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Human Molecular Genetics Advance Access originally published online on September 12, 2007
Human Molecular Genetics 2007 16(23):2933-2943; doi:10.1093/hmg/ddm254
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Reduced life span with heart and muscle dysfunction in Drosophila sarcoglycan mutants

Michael J. Allikian1, Gira Bhabha1, Patrick Dospoy1, Ahlke Heydemann1, Pearl Ryder1, Judy U. Earley1, Matthew J. Wolf3, Howard A. Rockman3,4,5 and Elizabeth M. McNally1,2,*

1 Department of Medicine and 2 Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA and 3 Department of Medicine, 4 Department of Cell Biology and 5 Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27110, USA

* To whom correspondence should be addressed: Department of Medicine, Section of Cardiology, University of Chicago, 5841 S. Maryland, MC6088, Chicago, IL 60637, USA. Tel: +1 7737022679; Fax: +1 7737022681; Email: emcnally{at}uchicago.edu

Received July 24, 2007; Accepted August 24, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
In humans, genetically diverse forms of muscular dystrophy are associated with a disrupted sarcoglycan complex. The sarcoglycan complex resides at the muscle plasma membrane where it associates with dystrophin. There are six known sarcoglycan proteins in mammals whereas there are only three in Drosophila melanogaster. Using imprecise P element excision, we generated three different alleles at the Drosophila {delta}-sarcoglycan locus. Each of these deletions encompassed progressively larger regions of the {delta}-sarcoglycan gene. Line 840 contained a large deletion of the {delta}-sarcoglycan gene, and this line displayed progressive impairment in locomotive ability, reduced heart tube function and a shortened life span. In line 840, deletion of the Drosophila {delta}-sarcoglycan gene produced disrupted flight muscles with shortened sarcomeres and disorganized M lines. Unlike mammalian muscle where degeneration is coupled with ongoing regeneration, no evidence for regeneration was seen in this Drosophila sarcoglycan mutant. In contrast, line 28 was characterized with a much smaller deletion that affected only a portion of the cytoplasmic region of the {delta}-sarcoglycan protein and left intact the transmembrane and extracellular domains. Line 28 had a very mild phenotype with near normal life span, intact cardiac function and normal locomotive activity. Together, these data demonstrate the essential nature of the transmembrane and extracellular domains of Drosophila {delta}-sarcoglycan for normal muscle structure and function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The dystrophin glycoprotein complex (DGC) is found at the plasma membrane of striated muscle and is the target of genetic mutations that lead to human muscular dystrophy and cardiomyopathy. DGC consists of dystrophin, dystroglycan, sarcoglycans, sarcospan, dystrobrevins and syntrophins (1,2). It is nonuniform in its distribution along the sarcolemma and is concentrated at the costameres over the Z disk in skeletal muscle (3,4). Dystrophin is a wholly cytoplasmic protein that binds directly to the transmembrane protein dystroglycan and to cytoskeletal actin providing a mechanically strong linkage from the sarcolemma to the Z disk (5). Loss of function mutations in the genes encoding dystrophin and {alpha}-, ß-, {gamma}- and {delta}-sarcoglycan lead to X-linked and autosomal recessive forms of muscular dystrophy and cardiomyopathy. In common to these genetic forms of muscle degeneration is the loss of the sarcoglycan complex, a key component for maintaining muscle membrane integrity along with dystroglycan and dystrophin.

Within the DGC, the sarcoglycan component is composed of a series of single pass transmembrane proteins (6,7). There are six sarcoglycans, {alpha}–{zeta}, in mammals; the major sarcoglycan complexes found in mammalian skeletal and cardiac muscle are {alpha}-, ß-, {gamma}- and {delta}-sarcoglycan (7). Mutations in the gene encoding {gamma}-sarcoglycan produce limb girdle muscular dystrophy 2C, and many patients carry a common founder mutation that ablates expression of {gamma}-sarcoglycan (8). {delta}-Sarcoglycan gene mutations are a less frequent cause of muscular dystrophy and cardiomyopathy, but produce a similar constellation of defects as seen with {gamma}-sarcoglycan gene mutations that include both progressive muscle wasting as well as progressive cardiac dilation and dysfunction (9–11). Additionally, mutations in the {delta}-sarcoglycan gene have also been associated with inherited dilated cardiomyopathy that occurs in the absence of overt skeletal muscle disease (12). A murine model of a {delta}-sarcoglycan gene mutation, S151A, recapitulates the cardiomyopathic phenotype and demonstrates the causative nature of gene mutations for cardiac function (13). The Syrian hamster model, BIO 14.6, is a small animal model of cardiomyopathy and muscular dystrophy and is associated with a large deletion encompassing the 5' end of the hamster {delta}-sarcoglycan gene (14). Interestingly, genetic substrains of this model develop either hypertrophic cardiomyopathy or dilated cardiomyopathy (15).

Both {gamma}- and {delta}-sarcoglycans, type II transmembrane proteins, are highly related with short cytoplasmic domains that contain conserved serine/threonine or tyrosine residues that undergo phosphorylation. Muscle engineered to lack {delta}-sarcoglycan demonstrates enhanced membrane damage and loss of peak force when subject to contraction protocols (16) similar to what has been described for dystrophin mutant muscle (17–19). In contrast, muscle in which the {gamma}-sarcoglycan gene was targeted did not show enhanced membrane damage after contraction. This lack of mechanical deficit in {gamma}-sarcoglycan null muscle stands in contrast to what has been described for {delta}-sarcoglycan null muscle and is consistent with a multifunctional sarcoglycan complex in mammalian muscle where, despite sequence similarity, the loss of {delta}- and {gamma}-sarcoglycans lead to differential effects on muscle. Despite these mechanical differences, the phenotype in mice lacking {gamma}-sarcoglycan (Sgcg null) or {delta}-sarcoglycan (Sgcd null) is virtually indistinguishable. Both models display abnormal uptake of the vital tracer Evans Blue dye and both develop progressive fibrofatty infiltration of muscle (20,21). Each model displays a similar cardiomyopathic process of progressive dilation that is evident between 6 and 12 months of age (21). As in these murine models, most of the {gamma}- and {delta}-sarcoglycans' primary gene mutations destabilize the protein and the nonmutant sarcoglycan subunits are secondarily destabilized. The trafficking of {gamma}- and {delta}-sarcoglycan differ where {delta}-sarcoglycan forms the core of the sarcoglycan subunit during assembly and {gamma}-sarcoglycan is added as a later component (16,22). However, destabilizing this assembly at multiple levels leads to the phenotype of progressive cardiac and skeletal muscle dysfunction.

Sequence comparisons have been used to identify the orthologous genes encoding the DGC that are present in Drosophila melanogaster (23) and suggest conservation of function in invertebrate muscle. In the D. melanogaster genome, there are only three sarcoglycan subunits. There is a single subunit related to {alpha}- and {epsilon}-sarcoglycans, and a single ß-sarcoglycan-like subunit. There is one single subunit that is equally related to {gamma}-, {delta}- and {zeta}-sarcoglycans and, in this report, we will refer to this as {delta}-sarcoglycan. The simpler system in Drosophila with three sarcoglycan subunits as opposed to the six in mammals may indicate that multiple functions unique to individual mammalian sarcoglycan subunits are assumed by single subunits in Drosophila muscle. We report here the generation of a Drosophila model of muscular dystrophy and cardiomyopathy associated with an allelic series of deletions that affect the {delta}-sarcoglycan locus. These {delta}-sarcoglycan-deficient flies display progressive muscle dysfunction along with defective function of the heart tube, the equivalent of human cardiomyopathy. The animals display age-dependent muscle and mobility defects as well as a shortened life span. Interestingly, we found abnormal sarcomere structure in muscle from Drosophila {delta}-sarcoglycan mutants suggesting an essential role for this transmembrane protein in sarcomere organization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Generation of Drosophila {delta}-sarcoglycan mutant
In mammals, the sarcoglycan complex of striated muscle includes {alpha}-, ß-, {gamma}- and {delta}-sarcoglycans. There are three sarcoglycan genes in the Drosophila genome and only one gene related to the {gamma}-, {delta}- and {zeta}-sarcoglycan genes in mammals. A schematic of the predicted topology of the protein encoded by the Drosophila {delta}-sarcoglycan gene is shown in Figure 1. This sequence is equally related to both mammalian {gamma}- and {delta}-sarcoglycans and encodes conserved residues including the C-terminal cysteine residues, putative sites for tyrosine phosphorylation and the site that has been shown to be glycosylated in mammalian {gamma}- and {delta}-sarcoglycans (24). To create {delta}-sarcoglycan-deficient D. melanogaster, imprecise P element excision mutagenesis was carried out on insertion line KG5430 (Bloomington Stock #13543). This line was generated as part of the BDGP gene disruption project (25) and contains a P element inserted 105 bp upstream from the start of the first exon of the Drosophila {delta}-sarcoglycan gene (Scg{delta}) in a region near promoter elements (Fig. 2). The gene encoding Drosophila {delta}-sarcoglycan is composed of six exons. Exon 1 encodes the 5' untranslated region, and exon 2 encodes the initiator methionine, the cytoplasmic domain and the transmembrane domain.


Figure 1
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  		Figure 1. Schematic of the predicted protein topology of Drosophila {delta}-sarcoglycan. Residues identical among mammalian {gamma}- and {delta}-sarcoglycans and the Drosophila sequence are shown in blue green. Those residues shared only between mammalian {gamma}-sarcoglycan and the Drosophila sequence are shown in yellow, and those between mammalian {delta}-sarcoglycan and the Drosophila sequence are shown in pink. The region in grey is unique to the Drosophila sequence.

 


Figure 2
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  		Figure 2. Generation of {delta}-sarcoglycan deletion mutants in Drosophila. (A) Imprecise P element excision was used to generate deletions that extend into the {delta}-sarcoglycan locus. PCR of genomic DNA was used to map the deletions in lines 28, 840 and 169. Products are derived from primer sets that amplify a positive control, {delta}-sarcoglycan exon 4 and {delta}-sarcoglycan exon 5. (B) Southern blotting indicates the presence of deletions. Analysis of wild-type (WT = Or–R–S or KG5430), line 28 (deleted into exon 2), line 840 (deleted through exon 4) and line 169 (deleted through the entire {delta}-sarcoglycan gene) are shown. (C) RT–PCR demonstrates that line 28 contains a deletion that extends into the cytoplasmic domain removing the extension that is present in Drosophila sarcoglycan. (D) The grey bars indicate the sizes of the deletions in lines 28, 840 and 169 based on PCR mapping and Southern blotting. The location of the P element in the genome is given and represented by name (KG5430). The positions of the primers in exons 4 and 5 primer that were used for PCR mapping breakpoints are shown. Positions of the predicted start codon (ATG) and direction of transcription, location of the transmembrane (TM) domain and region of genomic DNA used as a probe for Southern blotting are also shown. H3 indicates HindIII sites used for Southern blotting experiments. The position of the primers used for RT–PCR in C are indicated as the RT1 and RT2 primer sets.

 
We characterized three independent excision lines of varying deletion sizes that were confirmed by PCR analysis of genomic DNA and Southern blotting (Fig. 2). Line 28 contained the smallest deletion (3.9 kb) and extended into exon 2 into a region that encodes the cytoplasmic domain of the {delta}-sarcoglycan protein. RT–PCR analysis of line 28 confirmed expression of the transmembrane domain indicating a truncation of the amino terminal, cytoplasmic domain (Fig. 2). On the basis of sequence analysis of the deletion and RT–PCR products, the methionine closest to the membrane is available as a translational initiator. A second deletion line, Line 840, had ~6.5 kb deleted and extended through exon 4 of the Drosophila {delta}-sarcoglycan gene. This deletion eliminates the entire cytoplasmic domain and transmembrane domain and portions of the extracellular domains and serves as a functional null of {delta}-sarcoglycan (Fig. 2D) since no mRNA was detected by RT–PCR (Fig. 2). The third line, Line 169, contains a large deletion that extends beyond the 3' end of the {delta}-sarcoglycan gene. Mapping the 3' end of the deletion in line 169 was complicated by the presence of repeated DNA sequences (data not shown).

Using an antibody we generated that was directed against the C-terminus of {delta}-sarcoglycan, in normal muscle from the control Oregon R strain (Or-R-S), we found that the expression of {delta}-sarcoglycan was concentrated into costameric structures similar to those seen in mammalian muscle (Fig. 3). Using this antibody, we found that {delta}-sarcoglycan expression was appreciably reduced in the adult flight muscles of line 28 and was absent in line 169 (Fig. 3).


Figure 3
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  		Figure 3. Loss of {delta}-sarcoglycan protein from adult flight muscle in Drosophila mutant lines. An antibody was raised to the C-terminus of {delta}-sarcoglycan (D-{delta}sg) and was used to demonstrate the normal expression of {delta}-sarcoglycan in wild-type (Oregon R) adult Drosophila muscle. The secondary antibody used was Alexa 488-conjugated goat anti-rabbit secondary antibody [(A), (D) and (G)]. Texas Red phalloidin [shown in (B), (E) and (H)] marks sarcomeric actin, and the merged images are seen in the last column [(C), (F) and (I)]. D-{delta}sg is seen in costameric pattern in register with the sarcomeric Z band in wild-type (Or–R–S) flight muscle. D-{delta}sg staining is reduced in line 28 and absent in line 169. The scale bar represents 5 µm.

 
Abnormal heart function in {delta}-sarcoglycan deletion lines
One of the hallmarks of sarcoglycan mutations in mammals is the cardiomyopathy that derives from loss of the sarcoglycan complex in cardiomyocytes (21,26). Flies from each genotype were analyzed blinded to genotype at 7 days of age by optical coherence tomography (OCT). Data are shown for female flies (Fig. 4). Lines 169 and 840, harboring the most extensive deletions through the {delta}-sarcoglycan gene, displayed a dilated and poorly contractile heart tube with significantly enlarged end systolic and end diastolic diameters (Fig. 4). Line 28, containing the partial deletion of {delta}-sarcoglycan, did not display an increased end diastolic diameter compared with control. There was an increase in end systolic diameter for line 28, but this was not significantly different from KG5430, the P element containing line used to generate the deletion mutants. Fractional shortening was also significantly reduced in both males and females (Table 1). We did not detect a difference in the level of mRNA expression in KG5430 relative to Or–R–S (data not shown). However, modest decrease in {delta}-sarcoglycan mRNA expression, mediated by the presence of the P element and below the limit of detection of our assay, may lead the observed to subtle decrements in heart function in the P element containing parent line.


Figure 4
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  		Figure 4. Reduced cardiac function in Drosophila {delta}-sarcoglycan mutants. (A) Representative images from wild-type (top tracing), the P element containing line KG5430 (bottom tracing) and the three mutant Drosophila lines 28, 840 and 169. Markedly impaired function is seen for lines 840 and 169, the two lines with deletions spanning the Drosophila {delta}-sarcoglycan locus. Line 28 has near normal function. (B) End diastolic diameter is significantly larger in lines 840 and 169 while it is normal in line 28. End systolic diameter is larger for lines 840 and 169. End systolic diameter is increased in line 28 compared with w1118, but is not significantly different from the P element containing parent line KG5430 suggesting that subtle changes in expression mediated by the presence of the P element may mildly impair heart function in KG5430. *P < 0.01 compared with w1118 using Student’s t-test.

 


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  		Table 1. Reduced fractional shortening in Drosophila sarcoglycan mutants

 
Loss of {delta}-sarcoglycan in Drosophila results in progressive impaired locomotion
Larval locomotive ability of each Drosophila {delta}-sarcoglycan gene deletion line was assessed and was unchanged relative to controls (data not shown) indicating that locomotive ability was normal early in development. To assess mature muscle function, we employed a negative geotaxis assay to determine whether the loss of {delta}-sarcoglycan affected skeletal muscle performance in Drosophila. This assay measures the number of flies able to ascend a threshold over time (27,28). We found that at 2 days of age, neither deletion line 28 nor 840 differed significantly from line KG5430 (Fig. 5). By 20 days of age, both males and females of line 840 differed significantly in their ability to perform in the assay relative to lines 28 and KG5430 (Fig. 5). Thus, sarcoglycan loss does not affect muscle function early in development, but does impair muscle function in older animals. As in humans with sarcoglycan gene mutations, muscle function worsens with time. Because line 28 has normal function, these data indicate that the elongated cytoplasmic extension of the Drosophila {delta}-sarcoglycan sequence (shown in Fig. 1) is dispensable for normal muscle function.


Figure 5
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  		Figure 5. Locomotion is progressively impaired in Drosophila {delta}-sarcoglycan mutants. A negative geotaxis-climbing assay was used to detect that flies from line 840 climb less well with age. Data for males are shown in black and for females are shown in grey. At 2 days of age, all lines are able to climb equally well. However, by 20 days line 840 is less able to climb relative to both KG5430 and line 28. Brackets seen in the 20-day graph indicate those lines for which statistically significant differences were seen when comparing like gender-matched datasets. One-way ANOVA tests indicate that (1) P < 0.001, (2) P < 0.05 and (3) P < 0.05.

 
Defective muscle attachment in {delta}-sarcoglycan mutant flies
We analyzed muscle histology in the dorsal median indirect flight muscles of adult Drosophila (Fig. 6). In deletion line 840, areas of disrupted muscle architecture were found within and at the edges of striated muscle. Adjacent to these tears, there was an enhanced eosinophilic staining consistent with in vivo sarcomere disruption and contraction band necrosis. This abnormal muscle histology was also apparent in deletion line 28 although apparently to a degree not sufficient to produce impaired muscle function. No defects were detected in the control Or–R–S or KG5430 lines. In deletion lines 840 and 28, the muscle appeared normal at young ages (2 days, data not shown). These findings are consistent with the progressive muscle degeneration that is seen from the loss of sarcoglycan in humans.


Figure 6
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  		Figure 6. Muscle degeneration in {delta}-sarcoglycan mutant Drosophila lines. Hematoxylin and eosin staining is shown for 30–32-day-old adult flies with evidence of tears or disruptions in the flight muscle of mutant lines 28 and 840. Control lines KG5430 and Or–R–S appeared normal. Arrows seen in figures of lines 28 and 840 indicate damaged areas.

 
Adult Drosophila flight muscle was also analyzed by transmission electron microscopy (TEM). Figure 7 shows tears in a flight muscle myofiber from line 840. These images were obtained from tissues of 20-day-old animals, an age consistent with the decline seen in their locomotion. In addition, sarcomere length was significantly reduced in deletion line 840 relative to the control line Or–R–S. The mean sarcomere length ± standard deviation was 2.67 ± 0.216 µm (n = 69) for line 840 while it was found to be 3.30 ± 0.126 µm for Or–R–S (n = 41, unpaired t-test P < 0.0001). In addition to ultrastructural destruction and shortened sarcomere length, the M line in mutant sarcomeres was disorganized relative to control (Fig. 7). Figure 7 shows the normal M line that is identified by the presence of dark glycogen deposition in TEM. In comparison, the M line in line 840 was grossly uneven compared with control whereas Z lines remained unperturbed in both mutant and control.


Figure 7
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  		Figure 7. Ultrastructural defects in mutant muscle. (A) Control (Or–R–S) flight muscle shows normal straight M-lines. (B) In contrast, line 840 flight muscle displays wavy M-lines (arrowhead) and a myofiber tear (arrow). Sarcomere length is shorter in mutant muscle compared with control. The scale bar represents 1 µm.

 
Mammalian dystrophic muscle is characterized by ongoing muscle regeneration that can be seen as small fibers with centrally placed nuclei. These regenerative features were not apparent in Drosophila sarcoglycan mutant muscle suggesting that muscle regeneration does not occur in response to later onset progressive muscle degeneration. Anti-phospho-histone H3 staining showed no increase in sarcoglycan mutant muscle, indicating a lack of muscle regenerative capacity in the fly (Supplementary Material, Fig. S1). During muscle development, anti-phospho-histone H3 staining was readily apparent but was absent in mutant muscle (not shown). Similarly, there was no increase in BrdU labeling compared with wild type. This is in contrast to the increase in BrdU staining seen in mammalian mutant sarcoglycan muscle that is not present in normal muscle (Supplementary Material, Fig. S2). The comparatively short life span of Drosophila versus mammals may support a regenerative process that is only present during the window of muscle development and incapable of contributing to muscle regrowth in the face of ongoing degeneration.

Reduced life span in {delta}-sarcoglycan mutant lines
We assayed life span of the individual sarcoglycan deletion lines and found that line 840 flies live significantly shorter than controls (Table 2). Line 28 also had a statistically significant shorter life span than controls; however, the decline in life span was more marked for line 840 compared with control. The difference in life span between line 28 and line 840 was significant. The reduction in life span in line KG5430 relative to the wild-type Or–R–S line indicates that the P element that inserted very near exon 1 of {delta}-sarcoglycan (Fig. 2) may be causing a partial reduction in the amount of {delta}-sarcoglycan produced in this line. There was no difference in the fecundity of the deletion lines (data not shown).


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  		Table 2. Reduced life span in {delta}-sarcoglycan mutant Drosophila

 
Drosophila {delta}-sarcoglycan can interact with mammalian sarcoglycans
To determine whether the Drosophila {delta}-sarcoglycan sequence could substitute for mammalian {delta}-sarcoglycan, we injected plasmid encoding the Drosophila {delta}-sarcoglycan gene under the control of a cytomegalovirus (CMV) promoter into murine muscle engineered to lack murine {delta}-sarcoglycan (Sgcd null). The direct injection technique yields relatively poor expression of the injected plasmid, but does provide a small number of myofibers at the injection site that will express the injected plasmid. In Sgcd null muscle, there is no expression of mammalian {delta}-sarcoglycan because of the excision of exon 2, which like the Drosophila gene encodes the cytoplasmic and transmembrane domains (16). In these mice, there is little to no expression of the remaining sarcoglycan subunits because of protein instability and an inability to assemble any sarcoglycan complex (16). One week after injection of the Drosophila {delta}-sarcoglycan gene, we could detect myofibers expressing {alpha}-, ß-, and {gamma}- sarcoglycans at the injection site along with Drosophila {delta}-sarcoglycan (Fig. 8). Each of these antibodies showed no staining in uninjected Sgcd null muscle (not shown) or in regions remote from the injection site. Therefore, this study demonstrates that the Drosophila {delta}-sarcoglycan sequence can function in the place of mammalian {delta}-sarcoglycan for assembling the sarcoglycan complex.


Figure 8
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  		Figure 8. Drosophila {delta}-sarcoglycan can substitute for mammalian {delta}-sarcoglycan. Scgd null muscle was injected with a plasmid expressing the Drosophila {delta}-sarcoglycan sequence under the control of the CMV promoter. Uninjected wild-type muscle and uninjected Sgcd null muscle are shown on the left, stained with an anti-mammalian {delta}-sarcoglycan antibody as described previously (53). This injection technique yields a few myofibers that will express the injected plasmid. At the injection site, myofibers expressing Drosophila {delta}-sarcoglycan were noted (upper right), and this expression was sufficient to allow the remaining mammalian sarcoglycans ({alpha}-, ß- and {gamma}-sarcoglycans) to assemble at the plasma membrane.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Drosophila is currently being used as a system to model a number of human diseases including Alzheimer’s disease (29–31), cancer (32–34), seizure (35), cardiomyopathy (36–38) and myotonic dystrophy (39). These and other studies support the idea that Drosophila is a suitable system to model human disease, albeit a much simpler system owing to its reduced genetic complexity and more limited physiology. Using D. melanogaster, we have successfully generated an invertebrate model of cardiomyopathy and muscular dystrophy that mirrors the degenerative process seen in humans with mutations in the orthologous {gamma}- or {delta}-sarcoglycan genes. The mutant fly lines were generated by P element excision of a neighboring transposon, and we show that deletion of the {delta}-sarcoglycan gene has detrimental effects on the flight muscles of adult flies, heart function and life span. The less severe phenotype in line 28 suggests that partial expression of {delta}-sarcoglycan can ameliorate both cardiac and skeletal muscle phenotypes that arise as a result of loss of this protein. The partial expression in line 28 reflects reduced total protein as well as expression of a protein that is predicted to lack the full cytoplasmic domain but retain the transmembrane and extracellular domains. Since {delta}-sarcoglycan, like {gamma}- and {zeta}-sarcoglycans, is a type II transmembrane protein, only those positively charged amino acids immediately proximal to the transmembrane domain are required for proper insertion in the plasma membrane. Moreover, on the basis of expression studies in heterologous cell types using mammalian sequences, a protein completely lacking the cytoplasmic domain is capable of assembling with other sarcoglycan subunits (22).

Recent data on Drosophila mutants lacking the large dystrophin isoforms found an increase in neurotransmitter release (40). Mutants in the Drosophila dystroglycan gene lead to early lethality similar to what was reported for targeted gene disruption of the mammalian dystroglycan gene (41,42). In early Drosophila development, dystroglycan is required to establish cell polarity in both epithelial cells and the oocyte. It was also shown that a loss of dystroglycan in Drosophila follicle cells causes a loss of dystrophin in these cells (43), and likewise by removing dystrophin the amount of dystroglycan is reduced. Therefore, the molecular linkage between dystroglycan and dystrophin is conserved in the fly. A recent study confirmed that interaction, and showed that the loss of either dystroglycan or dystrophin in Drosophila muscle, generated by RNA interference, results in a progressive muscular dystrophy phenotype (44). Dystroglycan loss has also been shown to cause a muscular dystrophy phenotype in zebrafish (45). Most recently, a zebrafish model of congenital muscular dystrophy was generated with a mutation in the gene encoding laminin {alpha}2, demonstrating conservation of the extracellular portion of the DGC (46). Interestingly, this model showed detachment of the muscle from the membrane similar to what was noted in our fly sarcoglycan gene mutations. Although it has never been shown that the sarcoglycan complex directly binds laminin {alpha}2, the similarity of these findings suggests that sarcoglycans participate directly or indirectly in attachment to the extracellular matrix.

The reduced sarcomere length in Drosophila {delta}-sarcoglycan deletion mutants has not been previously noted in models of sarcoglycan mutations in mammals. It has been suggested that reduced sarcomere length may be a protective adaptation (47) since shorter sarcomeres may produce lower force and therefore produce less muscle damage. Alternatively, the muscle fiber disruptions could be theorized to cause an increase in the amount of intracellular calcium that results in a hypercontraction and sarcomere shortening. The disorganized M line seen in Drosophila sarcoglycan mutants suggests an additional adaptation and supports an essential role for the extracellular matrix and costameric structures in determining both sarcomere length and structure. This feature has also not been specifically noted in human or mammalian sarcoglycan mutant muscle. In mammalian muscle, titin is a giant protein that spans from the Z band to the M line and is thought to be a molecular ruler for the sarcomere (48). Titin may be positioned to mediate costameric disruption that could, in turn, affect the M line.

The muscle degenerative phenotype in the fly causes reduced mobility as is characteristic of humans with sarcoglycan mutations. This phenotype will lend itself to suppressor screens. There was a notable absence of enhanced regeneration in Drosophila sarcoglycan mutants, suggesting that the satellite cells found in mammalian muscle and responsible for repair in mature myofibers are not active in adult Drosophila. Because these Drosophila sarcoglycan mutants display the degenerative aspects of the muscular dystrophy phenotype without evidence for ongoing regeneration, it is likely that suppressive mutants would be those that stabilized the plasma membrane of muscle in the absence of the DGC. The reduction in life span seen in Drosophila sarcoglycan mutants also mirrors effectively what is seen in humans and can also be used to test interventions to improve cardiac and muscle degenerative disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Drosophila stocks and genetics
All stocks used in this study were obtained from the Bloomington Stock Center (Bloomington, IN; http://www.flystocks.bio.indiana.edu/). Stocks were maintained on cornmeal–agar–molasses media and kept at 25°C on 12:12 light–dark cycle. The Bloomington stock used for imprecise excision of the P element near {delta}-sarcoglycan was BL#13543 and was originally created as part of the BDGP gene disruption project (25). Standard protocols were used for performing the excision (41,49,50). Briefly, the Bloomington P element containing line 13543 was crossed to a {Delta}2–3 transposase line. F1 yellow– flies were chosen as candidate deletions, balanced, then homozygosed and deletion sizes determined by PCR and sequencing. Approximately 900 different lines were analyzed. Deletions were confirmed by Southern blotting using standard protocols.

Polyclonal antibody generation
The peptide sequence corresponding to amino acids 290–305 of the Drosophila {delta}-sarcoglycan sequence (CG14808-PA, Genbank Accession# AAG17402 [GenBank] ) was chosen for antibody production. The 16 amino acid peptide sequence used was PHVRAEPGRELRLESP. The polyclonal antibody was linked to KLH and injected into rabbits (Bethyl Laboratories, Inc., Montgomery, TX). The antibody was then affinity purified using the synthesized peptide and confirmed by ELISA.

Immunofluorescence microscopy
For immunofluorescence microscopy, flies of the indicated genotype were arranged in tissue-freezing media (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA) and placed in liquid nitrogen-cooled isopentane. Flies were positioned to obtain sagittal cryosectioning of 6–7 µM. The sections were fixed in 4% paraformaldehyde for 2 min and rinsed in phosphate-buffered saline (PBS). Blocking was performed using 5% heat-inactivated fetal bovine serum in PBS for 1 h at room temperature.

Anti-phospho-histone H3 staining was performed as described previously (51). Briefly, the cryosectioned tissue was fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. The tissues were then washed 5 x 10 min in PBST (PBS/0.1% Triton X-100), blocked for 40 min in PBST/1% BSA (PBSTB) and incubated overnight at a dilution of 1:200 with polyclonal anti-phospho-histone H3 (Ser10) antibody (Upstate). The tissues were then washed 5 x 10 min in PBST and re-blocked in PBSTB for 40 min at room temperature. Finally, the tissue was incubated with a 1:2000 dilution of goat anti-rabbit Alexa Fluor-568 and a 1:500 dilution of FITC-conjugated phalloidin (Invitrogen, Carlsbad, CA). The tissues were put through a final 5 x 10 min wash with PBST and mounted using ProLong Gold mounting media (Invitrogen). The Drosophila {delta}-sarcoglycan antibody was used at a concentration of 1:500, diluted in blocking solution and incubated overnight at 4°C. The slides were washed 3 x 10 min each in PBS on ice. Alexa Fluor-488 conjugated to goat anti-rabbit secondary antibody (Invitrogen) was diluted to 1:2500 in blocking solution and added to the slides, which were then incubated for 2 h at room temperature with Texas Red Phalloidin (Invitrogen) at a concentration of 1:500. Slides were washed as before and mounted in ProLong Gold mounting media (Invitrogen). Images were taken on a Zeiss LSM510 Confocal Microscope.

Optical coherence tomography
Optical coherence tomography analysis of Drosophila lines was performed as described previously (38). Each line was assayed at 7 days of age and the testers were blinded to genotype.

Negative geotaxis assay
The negative geotaxis assay was carried out as described previously (27), with modifications as indicated. Male and female flies of each genotype were separated and collected. Flies were kept at no more than 40 flies per vial and were transferred to fresh food every 2 days. On days when the flies were assayed, random vials from each line and gender were chosen to test. The flies were immobilized using CO2, and 10 groups of 10 flies each were placed into glass 100-ml graduated cylinders. Flies were allowed to recover for 10 min before testing. Each graduated cylinder was assayed by gentle tapping to engage the negative geotactic response of the flies. The number of flies able to cross a line 80-mm from the base of the cylinder was recorded at 10, 20, 30 and 40 s. Each assay was repeated twice. This was repeated for each gender and genotype for time points of 2, 10, 20, 30 and 40 days of age. Data were analyzed with Prism 4.0c statistics software.

Life span assay
Flies of each genotype were collected at 1-day posteclosion and separated by gender. Vials were kept to a maximum of 40 flies, and combined as flies died to keep an approximate constant density. Flies were kept at 25°C and transferred into fresh food vials every other day and the number of dead were recorded. A minimum of 700 flies was used per gender and per genotype. Kaplan–Meier statistical analysis was performed, and the data are presented as median survival with associated P-values. The percent change between individual control (C) and experimental (E) lines were calculated as ((E–C)/C) x 100.

Transmission electron microscopy
Electron microscopy was performed essentially as described (52). Briefly, whole flies were placed dorsal side up on a spot of OCT tissue freezing medium. The slide was dipped in liquid nitrogen and then bisected sagitally using a liquid nitrogen-cooled razor blade. The sections were then fixed in 2.5% gluteraldehyde in 0.1 M NaH2PO4 buffer (pH 7.4) overnight at 4°C. They were then postfixed in 2% osmium tetroxide for 2 h at room temperature, dehydrated through an ethanol series and embedded using Epon resin.

Direct injection in to sgcd muscle
The Drosophila {delta}-sarcoglycan sequence was ligated into pcDNA3.1 and plasmid was purified using a Qiagen column. One hundred micrograms of plasmid was injected midbelly in the gastrocnemius muscle of Sgcd null mice (16). One week after injection, muscle was harvested and sectioned in its entirety as described previously (53). Every fifth section was stained with an anti-{gamma}-sarcoglycan antibody. Sections adjacent to the region of {gamma}-sarcoglycan were then examined for expression of the remaining sarcoglycan subunits.

Supplemental methods for data not shown
Fecundity assay
Fecundity was assayed according to the previously published protocols (54). Briefly, groups of five virgin females and five males were placed into vials. Four replicate groups were set up per genotype. The flies were transferred to fresh food vials every other day until all parental females died. Total numbers of resulting F1 progeny from each line were counted.

Larval locomotion assay
Larval locomotion for each genotype was performed exactly as described previously (27). Briefly, wandering third instar larvae were collected and tested individually on a 1.5% sucrose/agarose plate with 0.5-cm gridlines marked on it. The number of lines crossed by the larvae in 5 min was recorded, and the number of larvae used was between 13 and 18. One-way ANOVA analysis of the data was done using InStat (Graph Pad).

BrdU incorporation in muscle
Incorporation of 5-bromo-2-deoxyuridine (BrdU, Sigma) was done as follows. Adult Or–R–S and line 840 males and females were cultured on standard fly media vials augmented with 200 µl of 6 mg/ml BrdU plus 20% sucrose. Flies were cultured at 25°C and transferred to new media every 2 days for 8 days to achieve maximal labeling. Flies were frozen at 38 days of age, cryosectioned and stained as outlined above using the anti-BrdU monoclonal antibody at 1:100 (BD Biosciences). For BrdU incorporation into the mouse, a 3-month-old {delta}-sarcoglycan mutant animal was intraperitoneally injected with 50 mg/kg BrdU in PBS. The animal was sacrificed after 2 h and the quadriceps muscle was harvested and snap frozen in liquid nitrogen-cooled isopentane. Cryosections were cut at 7 µm, and the tissue was fixed in 4% paraformaldehyde. The sections were then washed three times for 10 min each in PBS. The tissue was then subjected to 2 M HCl for 20 min at 37°C. The slides were washed as discussed before and then blocked in blocking solution (5% heat inactivated fetal bovine serum/PBS). The tissue was incubated with anti-BrdU antibody (1:100) in blocking solution for 1 h at room temperature and washed as discussed above. Goat anti-mouse Alexa 488 (Invitrogen) was used at 1:2000 diluted in blocking buffer. The slides were washed again and mounted in ProLong Gold mounting media (Invitrogen).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.

Conflict of Interest statement. None declared.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
M.J.A. was supported by the Muscular Dystrophy Association. E.M.M. was supported by the NIH (HL61322) and the Muscular Dystrophy Association.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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