Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4
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ABSTRACT |
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Direct gene
transfer into skeletal muscle in vivo presents a convenient
experimental approach for studies of adult muscle gene regulatory
mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential
expression of fast myosin heavy chain and slow myosin light chain and
troponin I (TnIslow) gene constructs in muscles enriched in the
appropriate fiber type. We now report a troponin I fast (TnIfast)
direct gene transfer study. We injected into the mouse soleus muscle
plasmid DNA or recombinant adenovirus carrying a TnIfast/
-galactosidase (
-gal) reporter construct that had previously been
shown to be expressed specifically in fast fibers in transgenic mice.
Surprisingly, microscopic histochemical analysis 1 and 4 wk
postinjection showed similar TnIfast/
-gal expression in fast and
slow fibers. A low but significant level of muscle fiber segmental
regeneration was evident in muscles 1 wk postinjection, and
TnIfast/
-gal expression was preferentially targeted to regenerating
fiber segments. This finding can explain why TnIfast constructs are
deregulated with regard to fiber type specificity, whereas the myosin
constructs previously studied are not. The involvement of regenerating
fiber segments in transduction by plasmid DNA and recombinant
adenoviruses injected into intact normal adult muscle is an
unanticipated factor that should be taken into account in the planning
and interpretation of direct gene transfer experiments.
in vivo gene transfer; troponin I; plasmid DNA injection; recombinant adenovirus
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INTRODUCTION |
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DIRECT GENE TRANSFER into skeletal muscle in vivo using either plasmid DNA (43) or recombinant viruses (30) has medical applications in vaccination and gene therapy and also has been widely used in studies of developmental and physiological regulation of muscle gene expression (4, 8, 11, 18, 23, 36). However, we know little of the cellular mechanisms involved in direct gene transfer and what impact these mechanisms may have on the experimental applicability of the method for particular aspects of adult muscle gene expression.
Adult muscle fibers are highly differentiated cells falling into physiologically and biochemically specialized classes, the fast and slow fiber types, which have distinct gene expression profiles (reviewed in Refs. 6, 28, 33). The molecular mechanisms that drive fiber-type-specific gene expression are largely unknown, although recent progress has been made in identifying relevant regulatory DNA sequences and DNA-binding proteins (7, 10, 26, 34). Transgenic mice have been extensively used in these studies because there are no effective cell culture models of mature adult fast and slow fibers. Direct gene transfer in vivo provides an attractive experimental alternative to laborious transgenic mouse studies. Injection of plasmid DNA or recombinant adenovirus into muscle results in expression of the transgene in a small number of fibers (30, 43). Notwithstanding a relatively low transduction efficiency, direct gene transfer can be a simple and effective approach to gene expression analysis in adult muscle fibers. Moreover, the transduction efficiency can be increased considerably by inducing extensive muscle fiber regeneration (1, 38, 39), although this could in principle interfere with normal adult muscle fiber regulatory mechanisms. Several direct gene transfer studies have reported preferential expression of fast (36) or slow (11, 18, 23) contractile protein gene constructs in muscles enriched in the appropriate fiber type.
We now report a direct gene transfer study of a fast-fiber-specific
gene encoding a troponin I isoform, TnIfast. We used a TnIfast/-galactosidase (
-gal) reporter gene construct known from
previous transgenic mouse studies to contain all the
cis-regulatory information required to direct
fast-fiber-specific expression. Fiber type expression was assessed 1 and 4 wk postinjection by microscopic histochemical analysis of
reporter
-gal expression in fast and slow fibers of the mouse soleus
muscle. Our initial hope was that the transferred construct would show
fast-fiber-specific expression, thereby establishing direct gene
transfer as an appropriate method for functional analysis of TnIfast
gene regulatory elements. Unexpectedly, we found at both time points
that direct-transferred plasmid and adenoviral TnIfast/
-gal
constructs were expressed equally well in fast and slow fibers, i.e.,
expression was deregulated with respect to fiber type. Histochemical
analysis revealed the presence of regenerating muscle fiber segments 1 wk postinjection, although there had been no deliberate attempt to
induce necrosis/regeneration. Moreover, regenerating fiber segments
were preferentially targeted for both plasmid and adenoviral
transduction, and this can account for the observed deregulated
expression of TnIfast gene constructs in slow fibers. The unexpected
involvement of regenerating segments in transduction following
injection into normal adult muscle has implications for the use of
direct gene transfer methods in muscle gene regulatory studies,
particularly in the case of adult gene regulatory mechanisms that may
be perturbed by regeneration.
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MATERIALS AND METHODS |
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Animals. Normal (CD-1) and immunocompromised SCID (C.B-17/IcrCrl-scidBR) mice were obtained from Charles River Laboratories, St. Constant, Quebec, Canada. Transgenic mice carrying the TnILacZ1 transgene were from line 29 (15). Adult mice >6 wk old of both sexes were used. Animal housing and experimentation followed the guidelines of the Canadian Council for Animal Care in protocols approved by the McGill University Animal Care Committee.
TnIfast/-gal gene constructs.
We used the TnIfast/
-gal plasmid TnILacZ1 (15), which contains 530 bp of TnIfast 5'-flanking DNA, exon 1, intron 1, and the first
(untranslated) part of exon 2 of the quail TnIfast gene, linked to a
5.1-kb Sma I/EcoR I fragment of pRSVZ (23) containing promoterless Escherichia coli LacZ sequences and SV40 splicing and polyadenylation sequences, in the vector pBR322. As a control for
assessing plasmid uptake we used the
-gal parent plasmid pRSVZ, in
which expression is driven by a 524 fragment of the Rous sarcoma virus
(RSV) proviral genome including the 3' long terminal
repeat (LTR) (24). The recombinant adenovirus
AdTnILacZ(1), previously referred to as AVTnILacZ (21), consists of a
5.7-kb TnILacZ(1) construct inserted by homologous recombination into the E1 region of E1+E3-deleted human adenovirus type 5. The
TnIfast/
-gal insert in AdTnILacZ(1) is identical to that of
TnILacZ1, except that, because of adenovirus vector insert size
constraints, the bovine growth hormone gene polyadenylation sequence
[a 267-bp Xba I/BamH I fragment from pRc/RSV
(Invitrogen)] was used in place of SV40 sequences.
TnIslow/-gal gene construct.
The TnIslow construct BW188Z included the
95 promoter and
the upstream enhancer (USE) of the human TnIslow gene, as
in the TnIsUSE-95X1nucZ construct of Corin et al. (11), linked to the same LacZ and SV40 sequences used in TnILacZ1. A 0.3-kb Kpn
I/Hind III fragment from TnIsUSE-95X1nucZ, including the
TnIslow promoter and enhancer sequences, was cloned into the
corresponding sites in pBluescriptII SK+ (Stratagene), after which the
5.1-kb Sma I/EcoR I fragment of pRSVZ (see above) was
cloned into EcoR I and blunted Hind III sites.
Intramuscular injection. Supercoiled plasmid DNA was produced by (double) isopycnic banding in CsCl/ethidium bromide (31) and was resuspended in 0.15 M NaCl at 5-15 µg/µl. Recombinant adenovirus (5-10 × 1011 plaque-forming units/ml) was produced and purified as described in Ref. 1. For intramuscular injection of plasmid or virus, soleus muscles of anesthetized mice were exposed in a lateral approach to the hindlimb crural muscles by reflection of the gastrocnemius/plantaris muscle group, and volumes of 1-2 µl were injected using a drawn glass capillary pipette. Mice were killed 7, 21, or 28 days after injection, and soleus muscles were dissected from tendon to tendon and frozen for cryostat sectioning.
Histochemical analysis.
For -gal histochemistry, cryostat cross sections (10 or, in Fig.
1A, 40 µm) of frozen muscles were fixed 3 min
in 0.25% glutaraldehyde in water at room temperature and were
incubated in phosphate-buffered saline containing 5 mM ferricyanide, 5 mM ferrocyanide, 2 mM MgCl2, 0.1 mg/ml
5-bromo-4-chloro-3-indoyl
-D-galactopyranoside
(X-Gal) at room temperature for 2-20 h. Optical
densities of stained muscle fibers were determined by video microscope
image analysis (Java software, Jandel), using a reference series of
standard neutral density filters for calibration (15). Myosin ATPase
histochemistry was done on 10-µm sections after preincubation at pH
4.6 (5). By this method slow fibers are strongly stained, whereas fast fibers show little or no staining. Myosin heavy chain isoforms were
identified using monoclonal antibodies BA-D5 (type I, i.e., slow),
SC-71 (adult fast IIA) (32), and 47A (embryonic) (29) on unfixed frozen
sections. Detection was by biotin-conjugated secondary antibodies and
peroxidase-coupled streptavidin (Vector Laboratories). Statistical
analysis was done at the VassarStats website created and maintained by
Richard Lowry, Department of Psychology, Vassar College, Poughkeepsie,
New York.
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RESULTS |
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Direct-transferred TnIfast/-gal plasmid DNA and
recombinant adenovirus are expressed in both fast and slow muscle
fibers.
Our experimental gene constructs were based on promoter and regulatory
sequences from the quail TnIfast gene. We have previously shown that
the TnIfast
-gal reporter construct TnILacZ1 is expressed in fast
but not in slow fibers in transgenic mouse skeletal muscle (15).
Preparatory to our direct gene transfer experiments, we investigated
TnILacZ1 germ-line transgene expression in the soleus muscle; X-Gal
histochemistry showed
-gal expression in fast but not in slow fibers
(Fig. 1). By quantitative
microdensitometry, X-Gal staining optical densities of fast fibers were
found to be 4.4-fold higher than the apparently background optical
densities of slow fibers. Thus the TnILacZ1 construct contains all the
necessary regulatory cis elements to direct fast vs. slow
fiber-type-specific expression in the soleus muscle.
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Preferential transduction of regenerating segments.
In cross sections of plasmid- or adenovirus-injected muscles 7 days postinjection, most fibers had the typical microscopic appearance and ATPase histochemical reactions of normal fast or slow
muscle fibers. However, a minority, ranging up to ~15%, were morphologically/histochemically atypical. Many of these had the characteristics of regenerating muscle fibers; they were small, had
centrally, rather than peripherally, located nuclei, and expressed the
embryonic isoform of myosin heavy chain (Fig.
5). Their ATPase histochemical staining was
intermediate between the strong staining of typical slow fibers and the
extremely weak staining of typical fast fibers and often showed the
myotube appearance characteristic of newly formed or regenerated muscle
fibers. Regenerating fibers were found throughout the muscles,
scattered individually or in small clusters.
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Long-term expression of TnILacZ1 plasmid in fast and slow fibers.
Because our results at 7 days postinjection revealed preferential
transduction of regenerating fiber segments, we wished to determine
whether fast-fiber-specific expression of TnIfast/-gal might emerge
in longer-term experiments that would permit the transduced
regenerating fiber segments to undergo additional maturation. However,
very little
-gal expression was detected in longer-term experiments
in normal mice. In an extensive experimental series in CD1 mice at 3 wk
postinjection, we found only a single
-gal-expressing fiber when we
should have expected to see scores. Others have observed that plasmid-
or adenovirus-mediated gene expression is transient in normal animals,
apparently due to immunological reaction against the reporter gene
polypeptide (9, 37, 40), and that longer-term expression can be
attained using immunocompromised animals (2, 27). We repeated the
experiments in genetically immunodeficient SCID mice and then readily
detected TnIfast/
-gal-expressing fibers 4 wk postinjection. None of
the
-gal-expressing fibers expressed detectable embryonic myosin
heavy chain, indicating that regeneration/maturation had proceeded
beyond the stage observed at 7 days postinjection. Nonetheless, similar
numbers of fast and slow fibers (Table 1, and see Fig. 6) expressed
-gal, and the
-gal-staining optical densities of fast and slow
fiber profiles were similar (data not shown). Thus we did not see
evidence that regenerated slow muscle fibers repress TnIfast/
-gal
expression on further maturation, although it is possible that stable
-gal protein or mRNA synthesized early during regeneration could
obscure a subsequent transcriptional repression.
Expression of a TnIslow construct in fast and slow fibers.
The TnI gene family includes members expressed specifically in both
fast (TnIfast) and slow (TnIslow) muscle fibers. Previous direct gene
transfer studies of TnIslow constructs, based on biochemical assay of
reporter enzymes in rat muscle homogenates, showed approximately sixfold greater expression of direct-transferred TnIslow constructs in
soleus muscle than in extensor digitorum longus muscle at 5 days
postinjection, indicating slow fiber preferential expression (11). To
assess fiber-type-specific expression of TnIslow constructs at the
individual cell level, we injected construct BW188Z in which -gal
expression was driven by human TnIslow enhancer and promoter elements
that are known by microscopic analysis to drive slow-fiber-specific
expression in transgenic mice (11). When we examined expression at 7 days postinjection, we detected
-gal expression in both fast and
slow fibers (Table 1). However, there was preferential expression in
slow fibers; almost three times as many slow fibers as fast fibers
showed
-gal-staining (significant by
2 test,
P < 0.05), and their average staining intensity was twice as
great (significant by Mann-Whitney U-test, P < 0.025). Thus the TnIslow construct showed some deregulated expression
in the inappropriate fiber type, although it differed from TnIfast, in that fiber type deregulation was not so extensive as to completely override preferential expression in the appropriate fiber type.
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DISCUSSION |
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The TnILacZ1 construct is expressed in fast but not slow muscle fibers
in the soleus and other mouse muscles when present as a germ line
chromosomal transgene (Fig. 1 and Ref. 15). Thus all the cis
regulatory elements required to direct fast-fiber-specific expression
are present in the TnIfast/-gal plasmid and adenovirus constructs
used in the present study. However, despite the presence of these
regulatory elements, we found that the TnIfast constructs were
expressed with similar efficiencies in slow and fast fibers when
direct-transferred by intramuscular injection in adult
animals. Slow and fast fibers were present in the
-gal-expressing
muscle fiber subpopulation in about the same ~1:1 ratio as they are
found in the soleus muscle as a whole, and quantitative levels of
-gal expression were similar in the two fiber types.
Because of the intrinsic inefficiency of the direct-transfer method,
the average numbers of -gal-expressing fibers per muscle in our
study were small, on the order of 1-10. However, there is no
reason to doubt that the
-gal-expressing fibers observed, though few
in number in any individual muscle, are typical of the fibers that are
responsible for reporter gene expression in other published studies
using the direct-transfer method in normal adult muscle. By studying a
large number of muscles, we accumulated sufficient data to reliably
establish the character of the
-gal-expressing fiber population.
The unexpected deregulated expression of TnIfast/-gal constructs in
slow fibers in our study is likely related to inadvertent muscle fiber
segmental necrosis/regeneration. We found a low but significant level
of segmental regeneration in plasmid- and adenovirus-injected muscles
and preferential targeting of TnIfast/
-gal gene expression to
regenerating fiber segments. A large fraction of
-gal-expressing fibers showed features of regeneration, and regenerating fiber profiles
were considerably (~4-fold) overrepresented in the
-gal-expressing fiber subpopulation compared with their abundance in the muscle as a
whole. The presence of regenerating fibers in plasmid DNA-injected muscles has been observed before (12, 27), although their segmental
nature and the preferential targeting of transferred gene expression
have not been noted. Preferential transduction of regenerating fiber
segments is consistent with studies showing that transduction by
plasmid DNA or recombinant adenovirus is dramatically increased
by conditions that induce extensive muscle fiber regeneration (1, 38,
39). Presumably, activated satellite cells and myoblasts, and/or
newly formed fibers, are better able to take up and express
plasmid/viral constructs than are fully mature muscle fibers.
Preferential transduction of regenerating segments can account for at
least some of the deregulated expression of TnIfast/-gal constructs
in slow as well as in fast fibers. The endogenous TnIfast gene is known
to be activated during myoblast differentiation (16, 20) and to be
expressed in regenerating fast and slow muscle (35). Thus regenerating
segments of both fast and slow fibers would be expected to contain
transcription factors capable of driving TnIfast/
-gal gene
expression. We have observed a weak but detectable activation of germ
line transgene TnILacZ1 expression in regenerating slow muscle fiber
segments in soleus muscle injected with a non-
-gal-encoding plasmid
(data not shown).
Segmental regeneration implies muscle fiber segmental necrosis and repair, a process frequently observed in muscle pathology. We found that sham injection of vehicle only (0.15 M NaCl) induced similar levels of regeneration as did DNA injection (data not shown), so that physical disruption associated with the introduction of the injection needle and significant liquid volumes appears to be the predominant cause of necrosis/regeneration. Whatever the mechanism, necrosis occurs early on and after several weeks postinjection regenerating fiber segments are no longer found. Other studies have established that, while actively regenerating fibers are evident at 1 wk following acute muscle injury, regeneration is essentially complete by 3-4 wk (41).
Despite the transient nature of regeneration, we continued to find
-gal-stained slow muscle fibers 4 wk after injection of TnIfast/
-gal constructs. The continued presence of
-gal in slow fibers could reflect the presence of stable
-gal mRNA and/or protein
synthesized during an earlier phase of regeneration. Alternatively, there may be ongoing deregulated TnIfast/
-gal gene expression in
mature, fully regenerated slow fibers, perhaps as a persistent juvenile
feature akin to the central nucleation that permanently marks
regenerated rodent muscle fibers (19).
Fiber-type-specific gene expression. Previous direct gene transfer studies of fiber type regulation have shown apparently proper fast/slow regulation of constructs based on fast myosin heavy chain IIB (36) and on slow myosin light chain (MLC-1s/v [18] and MLC2s [23]) and troponin I (TnIslow [11]) genes. In contrast, our results show similar expression of TnIfast constructs in slow and fast fibers. This difference reflects, in part, gene-to-gene variation in the extent to which fiber-type-specific expression is deregulated during muscle regeneration. Endogenous genes encoding MLC-1s/v (also called MLC1s b) and MLC2s are known to maintain slow muscle specificity throughout the regeneration process and are not expressed, even transiently, in regenerating fast muscle (13, 35). The endogenous fast myosin heavy chain IIB gene shows only an exceedingly transient deregulated expression (< 24 h duration) during slow muscle regeneration (17). Direct-transferred constructs based on these genes would be expected to show little or no regeneration-associated deregulation compared with TnIfast, which is expressed in regenerating slow muscle over a period of ~10 days (13). Indeed, the MLC-1s/v and MLC2s gene transfer studies showing slow muscle-enriched expression were carried out not in intact adult muscles but in regenerating muscles following toxin-induced total necrosis, to increase the transduction efficiency (18, 23). It is likely that different kinds of regulatory mechanisms control regeneration-deregulated (e.g., TnIfast) and regeneration-resistant (e.g., MLC-1s/v, MLC2s) fiber-type-specific gene expression.
Regarding TnIslow, the endogenous gene is known to be expressed in regenerating fast as well as slow muscle (13, 35) so some deregulated expression of direct-transferred TnIslow constructs in fast fibers might be expected. In our experiments we did in fact observe significant TnIslow/Implications for use of the direct transfer technique. Our results reveal an unanticipated aspect of direct gene transfer that has important implications for its experimental use, namely, that intramuscular injection of plasmid DNA or recombinant adenovirus does not strictly introduce genes directly into mature adult muscle fibers. Even in the absence of deliberate attempts to induce regeneration, injection of plasmid DNA or adenovirus into adult muscle by itself induces segmental regeneration. Although regenerating segments are a minority, we find they are transduced more efficiently than mature fiber segments, so that much of the transferred gene's transcriptional activity may be in regenerated segments. Moreover, diffusion within the muscle fiber of reporter gene mRNA or protein into adjacent normal segments may give a misleading impression of transferred-gene expression in normal adult fibers.
The impact of regeneration varies greatly among muscle genes and is a factor that should be taken into account in planning or interpreting direct gene transfer experiments. In the case of regulatory mechanisms that are relatively unperturbed by regeneration, direct gene transfer by intramuscular injection of plasmid or recombinant adenovirus presents an effective experimental alternative to germ line transgenesis for molecular genetic studies such as cis-element mapping. However, the same is not true for mechanisms that are extensively perturbed by regeneration, such as those that drive fiber-type-specific expression of the TnIfast gene, and perhaps other aspects of adult muscle gene regulation as well. Direct gene transfer methods that induce and target regenerating segments are not applicable to the functional analysis of fiber type regulatory elements of the TnIfast gene. Transgenic mice remain an alternative approach; however, the development of convenient direct gene transfer methods that do not induce regeneration, or that do not preferentially target regenerating fibers, would facilitate progress. Recombinant adenoassociated virus vectors merit attention in this regard, as recent reports suggest efficient direct gene targeting of adult muscle fibers (14, 22). Electrically augmented plasmid direct transfer (3, 25) may provide an additional avenue, if regeneration is found to play a less prominent role in this process than is the case in conventional plasmid direct transfer. ![]() |
ACKNOWLEDGEMENTS |
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Bob Wade provided the TnIsUSE-95X1nucZ construct. He also provided inspiration and a fine example, both in science and in life. He will be missed. We thank Peter Merrifield and Stefano Schiaffino for kindly supplying antibodies.
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FOOTNOTES |
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This work was supported by a research grant from the Medical Research Council of Canada to K. E. M. Hastings, a Killam Fellow of the Montreal Neurological Institute.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. E. M. Hastings, Montreal Neurological Institute, McGill Univ., 3801 University St., Montreal, Quebec, Canada H3A 2B4 (E-mail: cxph{at}musica.mcgill.ca).
Received 1 June 1999; accepted in final form 13 January 2000.
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