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Human Molecular Genetics Advance Access originally published online on March 9, 2005
Human Molecular Genetics 2005 14(8):1029-1040; doi:10.1093/hmg/ddi095
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Muscle-specific BCL2 expression ameliorates muscle disease in laminin {alpha}2-deficient, but not in dystrophin-deficient, mice

Janice A. Dominov1,2,*, Amanda J. Kravetz1, Magdalena Ardelt1, Christine A. Kostek1, Mary Lou Beermann1 and Jeffrey B. Miller1,2

1Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA and 2Department of Neurology, Harvard Medical School, Boston, MA 02115, USA

* To whom correspondence should be addressed. Tel: +1 6176587739; Fax: +1 6179721761; Email: dominov{at}bbri.org

Received December 18, 2004; Revised February 17, 2005; Accepted February 28, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To examine the role of apoptosis in neuromuscular disease progression, we have determined whether pathogenesis in dystrophin-deficient (mdx) and laminin {alpha}2-deficient (Lama2-null) mice is ameliorated by overexpression of the anti-apoptosis protein BCL2 in diseased muscles. The mdx mice are a model for the human disease, Duchenne muscular dystrophy (DMD), and the Lama2-null mice are a model for human congenital muscular dystrophy type 1A (MDC1A). For these studies, we generated transgenic mice that overexpressed human BCL2 under control of muscle-specific MyoD or MRF4 promoter fragments. We then used cross-breeding to introduce the transgenes into diseased mdx or Lama2-null mice. In mdx mice, we found that overexpression of BCL2 failed to produce any significant differences in muscle pathology. In contrast, in the Lama2-null mice, we found that muscle-specific expression of BCL2 led to a several-fold increase in lifespan and an increased growth rate. Thus, BCL2-mediated apoptosis appears to play a significant role in pathogenesis of laminin {alpha}2 deficiency, but not of dystrophin deficiency, suggesting that therapies designed to ameliorate disease by inhibition of apoptosis are more likely to succeed in MDC1A than in DMD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the genes that encode dystrophin and laminin {alpha}2 cause the human neuromuscular diseases Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy type 1A (MDC1A), respectively (reviewed in 1Go,2Go). Both diseases are characterized by progressive muscle weakness, loss of muscle fibers and premature death. However, in other respects, pathology is distinct in the two diseases, for example, regeneration of diseased myofibers is much more robust in DMD than in MDC1A. Disease progression in DMD and MDC1A depends on the type of mutation, with mutations that lead to complete loss of dystrophin or laminin {alpha}2 function causing more severe diseases than with mutations that lead to partial loss of function (3Go–5Go).

Despite the understanding of the molecular genetics of DMD and MDC1A, the molecular and cellular mechanisms that underlie the loss of muscle mass in these diseases are not fully understood. One possibility is that apoptosis plays a significant role in pathogenesis, because histological studies have found signs of apoptosis in dystrophin-deficient and laminin {alpha}2-deficient muscles (reviewed in 6Go,7Go).

Pathogenesis in dystrophin-deficient and laminin {alpha}2-deficient muscles can be studied in mouse models. For example, mdx mice lack dystrophin and are used as models of DMD. Skeletal muscles in mdx mice appear relatively normal until ~2.5–3 weeks after birth, at which time evidence of apoptosis in muscle cells is first seen (8Go,9Go). Soon thereafter, mdx muscles show widespread necrosis of muscle fibers and infiltration of immune system cells including macrophages and T cells (10Go–12Go). Dystrophic myofibers degenerate and are rapidly replaced by satellite cells, which become activated to divide and then to repair or replace muscle fibers. These regenerated myofibers can be identified by their characteristic centrally located nuclei. In mice, these regenerated fibers, unless stressed by exercise (13Go,14Go), are relatively stable and undergo very slow degeneration/regeneration with little impact on lifespan. Satellite cell depletion ultimately occurs in aged mdx muscles (15Go–17Go).

Several mouse lines with mutations in the Lama2 gene encoding laminin {alpha}2 have been described, including both spontaneous and targeted mutations (18Go). Mice that lack laminin {alpha}2 show severe muscle loss, poor regeneration and a greatly shortened lifespan. A role for apoptosis in pathology of laminin {alpha}2 deficiency has been suggested by histological and in vitro studies. Kuang et al. (19Go) found that regenerating myofibers in muscles of Lama2-null mice exhibited relatively frequent signs of apoptosis. The myotubes formed from cells of laminin {alpha}2-deficient mouse myogenic cell lines die soon after formation in culture, but this death can be prevented by BCL2 (20Go–22Go). Finally, humans who lack laminin {alpha}2 expression similarly exhibit severely impaired muscle function, inflammatory pathology, shortened lifespan and muscle degeneration with evidence of apoptotic myofiber death (23Go–25Go).

Recently, two studies have extended the previous histological studies by experimentally examining the role of apoptosis in the pathology of mdx or Lama2-null mice. A transgenic approach was used to study mdx muscles that overexpress ARC (apoptosis repressor with caspase recruitment domain), a muscle-specific inhibitor of apoptosis (26Go). ARC has been suggested to interfere with both caspase-8-dependent and Bax-dependent apoptosis pathways (27Go,28Go). However, overexpression of ARC did not alter pathogenesis in mdx muscles (26Go). In the study of Lama2-null mice, on the other hand, both inactivation of the pro-apoptotic Bax and overexpression of the anti-apoptotic BCL2 from a MyoD promoter fragment significantly ameliorated Lama2-null disease progression (29Go).

Although these two recent studies suggested that apoptosis might be important in Lama2-null, but not in mdx, disease progression, the two studies used different experimental interventions, thus making it difficult to compare conclusions about the relative roles of apoptosis in the two diseases. In this study, therefore, we examined both mdx and Lama2-null mice using the same experimental interventions designed to inhibit apoptosis. We used two transgenes to overexpress BCL2 in mdx and Lama2-null specifically in skeletal muscles. BCL2 is not present at significant levels in normal muscle (30Go–32Go). From one transgene, BCL2 was expressed from a MyoD promoter fragment, which is active in both proliferating myoblasts and myofibers (33Go). We had previously shown that this transgene ameliorates Lama2-null pathology (29Go). From the other transgene, BCL2 was expressed from a MRF promoter fragment, which is expressed only in myofibers (34Go). With in vitro studies, we first found that overexpression of BCL2 from both of these transgenes delays the onset of apoptosis in muscle cells. Both transgenes also increased lifespan and ameliorated pathology in Lama2-null mice, but failed to affect mdx pathology. These results show that BCL2-mediated apoptosis pathways play a significant role in Lama2-null, but not in mdx, pathology. Therapeutic interventions to inhibit apoptosis may thus not be of benefit in DMD, but could be of considerable benefit in MDC1A.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Muscle-specific expression of BCL2 in transgenic mice
To directly test the function of BCL2 in myopathology, we use a transgenic mouse approach to overexpress human BCL2 (hBCL2) protein in diseased skeletal muscles and we then assessed whether transgene expression affected muscle cell survival and pathogenesis in vitro and in vivo. Two different transgenes were used for muscle-specific BCL2 expression. One transgene, termed MRF4-hBCL2, that we prepared and describe here for the first time, used an 8.5 kb fragment of the rat MRF4 promoter, which is expressed only in multinucleate myofibers (34Go) to drive muscle-specific expression (Fig. 1A). The other transgene, termed MyoD-hBCL2, was described previously (29Go) and used a 7 kb mouse MyoD promoter fragment, which is expressed in both proliferating myoblasts and multinucleate myofibers (33Go). We generated independent mouse lines from three MRF4-hBCL2 and two MyoD-hBCL2 founder mice, each of which contained a single transgene integration site determined by Southern blot analysis. Multiple founder lines were tested in various studies and results were consistent among founder lines.



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  		Figure 1. Transgenic expression of BCL2 specifically in skeletal muscles. (A) A DNA construct was prepared in which a fragment of the rat MRF4 promoter controlled expression of human BCL2 (hBCL2) cDNA. Arrows indicate primer binding sites for genotyping. This construct was used to produce transgenic mice expressing hBCL2 specifically in skeletal muscles. (B) Expression of transgenic hBCL2 was detected in limb muscle protein extracts (25 µg per lane) from MRF4-hBCL2 transgenic animals [Tg (+)], using a human BCL2-specific antibody (Ab-1). (C) hBCL2 protein was only observed in skeletal muscles of transgenic mice and not in heart or other tissues.

 
Antibodies specific for human BCL2 were used to examine transgene expression in tissues. The human BCL2 was expressed only in the skeletal muscles of transgenic mice and not in other tissues or in muscles of non-transgenic littermates (Fig. 1B and C) (29Go). Analysis of RNA expression using northern blots confirmed expression of transgenes in muscles of transgenic animals (data not shown). Muscles from transgenic mice were immunostained with a human BCL2-specific antibody to determine location of expression within muscle tissues. Both MRF4-hBCL2 and MyoD-hBCL2 muscles exhibited a low level, slightly granular staining pattern throughout the myofibers, consistent with a localization within the cytoplasm and possibly in association with mitochondria, as would be expected for endogenous BCL2 (data not shown) (35Go). Although there was occasionally some variation in intensity among fibers, no clear fiber-type differences were consistently observed.

Mice expressing MRF4-hBCL2 and MyoD-hBCL2 transgenes exhibited normal gross morphology, behavior and fertility. Therefore, the level of protein expressed in these animals was not detrimental to their overall physiology and did not give rise to uncontrolled growth or tumors.

MRF4-hBCL2 and MyoD-hBCL2 transgenes promote survival of muscle cells in vitro
To test whether the hBCL2 expressed in muscle cells was functional as an apoptotic inhibitor, primary myoblast cultures from MRF4-hBCL2 and MyoD-hBCL2 mice were evaluated for their response to staurosporine (STS), a protein kinase inhibitor that induces apoptotic death through the mitochondrial pathway. Limb muscles from individual transgenic animals [Tg(+)] or non-transgenic littermates [Tg(–)] were digested, and cell populations were purified to generate myogenic cell cultures typically containing >95% myogenic cells. Cells were allowed to differentiate for 2 days to promote myotube formation and expression of transgenes in the differentiated cells. To induce apoptosis, STS [or dimethyl sulfoxide (DMSO) as a solvent control] was added, and individual cultures were analyzed at various time points for evidence of apoptosis.

Apoptosis in response to STS treatment was delayed in muscle cultures derived from both MRF4-hBCL2 and MyoD-hBCL2 mice relative to wild-type sibling cultures (Fig. 2). Treatment of differentiated wild-type muscle cultures with 50 or 100 nM STS resulted in the appearance of the cleaved, active form of caspase-3 within 3 h, whereas the amount of activated caspase-3 was negligible in MRF4-hBCL2 transgenic cultures during the first 6 h of treatment, becoming apparent only by 12 h (Fig. 2A). Desmin expression, probed on the same western blots, served as an indicator of protein loading on gels, but was reduced in 12 h samples presumably because of degradation as cell death became extensive. Similar results showing delayed appearance of active caspase-3 were observed in muscle cultures of myogenic cells derived from MyoD-hBCL2 mice (data not shown).



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  		Figure 2. Inhibition of muscle cell apoptosis by expression of hBCL2 transgene. Staurosporine (STS) was added to induce apoptosis in differentiated myotube cultures derived from limb muscles of transgenic mice [Tg(+)] or non-transgenic littermates [Tg(–)], as described in Materials and Methods. (A) Expression of active caspase-3 was delayed in STS-treated myotube cultures from MRF4-hBCL2 transgenic mice when compared with that of non-transgenic mice. Western blots were analyzed using an antibody specific for the cleaved, active form of caspase 3, as well as an antibody specific for desmin to monitor protein loading. (B) The proportion of myotubes binding to FITC-VAD, which binds to multiple active caspases, was reduced in MRF4-hBCL2 transgenic cultures when compared with that of non-transgenic cultures. (C) The proportion of myotubes that bound Annexin V, a marker for cell surface PS, was reduced in MyoD-hBCL2 transgenic cultures when compared with that of non-transgenic cultures.

 
In separate experiments, caspase activation was monitored by cell staining with fluorescent FITC-VAD-FMK, a pan-caspase binder and inhibitor. Consistent with active caspase-3 results, the proportion of myotubes with detectable amounts of active caspases in response to STS treatment was lower in MRF4-hBCL2 transgenic cultures after 3 h than that in cultures of cells from non-transgenic littermates (Fig. 2B). Similarly, fewer FITC-VAD(+) myotubes were observed in STS-treated MyoD-hBCL2 transgenic cells than that in non-transgenic controls (data not shown).

We also examined the appearance of phosphatidylserine (PS) on the cell surface, which is an additional characteristic of cells in the early stages of apoptosis, using FITC-labeled Annexin V that binds to PS. Fewer myotubes in MyoD-hBCL2 transgenic cultures bound Annexin V than that in parallel wild-type cultures following exposure to STS (Fig. 2C). The decline in Annexin V binding at later time points reflects the death of cells in these cultures, as evident by an increase in membrane permeability to propidium iodide (PI) as previously shown (29Go). MRF4-hBCL2 transgenic myotube cultures also demonstrated delays in PS expression and PI uptake relative to wild-type cultures (data not shown).

Using multiple indicators of apoptosis, therefore, we demonstrated that the hBCL2 expressed in MRF4-hBCL2 and MyoD-hBCL2 transgenic muscles was functional and capable of inhibiting apoptotic death.

MRF4-hBCL2 and MyoD-hBCL2 transgenes expression does not significantly alter pathology in mdx muscle
To determine whether BCL2 overexpression in skeletal muscles would diminish mdx pathology, we crossed the MRF4-hBCL2 and MyoD-hBCL2 transgenic mice with mdx mice to generate mdx-affected animals (mdx/mdx or mdx/y, because the mdx locus is X-linked), which also expressed the MRF4-hBCL2 or MyoD-hBCL2 transgene. These Tg(+) mdx animals were compared with non-transgenic mdx littermates to determine whether the elevated expression of hBCL2 in Tg(+) animals had any significant effects on muscle pathology.

Immunoblotting confirmed that mdx muscles that carried either the MRF4-hBCL2 or MyoD-hBCL2 transgene contained substantially elevated human BCL2 levels, which were expressed from the transgene (Fig. 3). We also found that expression of the endogenous mouse BCL2 (mBCL2) was slightly elevated in mdx muscle tissue when compared with wild-type muscle that does not express detectable levels of this protein. The modest level of endogenous mouse BCL2 could have arisen because of expression within affected muscle fibers, in infiltrating macrophages or other immune system cells, or to a combination of both.



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  		Figure 3. Expression of endogenous and transgenic BCL2 protein in dystrophin-deficient mdx limb muscle. Limb muscle protein (100 µg per lane) from 4-week-old mdx-affected animals, with or without hBCL2 transgenes expressed, was analyzed on western blots using antibodies that specifically recognized mouse BCL2 (mBCL2) or human BCL2 (hBCL2). Normal thymus extract (Thy) was used as a positive control for mBCL2. Endogenous mBCL2 was nearly undetectable in wild-type muscles (WT) and was slightly elevated in all mdx samples, whereas the transgenic hBCL2 was found only in transgenic animals.

 
Expression of the MRF4-hBCL2 or MyoD-hBCL2 transgene did not alter overall growth of mdx-affected mice. Analysis of transgenic and non-transgenic mdx-affected mice at 4 weeks through 24 months of age revealed no significant differences in body mass because of hBCL2 expression (P > 0.05 for all pairwise comparisons, t-test) (Fig. 4). The masses of individual muscles (e.g. soleus and tibialis anterior) were also unaffected by transgene expression (data not shown).



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  		Figure 4. Muscle-specific expression of hBCL2 in mdx-affected mice does not alter animal growth. The mean body mass of mdx-affected mice (open bars), MRF4-hBCL2(+);mdx-affected (shaded bars) or MyoD-hBCL2;mdx-affected mice (black bars) at ~4 weeks (26–30 days), 10–11 weeks (70–80 days) and 18–24 months is shown. There were no significant differences (P>0.05, t-test) in mdx body mass because of muscle-specific hBCL2 transgene expression at any age studied (n, number of individual animals of each genotype).

 
Muscle tissues from transgenic and non-transgenic mdx-affected littermates at 3.5–5 weeks of age were examined for the extent of fiber regeneration by determining the proportion of centrally nucleated fibers (CNF). Additionally, the degree of macrophage infiltration, evident by the number of cells staining with Mac-1 antibody, was assessed as a secondary index of pathology, because significant macrophage infiltration occurs during the degeneration/regeneration process in these muscles.

CNF were evident in all mdx muscles studied (gastrocnemius, soleus, tibialis anterior, diaphragm, quadriceps) from both Tg(+) and Tg(–) mdx-affected animals (Figs 5A and 6). Macrophage infiltration was also evident in muscles of both Tg(+) and Tg(–) mdx-affected animals (Figs 5B and 6). The proportion of CNF within the mdx muscles was not significantly altered by expression of either the MRF4-hBCL2 or MyoD-hBCL2 transgene (Fig. 6). Although the proportion of CNF was slightly less in the MyoD-hBCL2 transgenic mice than that in the Tg(–) mdx animals, these differences did not reach statistical significance (P > 0.05, Mann–Whitney non-parametric test). The number of Mac-1(+) infiltrating macrophages also did not vary significantly among any of the Tg(+) or Tg(–) mdx-affected animals (Fig. 6). In addition, analysis of soleus muscles from 4-week-old Tg(+) or Tg(–) mdx-affected animals indicated that there were also no effects of hBCL2 expression on total muscle fiber number. The mean fiber number (±SE) in MyoD-hBCL2;mdx-affected soleus had 840±94 fibers (n=4), whereas Tg(–) mdx-affected soleus had 841±79 fibers (n=3) (not significant, P=0.98, t-test). We also observed no evidence of fiber-type proportion differences among these animals (data not shown).



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  		Figure 5. Pathology of dystrophin-deficient mdx muscle is not altered by expression of hBCL2. (A) H&E stained limb muscles sections from ~4-week-old non-transgenic [Tg(–)] mdx-affected mice and their hBCL2-expressing siblings indicated that the overall pathology and number of centrally nucleated regenerated muscle fibers were not significantly altered because of Tg expression. Bar=100 µm. (B) Similar muscle sections immunostained with an antibody that recognizes infiltrating macrophages (Mac-1) showed that the number of macrophages (arrows) was similar in hBCL2 transgenic and non-transgenic tissues.

 


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  		Figure 6. Quantitative analysis of pathology in mdx muscles with and without hBCL2 transgenes expressed. (A) The proportion of centrally nucleated regenerated fibers (CNF) and (B) the number of Mac-1 (+) cells were determined in several muscles from mdx-affected animals and hBCL2 transgenic mdx siblings. No statistically significant differences in these values because of transgene expression were observed. Black bars represent MyoD-hBCL2;mdx-affected mice, shaded bars represent MRF4-hBCL2(+);mdx-affected mice and open bars represent non-transgenic mdx-affected mice (n, number of individual animals scored).

 
As a further test of the role of BCL2-family-mediated pathways in mdx pathology, we also used cross-breeding to prepare mdx mice that lacked the pro-apoptosis protein Bax, which is normally expressed in skeletal muscle (36Go). For these experiments, mdx mice were crossed with C57BL/6-Baxtm1Sjk mice containing a targeted mutation in the Bax gene (37Go). We found that Bax inactivation did not change the proportions of regenerated CNF in mdx soleus muscles. At 6–7 weeks of age, Bax-null;mdx soleus had 46.6±4.4 SE% CNF (n=3), whereas Bax-expressing;mdx soleus had 40±10.2 SE% CNF (n=3) (P=0.59, t-test).

After the initial severe muscle degeneration and regeneration in mdx tissues, muscles continue to undergo gradual muscle turnover. Depletion of myogenic precursor cells has been postulated to occur with aging in mdx mice, as assayed by reduced proliferative capacity of muscle precursor cells from older mdx muscle (15Go). To determine whether MRF4-hBCL2 and MyoD-hBCL2 transgenes expression in mdx muscles might have preserved the proliferative capacity of myogenic precursor cells, we cultured myogenic cells from older (15.5–23-month-old) MRF4-hBCL2 and MyoD-hBCL2;mdx-affected animals at clonal density and determined the colony size distribution derived from each of these populations. As shown in Table 1, individual muscle progenitor cells from all older mdx-affected animals (mdx/mdx or mdx/y), mice produced fewer viable progeny and thus gave rise to smaller colonies than those from unaffected muscle (e.g. mdx/x or x/y). There was no difference in the average colony size between cultures derived from MRF4-hBCL2 or MyoD-hBCL2 transgenic and non-transgenic mdx-affected animals (P > 0.05, t-test, for all pairwise comparisons within litters). Thus, the detrimental effects of long-term degeneration and regeneration on satellite cell proliferation in mdx mice were not diminished by elevated levels of hBCL2.


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  		Table 1. Number of cells in colonies formed by cloned muscle progenitor cells from transgenic and non-transgenic mdx mice
 
MRF4-hBCL2 and MyoD-hBCL2 transgenes expression promotes survival and growth of laminin {alpha}2-deficient mice
To determine the role of BCL2-mediated pathways in laminin {alpha}2-deficient muscle pathology, we introduced the MRF4-hBCL2 and MyoD-hBCL2 transgenes into Lama2-null mice. The Lama2-null mice were homozygous for the dyW targeted insertion of a lacZ cassette, which disrupts normal laminin {alpha}2 expression (38Go). Only trace amounts of truncated laminin {alpha}2 are expressed in these mice (18Go), and they exhibit a severe pathology comparable to complete null mutants. Lama2-null mice appeared normal at birth, indicating that laminin {alpha}2 is not critical through fetal muscle development, but by ~10 days after birth, the Lama2-null mice are smaller than wild-type mice because of slower growth. Approximately two-third of the Lama2-null mice die by 4 weeks of age and none survive beyond 16 weeks (38Go). We compared pathology in Lama2-null mice that carried a MyoD-hBCL2 or MRF4-hBCL2 transgene with that in non-transgenic Lama2-null mice.

We found that BCL2 expression in skeletal muscles of Lama2-null mice resulted in a large increase in lifespan (Fig. 7). Of the Lama2-null mice that survived until weaning at 3 weeks, only 43% survived until 9 weeks (63 days) when mice were euthanized for analysis. In contrast, 100% of MyoD-hBCL2;Lama2-null and MRF4-hBCL2;Lama2-null mice survived at least until this age. All additional mice expressing hBCL2 that were kept past 9 weeks of age, survived for at least 13 weeks (n=8), with seven of these surviving past 4 months (an age where no Lama2-null survive) and three continuing to live past 5.5 months.



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  		Figure 7. Muscle-specific expression of hBCL2 enhances survival and growth of Lama2-null mice. (A) Survival of Lama2-null (red triangle, n=23 animals) post-weaning (at 3 weeks of age) declined dramatically over a 6 week time period, but all MRF4-hBCL2(+);Lama2-null (green square, n=7) and MyoD-hBCL2(+);Lama2-null (blue dot, n=18) mice survived during this period. (B) Transgenic hBCL2(+) Lama2-null mice were typically larger than non-transgenic Lama2-null littermates but remained smaller than WT animals. Shown are male siblings at ~3.5 weeks of age, when Tg -dependent differences in body size began to become apparent.

 
In addition to extending lifespan, the expression of MyoD-hBCL2 and MRF4-hBCL2 specifically in muscle tissue led to improved growth of Lama2-null mice, as evident in increased body mass (Figs 7 and 8). At 3 weeks of age when animals were weaned, Lama2-null mice were ~60% the size of wild-type or heterozygous Lama2+/– mice. There were no differences in body mass at this age associated with MyoD-hBCL2 or MRF4-hBCL2 expression. Beginning at ~3.5–4 weeks of age, Lama2-null mice expressing hBCL2 were significantly larger than non-transgenic Lama2-null animals. Although growth and survival of Lama2-null mice was improved as a result of muscle-specific hBCL2 expression, correction is only partial, as the mice did not achieve the size of normal healthy mice.



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  		Figure 8. Muscle-specific expression of hBCL2 leads to increased body mass of Lama2-null mice. The mean body mass of progeny from matings between Lama2+/– mice and MRF4-hBCL2(+);Lama2+/– or MyoD-hBCL2; Lama2+/– mice is shown. (A) At weaning (~3 weeks=20–24 days), there were no significant differences in Lama2-null body mass because of muscle-specific hBCL2 transgene expression. By ~4 weeks of age (27–32 days) and thereafter (~6 weeks=40–45 days), hBCL2-transgenic Lama2-null mice were larger than non-transgenic Lama2-null littermates. Open bars, non-transgenic mice; shaded bars, MRF4-hBCL2(+) mice; black bars, MyoD-hBCL2 (+) mice. Asterisk indicates statistically significant difference (P<0.05, t-test) between the indicated Tg(+);Lama2-null animals and age- and gender-matched non-transgenic Lama2-null mice (n, number of individual animals of each genotype). There was no difference in body mass between wild-type and heterozygous Lama2+/– mice regardless of Tg expression (data not shown). (B) Data from Lama2-null animals shown in (A), represented as the percentage of body mass normally found in wild-type mice of the matched age and gender.

 
Along with muscle pathology, laminin {alpha}2-deficient mice (and humans) develop abnormal motor nerve function, likely because of poor basal lamina formation by Schwann cells (39Go–42Go). Abnormal motor function becomes apparent in Lama2-null mice by 3–4 weeks after birth (e.g. reduced or unsteady movement, retraction of hindlimbs when suspended), with severe muscle contractures and hindlimb paralysis developing by ~7 weeks of age. Paralysis was not prevented by the expression of MyoD-hBCL2 or MRF4-hBCL2. Fixed muscle contractures and hindlimb muscle paralysis developed after 7 weeks in all Lama2-null mice regardless of hBCL2 transgene expression.

We also analyzed muscles from Lama2-null and MyoD-hBCL2;Lama2-null mice to determine whether muscle-specific hBCL2 expression altered the proportion of regenerated fibers or the total number of myofibers in these muscles. As expected from mdx muscle analysis, elevated levels of BLC2 protein were observed only in transgenic muscles and not in non-transgenic littermates (Fig. 9A). All Lama2-null muscles expressed only low levels of endogenous BCL2 protein. We found no significant difference in the proportion of centrally nucleated regenerated muscle fibers in muscles taken from 9-week-old MyoD-hBCL2;Lama2-null mice as compared to non-transgenic Lama2-null mice (Fig. 9B and C). Likewise, there were no significant differences in the number of Mac-1(+) infiltrating macrophages (data not shown). Thus, although hBCL2 might function to inhibit apoptosis in these muscles, the number of surviving regenerated fibers, as identified by the presence of CNF, was not altered by expression of hBCL2. In contrast, the total number of myofibers in Tg(+); Lama2-null muscles was increased when compared with non-transgenic Lama2-null muscles. For example, soleus muscles from 9-week-old MyoD-hBCL2;Lama2-null mice had significantly more total fibers (691±58 SE, n=3) than non-transgenic Lama2-null mice (482±55 SE, n=4) (P < 0.05, t-test).



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  		Figure 9. BCL2 expression and histological analysis of hBCL2 transgenic and non-transgenic laminin {alpha}2-deficient muscle. (A) Limb muscle protein (100 µg per lane) from 9-week-old Lama2-null animals, with or without MyoD-hBCL2 transgene expressed, was analyzed on western blots using antibodies that specifically recognized mouse BCL2 (mBCL2) or human BCL2 (hBCL2), with normal thymus extract (Thy) as a positive control for mBCL2. There was low-level endogenous mBCL2 expression in all Lama2-null samples, whereas higher levels of the transgenic hBCL2 were found only in transgenic animals. (B) H&E stained triceps brachii muscle sections from 9-week-old non-transgenic [Tg(–)] Lama2-null mice and their MyoD-hBCL2-expressing siblings indicated that the overall pathology was similar between transgenic and non-transgenic mice. Bar=100 µm. (C) Triceps brachii and diaphragm muscles sections from 9-week-old Lama2-null (open bars), MyoD-hBCL2 (+);Lama2-null (black bars) and Lama2+/– unaffected mice (third bar in diaphragm series) were scored for the proportion of centrally nucleated muscle fibers (CNF). There were no significant differences due to the expression of MyoD-hBCL2 (P > 0.05, t-test; n, number of animals scored).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we show that muscle-specific expression of BCL2 is sufficient to significantly ameliorate pathology in laminin {alpha}2-deficient, but not in dystrophin-deficient, mice. Apparently apoptotic myonuclei are found in many muscle diseases including the muscular dystrophies arising because of loss of dystrophin, laminin {alpha}2 or {gamma}-sarcoglycan (reviewed in 6Go,7Go). However, the extent to which apoptosis controls the progression of these diseases has not been fully understood. We have investigated whether muscle-specific overexpression of the apoptosis inhibitor BCL2 could ameliorate disease in two mouse models for degenerative muscle diseases. In dystrophin-deficient mice, which are a model for human DMD, we found no significant changes in pathogenesis due to muscle-specific overexpression of BCL2 (or because of the absence of the pro-apoptotic protein Bax). In contrast, muscle-specific overexpression of BCL2 (this study) or inactivation of Bax (29Go) led to a several-fold increase in lifespan and improved growth of Lama2-null mice. Therefore, BCL2-family-mediated apoptosis is a more significant contributor to pathogenesis in laminin {alpha}2 deficiency than that in dystrophin deficiency.

Two types of transgenic mice were produced and crossed with the disease model mice. MRF4-hBCL2 and MyoD-hBCL2 transgenic mice overexpress human BCL2 specifically in skeletal muscle fibers and not in other tissues (Fig. 1) (29Go). While the MyoD promoter fragment drives expression in both myoblasts and myofibers (29Go,33Go), the MRF promoter fragment drives expression only in mature myofibers [(34Go) and this study]. Skeletal muscle cells derived from the transgenic mice were more resistant to STS-induced apoptotic death than cells from wild-type mice (Fig. 2), demonstrating that transgenic skeletal muscle cells expressed elevated levels of functional human BCL2.

MRF4-hBCL2 and MyoD-hBCL2 transgenic mice were bred with mdx animals to generate transgenic and non-transgenic mdx-affected animals. Although transgenic mdx animals expressed BCL2 levels that were significantly higher than endogenous levels (Fig. 3), we found no significant differences in growth or extent of muscle pathology (e. g. proportion of regenerated fibers, macrophage infiltration, body mass or individual muscle mass or fiber number) due to the expression of either MRF4-hBCL2 or MyoD-hBCL2 (Figs 4–6). Analysis of muscle cell cultures from older mdx-affected animals indicated that the reduced proliferative capacity typically observed in muscle progenitor cells from older mdx muscle (15Go) was not altered by expression of either hBCL2 transgene (Table 1). Thus, consistent with the finding that overexpression of the apoptosis inhibitor ARC does not ameliorate mdx pathology (26Go), our results show that BCL2-mediated apoptosis is unlikely to be a significant contributor to pathogenesis in dystrophin-deficient muscle.

In contrast, we found that muscle-specific overexpression of BCL2 significantly increased longevity and growth of laminin {alpha}2-deficient mice (Figs 7 and 8). Thus, overexpression of BCL2 specifically in myofibers is sufficient to prolong survival of these mice. The transgenic Lama2-null mice did not attain normal body weight, however, and they developed typical Lama2-null hindlimb paralysis. The development of hindlimb paralysis, which is likely due to abnormal Schwann cell function and myelination defects in motor nerves (39Go–42Go), was expected because the muscle-specific MRF4 and MyoD promoter fragments would not drive hBCL2 expression in non-muscle cell types and thus would not affect motor nerves. In contrast, hindlimb paralysis was significantly diminished in Lama2-null mice that were also Bax-null, which is consistent with the deletion of the widely expressed Bax in motor nerve cells (29Go). Aberrant fiber sizes, shapes and fibrosis in all Lama2 –/–muscles, regardless of transgene expression, indicated that muscle degeneration was not completely overcome by overexpression of hBCL2 in these muscles.

In both disease models, muscle fiber degeneration at 3–5 weeks after birth is a prominent feature. However, regeneration of myofibers is much more extensive in mdx muscles than that in Lama2-null muscles (19Go,43Go). The percentage of regenerated (central nucleate) myofibers was not altered by overexpression of BCL2 in either mdx or Lama2-null muscles. Thus, the amelioration of Lama2-null pathology did not appear to be due to improved regeneration. However, there were significantly more myofibers in soleus muscles of MyoD-hBCL2;Lama2-null mice when compared with that of non-transgenic Lama2-null mice, suggesting some increase in overall fiber survival owing to hBCL2 expression.

Different mechanisms likely underlie the loss of myofibers in mdx and Lama2-null mice. The intracellular dystrophin protein interacts with both intracellular and membrane proteins to form a large dystrophin–glycoprotein complex (DGC), which provides a structural scaffold at the sarcolemmal membrane. Loss of dystrophin also causes loss of other proteins in the DCG. As reviewed by Petrof (44Go), diseases that disrupt the–DGC might induce muscle degeneration via pathways influenced by a number of parameters including mechanical weakness of the muscle cell membrane, inappropriate calcium flux, aberrant cell signaling, oxidative stress and ischemia. Loss of sarcolemmal integrity, as evident by permeability to Evans Blue dye and serum proteins, is a prominent feature of dystrophin-deficient muscle but not of laminin{alpha}2-deficient muscle (45Go). In addition to compromising plasma membrane structure, loss of dystrophin also appears to alter signaling in pathways critical for fiber survival and function, including neural nitric oxide synthase (nNOS), calmodulin and Grb2, among others (reviewed in 44Go,46Go,47Go). It remains to be determined how the different plasma membrane pathologies and altered intracellular signals might be affected by altered BCL2 family expression, ultimately producing the different responses of dystrophin-deficient and laminin {alpha}2-deficient muscles to apoptosis inhibition.

Disruption of signaling pathways might also contribute to pathogenesis in laminin {alpha}2-deficient muscle. Laminin {alpha}2 (as part of laminin-2 or merosin) binds to both {alpha}-dystroglycan (a component of the DGC) and {alpha}7ß1 integrin complex on the muscle membrane (48Go,49Go). In vitro studies have shown that disruption of the interactions of {alpha}-dystroglycan with laminin {alpha}2 leads to disrupted PI3K/AKT signaling and apoptotic death of myotubes and that activated AKT can restore myotube survival (50Go). Other studies have shown that disruption of laminin-2/4 binding to integrin {alpha}7ß1D leads to apoptosis involving Src tyrosine kinase signaling (p60Fyn) and a p38a SAPK-dependent pathway (51Go). In vitro, laminin 2-deficient myotubes exhibit altered levels of BCL2, BAX and several related apoptotic regulators, and these myotubes can be rescued from apoptotic death by BCL2 expression (21Go,22Go). Thus, apoptosis regulation in Lama2-null mice by BCL2 and related proteins might involve modulation of a number of signaling pathways and possibly additional apoptotic regulatory pathways.

On the basis of our results, it appears that modulation of apoptotic pathways as a therapeutic intervention might be more successful in treatment of some muscle diseases, such as MDC1A, than others such as DMD. Apoptotic death of laminin {alpha}2-deficient muscle might be regulated by different mechanisms at different phases of the pathology (e.g. early inflammatory phase and primary degeneration or later survival of regenerated fibers). It might be possible to reduce the pathology of laminin {alpha}2-deficient muscle further by inhibiting not only BCL2 -dependent pathways but also alternate apoptotic pathways or combinations of these pathways. Ultimately, critical muscle survival mechanisms could be identified and lead to strategies for the treatment of human congenital muscular dystrophy and possibly other degenerative muscle diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
hBCL2 constructs and transgenic mouse production
A cDNA clone containing the complete coding sequence for human BCL2 (hBCL2) [pB4 (52Go), American Type Culture Collection] was digested with EcoRI, and the 0.91 kb cDNA insert was ligated into two expression vectors containing promoters for muscle-specific regulatory factors. In one construct, the rat 8.5 kb MRF4 promoter-lacZ expression plasmid gMRF4a-8.5-NL [–8500 to +71 bp relative to the rat MRF4 transcription start, from Dr Stephen Konieczny (34Go,53Go)] was digested with KpnI and StuI to remove a 3.2 kb lacZ fragment, which was then replaced with the 0.91 kb hBCL2 sequence in the proper orientation for full protein expression. The resulting MRF4-hBCL2 expression construct contained a SV40 poly A sequence and was in the vector pNL (54Go). This MRF4-hBCL2 expression construct was then used to generate a MyoD-hBCL2 expression construct (29Go) by removing the 8.5 kb MRF4 promoter sequence and replacing it with a 7.0 kb murine MyoD promoter sequence (–7 kb to +95 bp relative to the mouse MyoD transcription start), isolated as a KpnI–BamHI fragment from the plasmid pKCAT2 (from Dr Stephen Tapscott (33Go)]. These MRF4 and MyoD promoters and a similar MyoD promoter have been characterized both in vitro and in vivo and have been shown to mimic normal postnatal gene expression, driving expression of reporter genes exclusively in skeletal muscle cells (29Go,34Go,55Go).

The resulting expression constructs (Fig. 1A) were used to generate transgenic mice overexpressing hBCL2 specifically in muscle tissues. Transgenic mice were produced at the National Institute of Child Health and Human Development (NICHD) Transgenic Mouse Development Facility at the University of Alabama at Birmingham. Purified linear DNA fragments of MRF4-hBCL2 or MyoD-hBCL2 were injected into mouse zygotes (B6SJL hybrid) and transferred to pseudo-pregnant recipient females to generate transgenic mice. Resulting transgenic animals were subsequently maintained in crosses with C57Bl/6J mice (Jackson Labs). Progeny were genotyped using DNA prepared from tail biopsies (56Go) and primers that span the specific promoter/hBCL2 junction (Fig. 1A). To identify the MRF4-hBCL2 transgene, we used primer 1 (MRF4 promoter) (5'-tagatgttctggggagcactagc-3') and primer 2 (hBCL2-5'end) (5'-cactcgtagcccctctgcgacag-3'), (363 bp product); for identifying the MyoD transgene, we used primer 3 (MyoD promoter) (5'- tttccagctcccgggcttttaggc-3') and primer 2 (hBCL2–5'end) (364 bp product). PCR conditions were 94°C, 5 min, 30 cycles of 94°C, 1 min; 55°C, 1 min; 72°C, 1 min, then 10 min, 72°C, 1 min. Parallel PCR reactions to detect an unrelated endogenous mouse gene [e.g. OCT 3 (57Go)] were used to verify the DNA content in samples. Three founder lines of MRF-hBCL2 mice (1-1, 3-1, 7-8), and 2 founder lines of MyoD-hBCL2 (4–5 and 6–6), were established and used for analysis, each with a with single transgene integration site as determined by Southern analysis using a probe for the SV40 polyadenylation sequence (56Go).

Animals and tissues
Mice were maintained in a pathogen-free environment and all procedures followed guidelines specified by the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the USDA Animal Welfare Act. To obtain the specific genotypes described, transgenic mice were bred with mdx mice (C57/10ScSn-Dmdmdx, Jackson Laboratory), which carry a mutation in the X-linked dystrophin gene. PCR methods were used to determine mdx genotype as described (58Go) or using a modified forward primer for determining the mdx and wild-type allele (5'-agcttaggtaaaatcaatggattta-3') along with mdx-specific and wild-type-specific reverse primers. Transgenic mice were also bred with C57BL/6-Baxtm1Sjkmice containing a targeted mutation in the Bax gene (37Go) (Jackson Laboratory) and with laminin {alpha}2-mutant mice (Lama2dy–W/+ heterozygous mice, from E. Engvall). Progeny were genotyped using primers to distinguish Bax-null or Lama2dyW and corresponding wild-type alleles as described [respectively, Jackson Laboratory or Kuang et al. (38Go) except that LacZ antisense primer was 5'-gtcgacgacgacagtatcggcctcag-3'].

Animals were euthanized with CO2, then various tissues were collected and immediately frozen on dry ice. Tissue samples for histological analysis were mounted in OCT freezing compound and frozen in liquid nitrogen-cooled isopentane. Muscle tissues to be stained for hBCL2 were fixed in Bouin's fixative for 30–45 min, then rinsed several times in PBS with an overnight rinse in PBS. Tissue was incubated in 12.5% sucrose in PBS for 30 min, overnight in 25% sucrose in PBS at 4°C and blotted then frozen in OCT with liquid N2 cooled isopentane.

Protein analyses
Proteins were extracted from samples in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% NaDeoxycholic acid, 0.1% SDS) with protease inhibitors (complete with EDTA, Boehringer Mannheim) or with a protease cocktail (final 20 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mM PMSF), then a sample was quantified using a Biorad protein assay. The remaining protein was boiled in SDS–PAGE sample buffer for 5 min, then 25 µg protein was separated onto SDS–PAGE gels and analyzed by western blotting as described (36Go) using TBST buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) for rinses and 5% dry milk in TBST as blocking reagent prior to addition of the primary antibody. Blots were probed with a human BCL2 specific monoclonal antibody (Ab-1, 0.2 µg/ml, Oncogene Research Products or 6C8, 1.25 µg/ml, Pharmingen). Additional samples were analyzed on western blots for the expression of endogenous mouse BCL2 with hamster mAb 3F11 (from S.J. Korsmeyer), as previously described (59Go).

Cell culture
Myogenic cell cultures were generated from the limb muscles of sibling transgenic [Tg(+)] or non-transgenic [T(g–)] mice using methods previously described (36Go) in which highly purified myogenic cells were purified from the 50–70% interface of Percoll density columns. Cells were cultured in primary culture medium [DMEM (GIBCO) with 15% horse serum (HyClone), 3% chicken embryo extract, 2 mM L-glutamine, 10 mM HEPES pH 7.4, 100 U/ml penicillin, 1 mM pyruvate) on E-C-L -coated (Upstate Biotechnologies) plates as described (36Go) and fed daily. For clonal assays, freshly isolated cells were allowed to attach to plates overnight, then trypsinized the next day, counted and cultured on a coated dish at a clonal density of 100 cells/10 cm diameter (~1.7 cells/cm2). This protocol allowed an accurate viable cell count of initial populations prior to any significant cell division in vitro. Cells were allowed to grow undisturbed for 8 days, then were fixed with 1% paraformaldehyde, immunostained for desmin expression to identify myogenic colonies and the number of cells per myogenic colony was scored as described previously (59Go).

For induction of apoptosis, primary cultures were seeded on 35 mm E-C-L-coated plates, grown to near confluence in primary culture medium and then allowed to differentiate into myotubes for 2 days in differentiation medium (DM) (DMEM with 2% horse serum, 2 mM L-glutamine, 10 mM HEPES pH 7.4, 100 U/ml penicillin, 1 mM pyruvate). Apoptosis was induced by the addition of STS in DMSO at a concentration of 10–100 nM in sterile DM with control cultures receiving an equivalent volume of DMSO. Cells were analyzed (discussed subsequently) at various times up to 24 h to monitor the apoptotic progression in these cells.

Apoptosis assays
Cell cultures induced to undergo apoptosis by the addition of STS were analyzed for the expression of markers of the apoptosis pathway. Expression of activated caspases was monitored using two assays. First, the expression of active caspase-3 was analyzed on western blots. Cells treated with STS for various times were collected from plates in PBS with a rubber policeman, centrifuged at 350 g, then incubated in lysis buffer (50 mM Tris HCl, pH 8.0, 120 mM NaCl, 0.5% NP40, 50 µg/ml PMSF, 15 µg/ml aprotinin, 5 µg/ml leupeptin, 0.2 mM Naorthovanadate) for 45 min. at 4°C. Following centrifugation (10 000 g, 10 min, 4°C), a sample was removed for protein quantitation and the rest diluted with an equal volume of 2xSDS–PAGE sample buffer, boiled and 25 µg protein was analyzed by western blotting as described earlier using an antibody against the 17 kDa fragment of cleaved caspase-3 (Cell Signaling Technology, at 30 ng/ml). The same blots were also probed for desmin expression with a rabbit anti-desmin antiserum (ICN, 1/300 dilution) to demonstrate protein loading in each lane.

A second assay was used to detect the presence of active caspases in which STS-treated cells were stained with FITC-labeled VAD-FMK (CaspACE FITC-VAD-FMK, Promega), a cell permeable pan-caspase inhibitor that binds to active enzyme complexes. Procedures were as recommended by the manufacturer, and the proportion of cells binding FITC-VAD-FMK was determined by fluorescent microscopy.

In addition to monitoring caspase activity, the expression of phosphatidylserine (PS) on the surface of STS-treated cells was assessed as an early indicator of the apoptotic program. PS expression was measured using an Annexin V-FITC binding assay (Pharmingen) and manufacturers recommended methods.

Histology and immunohistochemistry
Frozen muscle tissues were cryosectioned (10 µM sections) and stained with hematoxylin and eosin (H&E) (Richard Allen Scientific). The proportion of CNF was assessed by counting all fibers in one to three complete, non-adjoining sections of each muscle tissue within the mid-region of each muscle. Muscle sections were also immunostained with an anti-Mac-1 antibody (anti-CD11b, mAb M1/70, Pharmingen, San Diego, CA, USA) as previously described (60Go), to determine the number of macrophages present within each muscle sample. Data was analyzed using t-test and non-parametric Mann–Whitney statistical analysis and InStat v 2.03 statistical analysis software (GraphPad Software, San Diego, CA, USA).


   ACKNOWLEDGEMENTS
 
This work was supported by grants to J.A.D. from National Institutes of Health (NIAMS) and to J.B.M. from the Muscular Dystrophy Association of the USA, the National Institutes of Health (NHLBI, NIAMS, NIEHS) and the United States Department of Agriculture. Transgenic mice were produced by the NICHD Transgenic Mouse Facility at the University of Alabama, Birmingham (contract no. N01-HD-5-3229). We thank Dr Eva Engvall (Burnham Institute, La Jolla, CA, USA) for providing Lama2dy–W/+ mice, Dr Stephen Tapscott (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) for the MyoD promoter fragment and Dr Stephen Konieczny (Purdue University, West Lafayette, IN, USA) for the MRF4 promoter fragment. We also thank Clifford Swap, Jonathan Nowak and Michelle Steffen for technical assistance.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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