morphological studies were performed on tissue sections of different age groups to examine the effect of ARC overexpression on the histopathology of dystrophic mdx skeletal muscle fibers. Hematoxylin and eosin staining of transgenic ARC/mdx mice confirmed the presence of a clear pattern of dystrophic muscle pathology including mononuclear cell infiltration, fibrosis, centrally located nuclei and necrotic fibers in quadriceps, diaphragm and soleus of 6-week-, 6-month-, 18-month- and 2-year-old mice (Fig. 4 and data not shown). To estimate myofiber degeneration and regeneration we counted centrally nucleated myofibers in quadriceps muscles of 6-week-old transgenic ARC/mdx, transgenic ARC/C57BL/10J, C57BL/10J and mdx littermates. At this age, quadriceps muscles from mdx mice, as well as transgenic ARC/mdx mice, displayed a high degree of central nucleation, 76% in mdx and 77% in Tg/mdx. C57BL/10J and transgenic ARC/C57BL/10J mice both displayed less than 1% centrally nucleated myofibers (Fig. 4A).

To evaluate the potential benefit of ARC overexpression in old mdx mice, we analyzed the morphology of 2-year-old transgenic ARC/mdx and mdx quadriceps and diaphragm muscles (Fig. 4B). Both mdx and transgenic ARC/mdx muscles demonstrated an advanced state of muscle degeneration characterized by substantial fat accumulation and fibrotic tissue. No obvious sign of phenotype amelioration was observed in mdx muscles overexpressing the ARC protein.

Caspase-3 activity and membrane permeability in mdx and ARC transgenic/mdx mice
ARC has been suggested to act as an inhibitor of apoptotic cell death by preventing activation of caspase-8 and caspase-2 (32). To compare the level of apoptosis in C57BL/10J, mdx and transgenic ARC/mdx muscle we analyzed active caspase-3 expression by immunofluorescence (Fig. 5). Caspase-3 represents the key effector caspase and is therefore a good indicator for cells undergoing apoptosis. Previously, TUNEL positive fibers were detected in mdx mice by several groups, suggesting the presence of a low level of apoptosis (24,25,27,47). mdx and transgenic ARC/mdx muscle showed a number of caspase-3 positive fibers, the vast majority of which appeared necrotic by hematoxylin and eosin staining. No obvious differences could be detected between mdx and transgenic ARC/mdx skeletal muscles. No active caspase-3 staining was observed in C57BL/10J muscles.



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  		Figure 5. Evans blue dye uptake and caspase-3 expression in mdx and transgenic ARC/mdx mice. H&E staining of quadriceps muscle sections of 8-week-old exercised mice demonstrate necrotic lesions in mdx and transgenic ARC/mdx characteristic of the mdx phenotype. Necrotic fibers take up Evans blue dye and express active caspase-3. The merged images show that the intensity levels and distribution of Evans blue dye in muscle fibers varies relative to the active caspase-3 staining, which may be due to the stage of fiber breakdown. Scale bar: 50 µm.

 
Evans blue dye is commonly used as a marker to distinguish degenerating and intact muscle fibers (7). Consequently, we analyzed Evans blue uptake in mdx and transgenic ARC/mdx mice. mdx as well as transgenic ARC/mdx mice displayed a large and variable number of Evans blue positive myofibers, the majority of which appeared to be necrotic. We did not observe an obvious difference in Evans blue uptake between mdx and transgenic ARC/mdx muscle (Fig. 5). We compared Evans blue localization with active caspase-3 localization to address the possibility that caspase-3 activation might be a consequence of membrane damage. Interestingly, all active caspase-3 positive fibers were also positive for Evans blue, but not all Evans blue positive fibers stained positively for activated caspase-3 (Fig. 5). Muscle fibers that showed co-localization of Evans blue and caspase-3, however, showed different distribution patterns and intensity levels of Evans blue and caspase-3 immunoreactivity. Fibers with intense Evans blue stain demonstrated weak caspase-3 staining and vice versa. This difference may correspond to the stage of apoptosis and/or necrosis in each muscle fiber.

Localization of caspase-3 and ARC
We co-stained serial sections from quadriceps muscle of transgenic ARC/mdx mice for active caspase-3 and ARC to compare their expression and localization pattern in muscle fibers (Fig. 6). Interestingly, caspase-3 positive fibers displayed faint or no ARC staining, suggesting that ARC is down-regulated or degraded in these fibers. Down-regulation could be a consequence of altered signaling and apoptosis in mdx muscle, while degradation could be a consequence of muscle fiber necrosis.



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  		Figure 6. Localization of ARC and active caspase-3 in transgenic ARC/mdx muscle sections. H&E staining of quadriceps muscle sections from 6-week-old transgenic ARC/mdx mice demonstrate typical morphological characteristics of dystrophy. Serial sections were stained with ARC and caspase-3 antibodies, showing that muscle fibers expressing active caspase-3 display diminished ARC expression. The asterix indicates orientation. Scale bar: 200 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Much progress has been made over the last decade in understanding the genetic and biochemical features of dystrophin and the DGC. However, little is known about the pathogenic mechanisms leading to the onset of muscular dystrophy and to progression of the disease. Several members of the DGC are associated with signaling molecules that provide a link to crucial signal transduction pathways (12–16). Disruption of these signaling cascades could alter metabolic pathways leading to increased susceptibility to oxidative stress, elevated calcium levels, altered mitochondrial function and eventually to apoptotic or necrotic cell death (19,21,24,25,48–51).

Several groups have characterized the expression of proteins involved in apoptosis in normal and diseased skeletal muscle tissue (28,52–54). Most apoptotic proteins are expressed at a low or non-detectable level in normal mice and demonstrate elevated expression only in degenerating and regenerating muscle fibers of mdx mice (28,55). We cloned the mouse isoform of the anti-apoptotic protein ARC and characterized its expression pattern in mice. ARC is the only anti-apoptotic protein known to be expressed at high levels in skeletal muscle (32). Interestingly, ARC is expressed mainly in oxidative fibers and colocalizes with the mitochondrial-specific marker cytochrome oxidase. Localization of ARC to mitochondria is regulated by phosphorylation of threonine 149 and only the phosphorylated, active form localizes to mitochondria (56). Slow oxidative fibers express type I MHC and display an oxidative metabolism with a great number of mitochondria. Fast fiber types IIa and IIx are capable of both oxidative and glycolytic metabolism and fast fiber type IIb are exclusively glycolytic (44). Mitochondria play a key role in responding to intracellular apoptotic signals by the release of cytochrome c followed by the activation of caspase-3 (33). ARC inhibits cytochrome c release from mitochondria and protects mitochondrial function from reactive oxygen species in H9C2 cells (36,38), suggesting its anti-apoptotic role in responding to intracellular signals. In addition, ARC was shown to interact with caspase-2 and caspase-8 and to inhibit apoptosis induced by caspase-8 (32). The inactive forms of caspase-2 and caspase-8 are located in mitochondria and are released into the cytoplasm upon stimulation (57,58). Thus, ARC may play an important role in preserving mitochondrial function and inhibiting apoptosis in skeletal muscle.

Interestingly, human ARC was shown to have an alternative splice product, named Nop30, that interacts with the splicing factor SRp30c (43). We were not able to find any evidence that Nop30 is expressed in mouse muscle. Our data is supported by the expression profile of SRp30c, which, in contrast to human muscle, is expressed at very low levels in mouse skeletal muscle (59). This lack of alternative splicing of the ARC gene in mouse skeletal muscle demonstrates one example of evolutionary divergence between humans and mice.

We characterized endogenous ARC expression in normal and mdx mice to explore the effect of a dystrophic background on ARC expression. ARC expression levels are similar in normal and mdx muscle. However, ARC localization is altered in the mdx background. Altered mitochondrial protein expression and localization could be responsible for differences in intracellular ARC localization in the C57BL/10J and mdx background. It was previously shown that respiratory chain-linked enzymes were downregulated in mdx muscle and oxidative phosphorylation was altered compared to normal muscle (49,51,60,61). To test if elevated levels of the anti-apoptotic and hypoxia-protecting protein ARC might alleviate any dystrophic phenotypic features, we generated transgenic mice that overexpressed ARC. Endogenous ARC was expressed at high levels in skeletal muscle, but mainly in oxidative fibers. We explored whether overexpression of ARC in all fiber types might protect dystrophic myofibers from apoptotic and/or necrotic cell death. Transgenic ARC was expressed uniformly in all fiber types and colocalized with mitochondria in oxidative fibers. To examine muscle fiber breakdown, we analyzed Evans blue uptake and active caspase-3 expression in transgenic ARC/mdx and mdx muscle. We found Evans blue uptake and active caspase-3 positive fibers in transgenic ARC/mdx and mdx muscle fibers, the majority of which appeared to be at various stages of necrosis. We did not observe any clear protection of myofibers in the ARC transgenic mice from the dystrophic pathology. One explanation could be that endogenous ARC is already functionally saturated in skeletal muscle, thus overexpression does not show an additional beneficiary effect.

However, the role of ARC in the apoptotic signaling cascade in skeletal muscle is not well understood. We detected reduced ARC expression in active caspase-3 positive fibers, which could be the cause, or the consequence, of fiber breakdown. Most cells contain a very complex, tightly regulated network of pro- and anti-apoptotic triggers, the balance of which can lead to continued cell life, or death. Altered expression of one apoptosis inhibitor or effector may be compensated by the upregulation of an antagonist to maintain a balance. The overexpression of ARC could have been counterbalanced by the up-regulation of a pro-apoptotic factor and therefore might have prevented the transgene from protecting muscle fiber breakdown. Alternatively, the overexpression of ARC could have effectively inhibited one apoptotic pathway, while muscle fiber breakdown in muscular dystrophy might occur through another, ARC-independent pathway. The elimination of one apoptotic stimuli could be superceded by other signals in favor of apoptosis or necrosis.

Although it has been shown in previous studies that apoptosis plays a role in dystrophic pathology, it remains unclear if apoptosis causes or is a secondary effect of muscle fiber breakdown (25,62). We detected active caspase-3 in dystrophic muscle, but the majority of the fibers appeared to be necrotic and had lost their membrane integrity, which would argue in favor of apoptosis being a secondary consequence resulting from loss of muscle fiber integrity. However, not all Evans blue positive fibers displayed clear staining for active caspase-3, and the intensity level and staining pattern within individual myofibers was variable, indicating that muscle fibers were at various stages of degeneration. These observations reinforce the idea that muscle cell death is a dynamic process and may reflect the increased susceptibility of myofibers to secondary triggers resulting from altered cell signaling, leading to active cell death. Interestingly, while we were able to detect some relatively normal appearing Evans blue dye-positive myofibers that did not express caspase-3, all caspase-3 positive fibers were at least weakly positive for Evans blue dye. These observations suggest a sequence of molecular events in dystrophic muscle in which an initial membrane damaging event allows the uptake of large extracellular molecules such as Evans blue dye, which is subsequently followed by up-regulation of caspase-3 and loss of ARC expression. Although apoptosis and necrosis represent different mechanisms of cell death, both may be intertwined. The ultimate fate of a cell may depend on the relative intensity of the secondary triggers and the energy status of the cell (29–31).

Nonetheless, we were not able to determine from these studies whether ARC over-expression failed to inhibit apoptosis in mdx muscles, or whether the apoptotic pathways regulated by ARC do not contribute to the dystrophic phenotype in mdx mice. The multiple functions of dystrophin and the DGC make it difficult to determine the extent that signaling failures contribute to muscle fiber death in mdx muscles. However, it was shown that altered signaling leads to impaired modulation of {alpha}-adrenergic vasoconstriction and functional ischemia in dystrophic muscle (17). While overexpression of ARC in the heart has been shown to protect from myocardial ischemia, overexpression of ARC in mdx skeletal muscles clearly did not alter the dystrophic phenotype (39,40). This observation could be explained by the fact that the dystrophic pathology results from alterations in multiple molecular pathways that together contribute to muscle fiber death. While the restoration of one signaling pathway may not be sufficient to ameliorate dystrophic pathology, a combination of treatments targeting mechanical, immunological and signaling pathways might be more effective (6,16,17,20,49,63,64).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Isolation of mouse ARC
The full length human ARC cDNA sequence was used to screen the expressed-sequence tag (EST) database of GenBank to find related sequences in mice. A 480 bp EST clone was identified. PCR primers based on this EST sequence, together with vector primers, were used to amplify the 5' and 3' ends of ARC directly from a mouse muscle cDNA library (41). The 5' and 3' ends of ARC were sequenced and cloned by recombinant PCR to generate a full-length mouse ARC cDNA.

Northern blot analysis
Full-length mouse ARC cDNA was labeled with radioactive {alpha}(17)-dCTP by random priming using a commercial kit (Rediprime kit, Amersham) and hybridized to a mouse multiple tissue northern blot (Clonetech) according to the manufacturer's instructions.

Chromosomal localization
A 144 bp intron located between base pair 475/476 of the mouse cDNA sequence was amplified from genomic C57BL/6J and M. spretus DNA. Direct sequence comparison of the PCR products revealed that bp 88 was different between the C57BL/6J and M. spretus strain, which leads to an ApoI restriction fragment length polymorphism. The intron was PCR amplified from the Jackson Laboratory interspecific backcross panel (C57BL/6JEixSPRET/Ei)F1xSPRET/Ei, called TJL BSS, followed by an ApoI digestion, which identified different haplotypes (65). The backcross haplotype data were analyzed by staff at the Jackson Laboratory. Raw data were obtained from the www.jax.org/resources/documents/cmdata.

ARC transgenic mice
The human ARC cDNA tagged at the C-terminus with a FLAG epitope (32) was cloned into an expression vector containing the human {alpha}-skeletal actin promoter, a splice acceptor from the SV40 VP1 intron and tandem SV40 polyadenylation signals (45). The human ARC expression construct was injected into SJL/J F2xSJL/J F2 embryos, and positive F0 mice were identified by PCR screening using specific primers for the expression construct. Two positive F0 mice were back-crossed onto the C57Bl/10J and mdx background. Further studies used primarily the line with the most uniform expression levels. For all studies, transgene negative/mdx and transgene negative/C57BL/10J littermates were used as controls.

Immunohistochemistry
Quadriceps and diaphragm muscle were frozen in liquid nitrogen cooled OCT embedding medium (Tissue-Tek) and stored at –80°C until use. Frozen sections were cut to a 5 µm thickness and mounted on silane-coated slides. For histochemical analysis, sections were fixed in methanol and stained with Gills no. 3 hematoxylin and eosin–phloxine (Fisher Scientific).

For immunostaining, sections were blocked with 1% gelatin in KPBS, then incubated for 2 h with the primary antibody diluted in KPBS with 0.2% gelatin and 1% donkey or goat serum. The following antibodies were used: anti-FLAG 1 : 500 (Sigma), anti-ARC 1 : 200 (Cayman), TRITC labeled anti-cytochrome oxidase subunit V 1 : 500 (Molecular Probes) and anti-caspase-3 1 : 500 (Pharmingen). After several washes the sections were stained with a FITC conjugated goat anti-rabbit (Alexa 488, Molecular Probes) secondary antibody for another hour, washed and mounted with Vectashield mounting media (Vector Laboratories). Sections were visualized with a Nikon E1000 microscope connected to a Spot-2 CCD camera. ImagePro software (Media Cybernetics) was used to determine the percentage of centrally located nuclei.

Western blot analysis
Quadriceps and diaphragm muscle were frozen in liquid nitrogen and stored at –80°C until use. Samples were homogenized (OMNI 5000) in lysis buffer (120 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 1 mM Na3VO3, 10 mM Na4P2O7, 10 mM NaF, 1% Triton, 10% glycerol, 50 mM Tris–HCl pH 8.0) plus protease inhibitor cocktail (Roche). The total amount of protein was determined using the Coomassie Plus Protein Assay Reagent (Pierce). Proteins were separated on a 15% polyacrylamide gel and immunoblotted with anti-FLAG 1/5000 (Sigma) and anti-ARC 1/2000 (Cayman) antibodies.

Evans blue assay
Evans blue dye (10 mg/ml in PBS) was injected into the tail vein of 6-week-old mdx, transgenic/mdx and C57BL/10J mice (50 µl/10 g body weight). After 3 h, mice were euthanized and quadriceps muscle were frozen in liquid nitrogen cooled OCT embedding media. Frozen, 5 µm thick sections were analyzed for Evans blue uptake by fluorescence microscopy (7).


   ACKNOWLEDGEMENTS
 
We thank Gabriel Nunez for kindly providing the human ARC cDNA, and Stephen Hauschka for helpful discussions. This work was supported by a grant from the National Institutes of Health (R01 AR40864) to J.S.C.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 2062215363; Fax: +1 2066168272; E-mail: jsc5{at}u.washington.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Monaco, A.P., Neve, R.L., Colletti-Feener, C., Bertelson, C.J., Kurnit, D.M. and Kunkel, L.M. (1986) Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature, 323, 646–650.[CrossRef][Medline]

  2. Emery, A.E. (1993) Duchenne muscular dystrophy—Meryon's disease. Neuromusc. Disord., 3, 263–266.[CrossRef][Medline]

  3. Ervasti, J.M. and Campbell, K.P. (1991) Membrane organization of the dystrophin–glycoprotein complex. Cell, 66, 1121–1131.[CrossRef][ISI][Medline]

  4. Karpati, G., Carpenter, S. and Prescott, S. (1988) Small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy. Muscle Nerve, 11, 795–803.[CrossRef][ISI][Medline]

  5. Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M. and Sweeney, H.L. (1993) Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl Acad. Sci. USA, 90, 3710–3714.[Abstract/Free Full Text]

  6. Dellorusso, C., Crawford, R.W., Chamberlain, J.S. and Brooks, S.V. (2001) Tibialis anterior muscles in mdx mice are highly susceptible to contraction-induced injury. J. Muscle Res. Cell Motil., 22, 467–475.[CrossRef][ISI][Medline]

  7. Straub, V., Rafael, J.A., Chamberlain, J.S. and Campbell, K.P. (1997) Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J. Cell Biol., 139, 375–385.[Abstract/Free Full Text]

  8. Brooks, S.V. (1998) Rapid recovery following contraction-induced injury to in situ skeletal muscles in mdx mice. J. Muscle Res. Cell Motil., 19, 179–187.[CrossRef][ISI][Medline]

  9. Lynch, G.S., Rafael, J.A., Chamberlain, J.S. and Faulkner, J.A. (2000) Contraction-induced injury to single permeabilized muscle fibers from mdx, transgenic mdx, and control mice. Am. J. Physiol. Cell Physiol., 279, C1290–1294.[Abstract/Free Full Text]

  10. Warner, L.E., DelloRusso, C., Crawford, R.W., Rybakova, I.N., Patel, J.R., Ervasti, J.M. and Chamberlain, J.S. (2002) Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin–glycoprotein complex and partially prevents dystrophy. Hum. Mol. Genet., 11, 1095–1105.[Abstract/Free Full Text]

  11. Brenman, J.E., Chao, D.S., Xia, H., Aldape, K. and Bredt, D.S. (1995) Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell, 82, 743–752.[CrossRef][ISI][Medline]

  12. Durbeej, M., Cohn, R.D., Hrstka, R.F., Moore, S.A., Allamand, V., Davidson, B.L., Williamson, R.A. and Campbell, K.P. (2000) Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E. Mol. Cell, 5, 141–151.[CrossRef][ISI][Medline]

  13. Hack, A.A., Cordier, L., Shoturma, D.I., Lam, M.Y., Sweeney, H.L. and McNally, E.M. (1999) Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proc. Natl Acad. Sci. USA, 96, 10723–10728.[Abstract/Free Full Text]

  14. Hack, A.A., Ly, C.T., Jiang, F., Clendenin, C.J., Sigrist, K.S., Wollmann, R.L. and McNally, E.M. (1998) Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin. J. Cell Biol., 142, 1279–1287.[Abstract/Free Full Text]

  15. Yang, B., Jung, D., Motto, D., Meyer, J., Koretzky, G. and Campbell, K.P. (1995) SH3 domain-mediated interaction of dystroglycan and Grb2. J. Biol. Chem., 270, 11711–11714.[Abstract/Free Full Text]

  16. Grady, R.M., Grange, R.W., Lau, K.S., Maimone, M.M., Nichol, M.C., Stull, J.T. and Sanes, J.R. (1999) Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies. Nat. Cell Biol., 1, 215–220.[CrossRef][ISI][Medline]

  17. Thomas, G.D., Sander, M., Lau, K.S., Huang, P.L., Stull, J.T. and Victor, R.G. (1998) Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle. Proc. Natl Acad. Sci. USA, 95, 15090–15095.[Abstract/Free Full Text]

  18. Sander, M., Chavoshan, B., Harris, S.A., Iannaccone, S.T., Stull, J.T., Thomas, G.D. and Victor, R.G. (2000) Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy. Proc. Natl Acad. Sci. USA, 97, 13818–13823.[Abstract/Free Full Text]

  19. Rando, T.A., Disatnik, M.H., Yu, Y. and Franco, A. (1998) Muscle cells from mdx mice have an increased susceptibility to oxidative stress. Neuromusc. Disord., 8, 14–21.[CrossRef][ISI][Medline]

  20. Disatnik, M.H., Chamberlain, J.S. and Rando, T.A. (2000) Dystrophin mutations predict cellular susceptibility to oxidative stress. Muscle Nerve, 23, 784–792.[CrossRef][ISI][Medline]

  21. Disatnik, M.H., Dhawan, J., Yu, Y., Beal, M.F., Whirl, M.M., Franco, A.A. and Rando, T.A. (1998) Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state. J. Neurol. Sci., 161, 77–84.[CrossRef][ISI][Medline]

  22. Chang, W.J., Iannaccone, S.T., Lau, K.S., Masters, B.S., McCabe, T.J., McMillan, K., Padre, R.C., Spencer, M.J., Tidball, J.G. and Stull, J.T. (1996) Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy. Proc. Natl Acad. Sci. USA, 93, 9142–9147.[Abstract/Free Full Text]

  23. Haycock, J.W., MacNeil, S., Jones, P., Harris, J.B. and Mantle, D. (1996) Oxidative damage to muscle protein in Duchenne muscular dystrophy. Neuroreport, 8, 357–361.[ISI][Medline]

  24. Tidball, J.G., Albrecht, D.E., Lokensgard, B.E. and Spencer, M.J. (1995) Apoptosis precedes necrosis of dystrophin-deficient muscle. J. Cell Sci., 108(Pt 6), 2197–2204.[Abstract]

  25. Matsuda, R., Nishikawa, A. and Tanaka, H. (1995) Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle. J. Biochem. (Tokyo), 118, 959–964.[Abstract/Free Full Text]

  26. Sandri, M., Minetti, C., Pedemonte, M. and Carraro, U. (1998) Apoptotic myonuclei in human Duchenne muscular dystrophy. Lab Invest., 78, 1005–1016.[ISI][Medline]

  27. Sandri, M., Podhorska-Okolow, M., Geromel, V., Rizzi, C., Arslan, P., Franceschi, C. and Carraro, U. (1997) Exercise induces myonuclear ubiquitination and apoptosis in dystrophin-deficient muscle of mice. J. Neuropathol. Exp. Neurol., 56, 45–57.[ISI][Medline]

  28. Tews, D.S. and Goebel, H.H. (1997) DNA-fragmentation and expression of apoptosis-related proteins in muscular dystrophies. Neuropathol. Appl. Neurobiol., 23, 331–338.[CrossRef][ISI][Medline]

  29. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. and Lipton, S.A. (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl Acad. Sci. USA, 92, 7162–7166.[Abstract/Free Full Text]

  30. Higuchi, M., Honda, T., Proske, R.J. and Yeh, E.T. (1998) Regulation of reactive oxygen species-induced apoptosis and necrosis by caspase 3-like proteases. Oncogene, 17, 2753–2760.[CrossRef][ISI][Medline]

  31. Eguchi, Y., Shimizu, S. and Tsujimoto, Y. (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res., 57, 1835–1840.[Abstract/Free Full Text]

  32. Koseki, T., Inohara, N., Chen, S. and Nunez, G. (1998) ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proc. Natl Acad. Sci. USA, 95, 5156–5160.[Abstract/Free Full Text]

  33. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. and Wang, X. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 91, 479–489.[CrossRef][ISI][Medline]

  34. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S. and Reed, J.C. (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J., 17, 2215–2223.[CrossRef][ISI][Medline]

  35. Hofmann, K., Bucher, P. and Tschopp, J. (1997) The CARD domain: a new apoptotic signalling motif. Trends Biochem. Sci., 22, 155–156.[CrossRef][ISI][Medline]

  36. Ekhterae, D., Lin, Z., Lundberg, M.S., Crow, M.T., Brosius, F.C. III and Nunez, G. (1999) ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circul Res., 85, e70–77.[ISI][Medline]

  37. Ekhterae, D., Platoshyn, O., Zhang, S., Remillard, C.V. and Yuan, J.X. (2003) Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents. Am. J. Physiol. Cell Physiol., 284, C1405–1410.[Abstract/Free Full Text]

  38. Neuss, M., Monticone, R., Lundberg, M.S., Chesley, A.T., Fleck, E. and Crow, M.T. (2001) The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function. J. Biol. Chem., 276, 33915–33922.[Abstract/Free Full Text]

  39. Chatterjee, S., Stewart, A.S., Bish, L.T., Jayasankar, V., Kim, E.M., Pirolli, T., Burdick, J., Woo, Y.J., Gardner, T.J. and Sweeney, H.L. (2002) Viral gene transfer of the antiapoptotic factor Bcl-2 protects against chronic postischemic heart failure. Circulation, 106, I212–217.[Medline]

  40. Gustafsson, A.B., Sayen, M.R., Williams, S.D., Crow, M.T. and Gottlieb, R.A. (2002) TAT protein transduction into isolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective. Circulation, 106, 735–739.[Abstract/Free Full Text]

  41. Lumeng, C., Phelps, S., Crawford, G.E., Walden, P.D., Barald, K. and Chamberlain, J.S. (1999) Interactions between beta 2-syntrophin and a family of microtubule-associated serine/threonine kinases. Nat. Neurosci., 2, 611–617.[CrossRef][ISI][Medline]

  42. Geertman, R., McMahon, A. and Sabban, E.L. (1996) Cloning and characterization of cDNAs for novel proteins with glutamic acid-proline dipeptide tandem repeats. Biochim. Biophys. Acta, 1306, 147–152.[Medline]

  43. Stoss, O., Schwaiger, F.W., Cooper, T.A. and Stamm, S. (1999) Alternative splicing determines the intracellular localization of the novel nuclear protein Nop30 and its interaction with the splicing factor SRp30c. J. Biol. Chem., 274, 10951–10962.[Abstract/Free Full Text]

  44. Schiaffino, S. and Reggiani, C. (1996) Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol. Rev., 76, 371–423.[Abstract/Free Full Text]

  45. Crawford, G.E., Faulkner, J.A., Crosbie, R.H., Campbell, K.P., Froehner, S.C. and Chamberlain, J.S. (2000) Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain. J. Cell Biol., 150, 1399–1410.[Abstract/Free Full Text]

  46. Harper, S.Q., Hauser, M.A., DelloRusso, C., Duan, D., Crawford, R.W., Phelps, S.F., Harper, H.A., Robinson, A.S., Engelhardt, J.F., Brooks, S.V. et al. (2002) Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat. Med., 8, 253–261.[CrossRef][ISI][Medline]

  47. Mukasa, T., Momoi, T. and Momoi, M.Y. (1999) Activation of caspase-3 apoptotic pathways in skeletal muscle fibers in laminin alpha2-deficient mice. Biochem. Biophys. Res. Commun., 260, 139–142.[CrossRef][ISI][Medline]

  48. Franco, A., Jr. and Lansman, J.B. (1990) Calcium entry through stretch-inactivated ion channels in mdx myotubes. Nature, 344, 670–673.[CrossRef][Medline]

  49. Chen, Y.W., Zhao, P., Borup, R. and Hoffman, E.P. (2000) Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J. Cell Biol., 151, 1321–1336.[Abstract/Free Full Text]

  50. Podhorska-Okolow, M., Sandri, M., Zampieri, S., Brun, B., Rossini, K. and Carraro, U. (1998) Apoptosis of myofibres and satellite cells: exercise-induced damage in skeletal muscle of the mouse. Neuropathol. Appl. Neurobiol., 24, 518–531.[CrossRef][ISI][Medline]

  51. Kuznetsov, A.V., Winkler, K., Wiedemann, F.R., von Bossanyi, P., Dietzmann, K. and Kunz, W.S. (1998) Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse. Mol. Cell Biochem., 183, 87–96.[CrossRef][ISI][Medline]

  52. Krajewski, S., Krajewska, M., Shabaik, A., Wang, H.G., Irie, S., Fong, L. and Reed, J.C. (1994) Immunohistochemical analysis of in vivo patterns of Bcl-X expression. Cancer Res., 54, 5501–5507.[Abstract/Free Full Text]

  53. Veal, E.A. and Jackson, M.J. (1996) Expression of programmed cell death-related genes in dystrophic mdx and control mouse muscle. Biochem. Soc. Trans., 24, 486S.[Medline]

  54. Sandri, M. and Carraro, U. (1999) Apoptosis of skeletal muscles during development and disease. Int. J. Biochem. Cell Biol., 31, 1373–1390.[CrossRef][ISI][Medline]

  55. Dominov, J.A., Houlihan-Kawamoto, C.A., Swap, C.J. and Miller, J.B. (2001) Pro- and anti-apoptotic members of the Bcl-2 family in skeletal muscle: a distinct role for Bcl-2 in later stages of myogenesis. Dev. Dyn., 220, 18–26.[CrossRef][ISI][Medline]

  56. Li, P.F., Li, J., Muller, E.C., Otto, A., Dietz, R. and von Harsdorf, R. (2002) Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC. Mol. Cell, 10, 247–258.[CrossRef][ISI][Medline]

  57. Qin, Z.H., Wang, Y., Kikly, K.K., Sapp, E., Kegel, K.B., Aronin, N. and DiFiglia, M. (2001) Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation. J. Biol. Chem., 276, 8079–8086.[Abstract/Free Full Text]

  58. Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Prevost, M.C., Alzari, P.M. and Kroemer, G. (1999) Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med., 189, 381–394.[Abstract/Free Full Text]

  59. Screaton, G.R., Caceres, J.F., Mayeda, A., Bell, M.V., Plebanski, M., Jackson, D.G., Bell, J.I. and Krainer, A.R. (1995) Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J., 14, 4336–4349.[ISI][Medline]

  60. Kemp, G.J., Taylor, D.J., Radda, G.K. and Rajagopalan, B. (1992) Bio-energetic changes in human gastrocnemius muscle 1-2 days after strenuous exercise. Acta Physiol. Scand., 146, 11–14.[ISI][Medline]

  61. Braun, U., Paju, K., Eimre, M., Seppet, E., Orlova, E., Kadaja, L., Trumbeckaite, S., Gellerich, F.N., Zierz, S., Jockusch, H. et al. (2001) Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice. Biochim. Biophys. Acta, 1505, 258–270.[Medline]

  62. Spencer, M.J., Walsh, C.M., Dorshkind, K.A., Rodriguez, E.M. and Tidball, J.G. (1997) Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity. J. Clin. Invest., 99, 2745–2751.[ISI][Medline]

  63. Spencer, M.J., Montecino-Rodriguez, E., Dorshkind, K. and Tidball, J.G. (2001) Helper [CD4(+)] and cytotoxic [CD8(+)] T cells promote the pathology of dystrophin-deficient muscle. Clin. Immunol., 98, 235–243.[CrossRef][ISI][Medline]

  64. Lynch, G.S., Hinkle, R.T., Chamberlain, J.S., Brooks, S.V. and Faulkner, J.A. (2001) Force and power output of fast and slow skeletal muscles from mdx mice 6–28 months old. J. Physiol., 535, 591–600.[Abstract/Free Full Text]

  65. Rowe, L.B., Nadeau, J.H., Turner, R., Frankel, W.N., Letts, V.A., Eppig, J.T., Ko, M.S., Thurston, S.J. and Birkenmeier, E.H. (1994) Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mamm. Genome, 5, 253–274.[CrossRef][ISI][Medline]


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