Human Molecular Genetics Advance Access originally published online on December 12, 2006
Human Molecular Genetics 2007 16(4):355-363; doi:10.1093/hmg/ddl453
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Nuclear sequestration of 
-sarcoglycan disrupts the nuclear localization of lamin A/C and emerin in cardiomyocytes
1 Department of Medicine, Section of Cardiology, 2 Committee on Developmental Biology and 3 Department of Human Genetics, The University of Chicago, Chicago, IL 60637, USA
* To whom correspondence should be addressed at: 5841 S. Maryland, MC6088, Chicago, IL 60637, USA. Tel: +1 7737022672; Fax: +1 7737022681; Email: emcnally{at}medicine.bsd.uchicago.edu
Received October 4, 2006; Accepted November 29, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sarcoglycan is a membrane-associated protein complex found at the plasma membrane of cardiomyocytes and skeletal myofibers. Recessive mutations of
-sarcoglycan that eliminate expression, and therefore function, lead to cardiomyopathy and muscular dystrophy by producing instability of the plasma membrane. A dominant missense mutation in the gene encoding -sarcoglycan was previously shown to associate with dilated cardiomyopathy in humans. To investigate the mechanism of dominantly inherited cardiomyopathy, we generated transgenic mice that express the S151A -sarcoglycan mutation in the heart using the 
-myosin heavy-chain gene promoter. Similar to the human -sarcoglycan gene mutation, S151A -sarcoglycan transgenic mice developed dilated cardiomyopathy at a young age with enhanced lethality. Instead of placement at the plasma membrane, -sarcoglycan was found in the nucleus of S151A -sarcoglycan cardiomyocytes. Retention of -sarcoglycan in the nucleus was accompanied by partial nuclear sequestration of ß- and 
-sarcoglycan. Additionally, the nuclear-membrane-associated proteins, lamin A/C and emerin, were mislocalized throughout the nucleoplasm. Therefore, the S151A -sarcoglycan gene mutation acts in a dominant negative manner to produce trafficking defects that disrupt nuclear localization of lamin A/C and emerin, thus linking together two common mechanisms of inherited cardiomyopathy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Inherited dilated cardiomyopathy is genetically heterogeneous with over 25 genes contributing to the development of this disease. There is pathologic overlap between the cardiomyopathy and the muscular dystrophies in that both are progressive, degenerative disorders with loss of cardiomyocytes or skeletal myofibers, respectively. Muscular dystrophies caused by sarcoglycan mutations usually occur as autosomal recessive disease with variable severity of skeletal muscle and cardiac muscle pathology (1). Dominantly inherited mutations were described in the gene encoding
-sarcoglycan, and among these was a mutation in which the serine at amino acid position 151 was mutated into an alanine, S151A (2). Affected individuals with S151A -sarcoglycan developed typical dilated cardiomyopathy in the second to third decades leading to death or cardiac transplantation.
Sarcoglycan is part of the larger dystrophin glycoprotein complex (DGC) and can be biochemically isolated as a subcomplex of the DGC (3,4). Each sarcoglycan subunit is a single-pass transmembrane protein that can be phosphorylated (3), suggesting signaling functions in addition to structural functions. -sarcoglycan is a type-II transmembrane protein, and like ß- and -sarcoglycans, lacks an amino terminal signal sequence and instead anchors in the plasma membrane by virtue of the positively charged residues just before its transmembrane domain. The carboxyl terminus of -sarcoglycan, containing S151A, resides in the extracellular domain where there is at least one site for asparagine-linked glycosylation. Loss of function mutations in the genes encoding -, ß-, - and -sarcoglycans lead to instability not only of the primary mutated gene product, but these mutations also lead to instability of the entire sarcoglycan complex indicating that trafficking of this complex is codependent (5,6). The S151A -sarcoglycan mutation stands in contradistinction to other sarcoglycan gene mutations because of its unusual autosomal dominant inheritance pattern and its associated phenotype of cardiomyopathy without much, if any, skeletal muscle disease. For this reason, we studied the mechanism of cardiomyopathy produced by the S151A -sarcoglycan mutation using transgenesis.
We now modeled S151A -sarcoglycan by overexpressing this protein in the heart. We generated five independent lines of transgenic mice expressing S151A -sarcoglycan under the control of the MHC promoter (7) at 1.5, 3, 4, 6 or 7 times normal -sarcoglycan levels. We also generated a single line of mice that overexpressed wild-type -sarcoglycan at 1.5 times normal levels on a wild-type background (8). S151A -sarcoglycan transgenic mice developed dilated cardiomyopathy and cellular hypertrophy at a young age. Similar to humans, the murine cardiomyopathy was severe and reduced life expectancy. We noted that lines expressing mutant but not wild-type -sarcoglycan showed nuclear sequestration of -sarcoglycan. This aberrant trafficking also lead to the sequestration of other sarcoglycan subunits but not other membrane-associated proteins such as caveolin-3, dystrophin or connexin 43. Nuclear retention of -sarcoglycan also mislocalized lamin A/C and emerin, two nuclear-membrane-associated proteins. Mutations in lamin A/C and emerin have been associated with inherited forms of cardiomyopathy. These findings molecularly link two independent genetic mechanisms of cardiomyopathy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic mouse lines
Five independent lines of transgenic mice were generated that expressed the
-sarcoglycan S151A mutation under the control of the MHC promoter (S151A Tg+). In addition, a control line expressing wild-type -sarcoglycan under the control of the MHC promoter on a wild-type background was used (DSG Tg+) (Fig. 1A). To avoid possible toxic effects, the transgene products were not epitope-tagged. All six transgenic lines were maintained on the C57BL/6J strain. Positive founders were identified with PCR amplification from tail DNA using transgene-specific primers. Immunoblots of microsomal membrane fractions with a -sarcoglycan-specific antibody were conducted to analyze expression levels (representative immunoblot, Fig. 1B). Quantitative immunoblot analysis was used to compare total -sarcoglycan protein expression in the S151A Tg+ mutant lines relative to non-transgenic littermate (S151A Tg–) controls. This analysis showed a range of total -sarcoglycan expression from 1.5x normal in line 1 to 7x normal in line 5. S151A Tg+ line 1 had an average expression of 1.51 times S151A Tg– with a standard deviation of 0.8; line 2 had 2.94 ± 1.87; line 3 had 4.2 ± 0.55; line 4 had 5.8 ± 1.72 and line 5 had 7.02 ± 2.07 (n = 4 of each). Despite variable expression levels, all of the mutant transgenic lines demonstrated a similar phenotype with one exception; the highest expressing line, line 5, did not display enhanced early lethality and reproduced according to Mendelian ratios (see below). The line overexpressing wild-type -sarcoglycan under the control of the MHC promoter expressed at 1.5x the level of non-transgenic mice (8). An antibody specific to -sarcoglycan revealed no change in the level of -sarcoglycan expression (data not shown).
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Survival
Fewer than expected S151A Tg+ mice survived to weaning at 3 weeks of age. Table 1 shows a reduction in the expected Mendelian frequency associated with the presence of the S151A
-sarcoglycan transgene in four of the five lines consistent with prenatal or perinatal lethality. Lines 1, 3 and 4 each showed a significant reduction in the number of transgene-positive animals that survived to weaning. Line 2 could not be sustained as it bred only once, and the transgene-positive pups did not survive. Line 5 has paradoxically produced the highest percentage of transgene-positive mice despite having the highest amount of S151A -sarcoglycan expression, thus lethality did not correlate with S151A -sarcoglycan protein overexpression levels. Data compiled from all five lines indicated that survival to 3 weeks was significantly reduced (P<0.05) in transgene-bearing animals compared with non-transgenic littermates.
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In addition to perinatal lethality, seemingly healthy S151A Tg+ adult mice often died suddenly. For example, three female animals from line 3 died at only 13–16 weeks old while delivering their first litters. S151A Tg+ males were also found to die suddenly, five positive males from line 1 (1.5x) died at 18 weeks of age during cage-bedding changes. These five mice were not housed in the same cage, and wild-type control littermates that were housed with the mutants survived. Due to the lethality associated with the S151A
-sarcoglycan transgene, only one line, line 5, has survived.
Histopathology from S151A -sarcoglycan transgenic animals
All of the S151A Tg+ lines displayed dilated cardiomyopathy at a young age. An example of this is shown in Figure 2. The littermate control (S151A Tg–) and the control transgenic (DSG Tg+) hearts were not dilated even at advanced ages (data not shown) (8). Using ImageJ, minimal diameter area calculations were utilized to assess the extent of cardiomyocyte hypertrophy. The S151A Tg+ animals had a trend towards increased cardiomyocyte diameter with an increase in the variability of cardiomyocyte size when compared with littermate controls (mutant average diameter 28.07 ± 12.11 versus 23.38 ± 5.14 µm for controls). In addition, heart weight to body weight ratios of 12-week-old mice were greater in mice expressing the S151A -sarcoglycan transgene compared with littermate control mice (5.29 ± 1.14 mg/g, n = 26 versus 4.54 ± 0.5 mg/g, n = 19, P = 0.012). The severity of these phenotypes including dilation, hypertrophy and heart weight to body weight ratios, was not correlated to transgene expression levels. Focal fibrosis, as assessed by Masson Trichrome staining, a common feature seen in -sarcoglycan null hearts (9) was not evident in any of the S151A Tg+ hearts (data not shown).
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Nuclear retention of the sarcoglycan complex
Normally the sarcoglycan complex, including
-sarcoglycan, is found at the plasma membrane. Immunofluorescence microscopy with an anti--sarcoglycan antibody on hearts from each of the mutant S151A -sarcoglycan lines revealed specific retention of -sarcoglycan within cardiomyocyte nuclei. An example of this is shown in Figure 3. In S151A -sarcoglycan Tg+ hearts, -sarcoglycan immunostaining was found concentrated with DAPI signals within nuclei of cardiomyocytes (Fig. 3, middle row of panels). The anti--sarcoglycan antibody detects expression from the transgene as well as the endogenous -sarcoglycan locus. In S151A -sarcoglycan Tg+ hearts, -sarcoglycan was preferentially concentrated in the nucleus with a fraction of -sarcoglycan immunoreactivity trafficking to the plasma membrane. We noted a reduction in -sarcoglycan immunoreactivity at the plasma membrane when comparing S151A Tg+ hearts with S151A Tg– or DSG Tg+ hearts. This reduction suggests that -sarcoglycan trafficking to the plasma membrane is impaired. We quantified the reduction of -sarcoglycan immunostaining at the plasma membrane finding that cardiomyocytes expressing S151A -sarcoglycan contained less -sarcoglycan at the sarcolemma than non-transgenic controls [11.06 ± 2.04 versus 16.01 ± 0.87 light units (LU), P = 0.001]. Non-cardiomyocyte nuclei remained free of -sarcoglycan staining consistent with the known expression pattern of endogenous -sarcoglycan and expression from the MHC promoter. Overexpression of wild-type -sarcoglycan did not produce mislocalization of -sarcoglycan (8), and non-transgenic controls expressed -sarcoglycan only at the plasma membrane (Fig. 3, bottom and top rows). Quantitation of the average color intensity along a line drawn through the nuclei revealed a statistical difference between -sarcoglycan expression in S151A -sarcoglycan cardiomyocyte nuclei and non-transgene nuclei (49.83 ± 12.56 versus 9.00 ± 1.55 LU in the control hearts, P = 0.0005). Interestingly, S151A Tg+ lines 1–4 had a higher percentage of cardiomyocytes nuclei with -sarcoglycan-positive nuclei than S151A line 5. The average percentage of -sarcoglycan-positive cardiomyocytes nuclei fields was 91.6 ± 8.4% compared with line 5 with an average of 63.6 ± 17% (P<0.0001, n = at least eight 40x microscopic fields counted per line). S151A Tg+ line 5, with the lower percentage of -sarcoglycan containing cardiomyocytes, was the only line that could be maintained.
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We also found retention of
-sarcoglycan in cardiomyocyte nuclei in all lines that expressed S151A -sarcoglycan. An example of this is shown from S151A Tg+ line 2 in Figure 4. Every nucleus that retained -sarcoglycan also retained -sarcoglycan that distributed to both the nucleus and the plasma membrane indicating that not all -sarcoglycan was sequestered in the nucleus (Fig. 4B). - and -sarcoglycan signals were found in close proximity to propidium iodide-stained chromosomal DNA shown in red (Fig. 4E). In these plots, -sarcoglycan plasma membrane staining is illustrated by the leading edge of the profile. By immunoblot, no change in -sarcoglycan was detected in microsomal fractions (data not shown). Co-staining with a ß-sarcoglycan-specific antibody revealed that ß-sarcoglycan was also partially retained in the -sarcoglycan containing nuclei in all mutant transgenic lines. The colocalization of -, ß-sarcoglycans and DAPI, indicating chromosomal content, is illustrated by the ImageJ line profile (Fig. 4F). The retention of the sarcoglycan components in the nuclei by the S151A -sarcoglycan transgene was specific for the sarcoglycan components. Imaging the intracellular location of other membrane-associated proteins such as dystrophin, caveolin 3 or connexin 43 showed normal positioning at the plasma membrane (data not shown).
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Nuclear membrane protein disruption by mutant
-sarcoglycanFigure 5 shows the distribution of
-sarcoglycan and the nuclear membrane protein lamin A/C in control littermates (left) and an S151A -sarcoglycan heart [line 5 (7x), middle column] and in the wild-type -sarcoglycan transgene (DSG Tg+, right panels). In most S151A Tg+ cardiomyocyte nuclei, -sarcoglycan was distributed uniformly throughout the nucleus, overlapping with the signal from DAPI-stained chromosomes (Fig. 5F–J). Interestingly, in all cases, lamin A/C localized with -sarcoglycan in S151A Tg+ cardiomyocyte nuclei, whether distributed throughout the nucleus or in a smaller number of nuclei, at the nuclear periphery. In nuclei of interstitial fibroblasts, where the mutant -sarcoglycan is not expressed, lamin A/C remained in its normal nuclear membrane location (asterisk, Fig. 5G). The distribution of lamin A/C relative to -sarcoglycan and DAPI are illustrated in the plots for S151A Tg– (Fig. 5E) and S151A Tg+ cardiomyocytes (Fig. 5J) from the nuclei bisected by the lines in Fig. 5D and I, respectively. In normal nuclei, there is a concentration of lamin A/C at the nuclear periphery seen as the two green peaks in the plot (Fig. 5E). In contrast in S151A Tg+ cardiomyocyte nuclei, lamin A/C was seen distributed throughout the nucleus along with -sarcoglycan (Fig. 5J). The average intensities of bisecting lines were compared between mutant nuclei (n = 6) and non-transgenic control nuclei (n = 6) and indicated an increased amount of lamin throughout the nucleus in the S151A -sarcoglycan mutant cardiomyocytes compared with controls (82 ± 31.48 versus 32.17 ± 7.17 LU, P = 0.0107).
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Emerin localization was also disrupted in S151A Tg+ hearts. In all S151A Tg+ mutant cardiomyocyte nuclei, emerin was found in close proximity to
-sarcoglycan, either throughout the nucleus (arrow, Fig. 6F) or at the nuclear periphery (arrowhead, Fig. 6F). In the S151A Tg– cardiomyocyte nuclei, emerin stains the nuclear membrane exclusively (Fig. 6B). Figure 6E and J indicate the difference in emerin localization of the S151A Tg– and S151A Tg+ nuclei indicated by the bisecting lines in Figure 6D and I, respectively. In S151A Tg+ mutant nuclei, emerin is localized throughout the nucleus in close proximity to -sarcoglycan and the chromosomes (Fig. 6J, green staining). The average intensities of bisecting lines were compared between mutant nuclei (n = 6) and non-transgenic control nuclei (n = 6) and indicated an increased amount of emerin throughout the nucleus in S151A -sarcoglycan mutant cardiomyocytes (151.42 ± 35.62 versus 63.67 ± 4.93 LU, P = 0.0016). Overexpression of wild-type -sarcoglycan did not disrupt emerin localization (data not shown).
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The sarcolemmal membrane is intact
Loss of the sarcoglycan complex from recessive loss of function mutations or from dystrophin mutations produces instability of the plasma membrane and increased membrane permeability. This membrane destabilization can be visualized by Evan's blue dye uptake exclusively in the damaged fibers in skeletal muscle and cardiomyocytes (10). We analyzed five S151A Tg+ hearts, two from line 1 (1.5x) and three from line 5 (7x), and found no dye positive fibers indicating an absence of plasma membrane leakage and a distinct mode of pathology compared with
-sarcoglycan hearts (data not shown).
Recent evidence has shown that sarcolemma disruption reduces the amount of full-length ß-dystroglycan within the plasma membrane of cardiomyocytes from the cardiomyopathic hamster (11). This reduction of ß-dystroglycan has also been observed in skeletal tissue of patients with Duchenne muscular dystrophy and sarcoglycan gene mutations but not in other myopathies (12). It has been hypothesized that the absence of the dystrophin and sarcoglycan complex exposes a cleavage site causing degradation of ß-dystroglycan (11). We examined the amount of full-length ß-dystroglycan expression as a means of assessing plasma membrane integrity of the S151A Tg hearts. The amount of ß-dystroglycan was found to be normal in S151A Tg+ cardiomyocytes from line 5 (96%) by immunoblot of microsomal fractions (Fig. 7). In support of the previous work, we found partial reduction of ß-dystroglycan in cardiomyocytes from -sarcoglycan null (82%) and mdx (69%) mice. Because of mortality, the other four lines could not be analyzed for ß-dystroglycan expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The data presented here reveal a novel model for cardiomyopathy that links two genetically distinct pathways for heart failure. We show that a serine to alanine mutation at amino acid position 151 of
-sarcoglycan causes an autosomal dominant cardiomyopathy. The engineered murine disease closely approximates the human disease (2). Like humans carrying this gene mutation, mice expressing this gene demonstrate cardiomyopathy and sudden death at a young age.
Five S151A -sarcoglycan transgenic-positive mouse lines were generated and compared with a transgenic line expressing wild-type -sarcoglycan and C57BL/6J littermate controls. No epitope tag was used in these constructs to avoid toxicity related to foreign protein expression. Wheeler et al. (8) described the phenotype of wild-type -sarcoglycan transgene expressed from the -myosin heavy-chain promoter. In this work, the Scgd–/– phenotype was rescued by the overexpression of -sarcoglycan from a transgene with no detectable adverse consequences. In contrast, all five of the S151A -sarcoglycan Tg+ mutant lines displayed cardiomyopathy as illustrated by increased heart weight to body weight ratio and reduced survival. Each S151A Tg+ line retained -sarcoglycan in the nucleus, partially retained ß- and -sarcoglycans in the nucleus, and disrupted lamin A/C and emerin localization. All five of the lines displayed these phenotypes equally with the exception that we could maintain line 5 and that this line did not display perinatal lethality and has survived. There was no correlation between the level of total -sarcoglycan expression and these phenotypes, and interestingly line 5, the surviving line, expressed the highest level of -sarcoglycan. We detected a smaller percentage of cardiomyocyte nuclei sequestering -sarcoglycan in S151A line 5 and believe that this explains the survival of this line. The mechanism by which this line spares nearly one-third of cardiomyocyte nuclei is unclear. The difference in the level of expression in lines 4 and 5 is minimal, and therefore it is unlikely to explain the molecular differences in sarcoglycan processing. The S151A -sarcoglycan mutation produces a myopathic process through a dominant negative mechanism that disrupts the nucleus and spares the sarcolemmal membrane. S151A -sarcoglycan perturbs trafficking of endogenous -sarcoglycan and to a lesser extent - and ß-sarcoglycans. The reduced sarcolemmal expression of -sarcoglycan in the S151A cardiomyocytes is consistent with the likely mislocalization of native, non-mutant -sarcoglycan. Alternatively, the endogenous -sarcoglycan may be targeted for degradation because of the reduced availability of sarcoglycan-binding partners. This degradation pathway is assumed to occur in null mutations where the mutation of one member leads to loss of the other complex members (5,6). The partial retention of some -sarcoglycan at the plasma membrane is sufficient to maintain integrity of the plasma membrane since we did not see dye uptake into these cardiomyocytes. Heterozygous gene carriers with loss of function alleles of Sgcd and heterozygous Sgcd mice do not display cardiomyopathy, indicating that 50% of protein levels is sufficient for normal function. The partial retention of the sarcoglycan complex at the sarcolemma and the normal level of full-length ß-dystroglycan in the S151A -sarcoglycan cardiomyocytes are consistent with an intact plasma membrane.
Humans with the S151A -sarcoglycan mutation did not have notable skeletal muscle involvement and instead displayed only cardiomyopathy (2). Thus, suggesting nuclear trafficking of the sarcoglycan complex may differ between cardiac and skeletal muscle in humans. Alternatively, nuclear retention of the sarcoglycan complex may not be pathologic in skeletal muscle. In S151A -sarcoglycan transgenic mice, expression of mutant sarcoglycan was directed and detected only in cardiomyocytes, so the absence of skeletal muscle pathology is expected. We did express mutant S151A -sarcoglycan in the skeletal muscle cell line C2C12 under the cytomegalovirus promoter and did not observe -sarcoglycan within the nucleus even after differentiation of the myoblasts into myotubes (data not shown). Interestingly, the orthologous S151A -sarcoglycan gene mutation was recently generated in Drosophila from the cardiac-specific tin-c promoter (13). This system also revealed a mutant phenotype in the heart. In the fly, there is only a single sequence encoding a //
-sarcoglycan-like protein, whereas in mammals these functions have diverged to three independent genes: -, - and -sarcoglycans.
The means of nuclear -sarcoglycan retained in S151A -sarcoglycan cardiomyocytes may involve unconventional mechanisms since this mutation does not create a novel nuclear localization signal nor are nuclear localization signals found within -sarcoglycan. Nuclear membrane retention has been noted for the common torsin A mutant that leads to dystonia (14). The deletion of glutamic acid 302/303 produces mislocalization of torsin A from the endoplasmic reticulum lumen to the nuclear membrane (15), and it has been suggested that this pathology overlaps with what may occur in the nuclear membrane diseases (16). Of interest, the mutant torsin A protein recruits wild-type torsin A to the nuclear envelope and is inherited in an autosomal dominant manner, both characteristics of the S151A -sarcoglycan mutation. In the case of S151A -sarcoglycan, we not only see sequestration of -sarcoglycan but also note retention of ß- and -sarcoglycans. An assembly model for the sarcoglycans has been demonstrated in primary skeletal muscle and in heterologous cell culture, respectively (17,18). In this model, - and ß-sarcoglycans first assemble in the endoplasmic reticulum followed by the addition of -sarcoglycan in the proximal Golgi apparatus and then the addition of -sarcoglycan in the distal endoplasmic reticulum (18). The data suggest that in the S151A -sarcoglycan mutant hearts there is either retroactive transport of partially assembled complex back to the nucleus or partial assembly of -, ß-and -sarcoglycans within the nucleus.
Mutations in the gene encoding lamins A and C are a relatively common cause of cardiomyopathy that may or may not be associated with skeletal muscle disease (19). Mutations in the gene encoding emerin are more rare but display a similar spectrum of striated muscle disease phenotypes as LMNA gene mutations (20). Thus, it is likely that the pathology from S151A -sarcoglycan is similar to LMNA gene mutations since S151A -sarcoglycan leads to mislocalization of lamins A and C as well as emerin. The mechanism by which lamin A/C mutations lead to disease may be multifactorial, and a mechanical deficit has been shown in fibroblasts from lamin A/C null mice (21). A similar mechanism may be occurring with S151A -sarcoglycan, but alternative pathologic mechanisms may also contribute to this. The nuclear membrane has many functions in post-mitotic cells such as cardiomyocytes including the regulation of gene expression and nuclear transport. Therefore, defects in these nuclear functions may be adversely affected by sarcoglycan protein retention. The finding that a plasma membrane protein defect can interfere with nuclear membrane function now links two independent cardiomyopathic pathways and indicates that sarcoglycan gene mutations may mechanistically overlap with lamin A/C gene mutations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic mouse lines
The S151A
-sarcoglycan construct was generated via PCR with the following mutant primers: 718G>Tf; GGA AAA TTG CTC TTT gCT GCa GAT GAC AGT G, 718G>Tr; C ACT GTC ATC tGC AGc AAA GAG CAA TTT TCC. The mutation is indicated by lowercase; in addition, a silent PstI restriction enzyme site mutation, underlined, was included in the primers. The resultant product was ligated to the -myosin heavy-chain promoter (22). No epitope tag was used in these constructs to avoid toxicity related to foreign protein expression. The transgene was digested with XhoI to liberate the insert for pronuclear injection as described previously (23). Transgenic animals carrying the wild-type -sarcoglycan driven by the -myosin heavy-chain promoter (wild-type -sarcoglycan transgene) were described previously (8). All transgene animals were created on a mixed 129/SVEM+/J and C57BL/6J background and then bred to C57BL/6J for multiple generations providing identical C57BL/6J strains during comparisons. All comparisons were performed with 12- to 16-week-old mice; representative data from both genders is shown due to no gender differences. All genotypes were identified with PCR and transgene-specific primers. Animals were housed, treated and handled in accordance with University of Chicago's Institutional Animal Care and Use Committee guidelines, the Animal Welfare Act regulations and the NIH Guide for the Care and Use of Laboratory Animals.
Immunoblot analysis
Heart microsomal preparations were prepared as described (24,25). Protein was quantified with the Bio-Rad kit (Hercules, CA, USA). The microsomal fractions were separated on a 12% SDS–PAGE gel and transferred to Immobilon P membranes (Millipore, Bedford, MA, USA). Equivalent loading was verified with 0.1% Ponceau in 5% acetic acid staining. Immunoblotting was preformed as described (6), using monoclonal ß-dystroglycan (Novocastra Laboratories, Newcastle upon Tyne, UK), or polyclonal - and -sarcoglycan antibodies (18). Four samples per line were analyzed by immunoblot and the averages and standard deviations are reported. Phosphoimager quantitation was performed on a Storm 860 (Molecular Dynamics, Piscataway, NJ, USA).
Histology and immunofluorescence microscopy
Hematoxylin and eosin staining was performed on paraffin-embedded hearts. All mouse hearts were cross-sectioned at one-quarter of the heart length from the apex. This allowed direct comparison of lumen size and ventricular wall thickness. Immunofluorescence microscopy was performed on the same heart regions to allow for direct comparisons. Animals were injected with 10 ug/ml Evans blue dye in PBS 15 h prior to harvest. Immunofluorescence microscopy was performed as described previously (26). Harvested hearts were mounted in OCT (Triangle Biomedical Sciences, Durham, NC, USA) medium and flash frozen in liquid-nitrogen-cooled isopentane. Seven-micrometer sections were prepared at –20°C, fixed for 5 min in ice-cold methanol, rinsed with PBS and blocked in 1% FBS diluted in PBS. Primary antibodies were diluted in the block solution and incubated on the sections for 2 h at room temperature. The following primary antibodies were utilized: polyclonal -sarcoglycan (18) at 1 : 50, monoclonal -sarcoglycan at 1 : 50, ß-sarcoglycan at 1 : 50, emerin (Novocastra Laboratories, Newcastle upon Tyne, UK) at 1 : 250 and lamin A/C (Abcam, Cambridge, MA, USA) at 1 : 50. Chromosomes were illuminated using Vectashield with DAPI or propidium iodide (Vector Laboratories, Burlingame, CA, USA) or Sytox Green (Molecular Probes, Eugene, OR, USA). Images were acquired on a Zeiss Axiocam (Carl Zeiss Inc., Thornwood, NY, USA) and recorded and processed using AxioVision 2.0 (Carl Zeiss), Adobe Photoshop and ImageJ 1.32j. ImageJ 1.32j was also utilized to demonstrate localization and relative quantity of proteins in the nuclei along a bisecting line (http://rsb.info.nih.gov/ij/). To quantify cardiomyocyte-specific nuclear proteins, camera exposure times were set manually at identical levels and then the LU within the nucleus along a nuclear bisecting line were averaged. For each quantitation, six mutant hearts were compared with six non-transgenic littermate hearts. Cardiomyocyte hypertrophy was quantified using the minor axis, from the best-fit ellipse algorithm from ImageJ 1.32j, to compute area (http://rsb.info.nih.gov/ij/). Cardiomyocyte hypertrophy was calculated on a total of 10 mutant animals (line 1 n = 3, line 2 n = 3 and line 4 n = 4) and compared with two non-transgenic littermates. Heart weight to body weight ratio was measured on all transgenic lines (line 1 n = 5, line 2 n = 5, line 3 n = 2, line 4 n = 6 and line 5 n = 8) and compared with a total of 19 non-transgenic littermates.
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ACKNOWLEDGEMENTS |
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This work was supported by the NIH 61322 (EMM), the Burroughs Wellcome Fund, the Heart Research Fund and the Muscular Dystrophy Association.
Conflict of Interest statement. None declared.
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REFERENCES |
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  M. J. Allikian, G. Bhabha, P. Dospoy, A. Heydemann, P. Ryder, J. U. Earley, M. J. Wolf, H. A. Rockman, and E. M. McNally Reduced life span with heart and muscle dysfunction in Drosophila sarcoglycan mutants Hum. Mol. Genet., December 1, 2007; 16(23): 2933 - 2943. [Abstract] [Full Text] [PDF]
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