Transient Production of �-Smooth MuscleActin by Skeletal Myoblasts During

Differentiation in Culture and FollowingIntramuscular Implantation

Matthew L. Springer, Clare R. Ozawa, and Helen M. Blau*

Baxter Laboratory for Genetic Pharmacology, Stanford University, Stanford, California

�-smooth muscle actin (SMA) is typically not present in post-embryonic skeletalmuscle myoblasts or skeletal muscle fibers. However, both primary myoblastsisolated from neonatal mouse muscle tissue, and C2C12, an established myoblastcell line, produced SMA in culture within hours of exposure to differentiationmedium. The SMA appeared during the cells’ initial elongation, persisted throughdifferentiation and fusion into myotubes, remained abundant in early myotubes,and was occasionally observed in a striated pattern. SMA continued to be presentduring the initial appearance of sarcomeric actin, but disappeared shortly there-after leaving only sarcomeric actin in contractile myotubes derived from primarymyoblasts. Within one day after implantation of primary myoblasts into mouseskeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, bothneonatal primary myoblasts and an established myoblast cell line appear tosimilarly reprise an embryonic developmental program during differentiation inculture as well as differentiation within adult mouse muscles. Cell Motil. Cy-toskeleton 51:177–186, 2002. © 2002 Wiley-Liss, Inc.

Key words: actin isoforms; myotubes; muscle differentiation; transient myogenic states

INTRODUCTION

Actin plays essential roles in both muscle and non-muscle cellular functions, ranging from large-scalemovements such as muscle contractility and cytokinesisto intracellular processes like mRNA localization.Among the multiple actin isoforms possessed by highervertebrates are three �-actin isoforms, encoded by sepa-rate genes, that are expressed predominantly in the mus-cle [Vandekerckhove and Weber, 1978] The two sarco-meric actin isoforms, �-skeletal and �-cardiac, are thepredominant isoforms in adult skeletal and cardiac mus-cle, respectively; while �-smooth muscle actin (hereafterreferred to as SMA) is found in blood vessel walls andother smooth muscle-containing tissues.

Despite their names and predominant expressionpatterns, the �-actin isoforms are expressed in diversemuscle and non-muscle cell types at various times duringdevelopment [McHugh et al., 1991]. Each of the twosarcomeric genes appears during both skeletal and car-

diac muscle development. Not only is the skeletal iso-form expressed in heart and the cardiac isoform ex-pressed in skeletal muscle, but the cardiac actin actuallyaccounts for the majority of �-actin in embryonic legmuscle during chick development [Gunning et al., 1983;Hayward and Schwartz, 1986; Vandekerckhove et al.,1986]. SMA is expressed transiently during embryonicdifferentiation of both skeletal and cardiac muscle but is

Contract grant sponsor: NIH; Contract grant numbers: HL65572,HD18179, AG09521, and CA59717; Contract grant sponsor: MuscularDystrophy Association of America.

*Correspondence to: Helen M. Blau, Ph.D., Baxter Laboratory forGenetic Pharmacology, Stanford University School of Medicine, Stan-ford, CA 94305-5175. E-mail: hblau@stanford.edu

Received 10 October 2001; accepted 28 November 2001

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cm.10022

Cell Motility and the Cytoskeleton 51:177–186 (2002)

© 2002 Wiley-Liss, Inc.

absent in these tissues in the adult [Babai et al., 1990;Ruzicka and Schwartz, 1988; Sawtell and Lessard, 1989;Woodcock-Mitchell et al., 1988]. This expression patternextends to satellite cells, as the satellite cells on singleisolated myofibers transiently express SMA during dif-ferentiation under the basement membrane [Yablonka-Reuveni and Rivera, 1994]. In addition, �-cardiac actin isexpressed during differentiation of human primary myo-blasts in culture [Gunning et al., 1987]. Moreover, adiverse pattern of �-actin expression has been observedin established cell lines derived from different muscletissues; for example, �-cardiac actin is the major isoformexpressed during differentiation of the C2C12 mouseskeletal muscle cell line [Bains et al., 1984]. Expressionof SMA has also been reported in myotubes derived fromboth C2C12 cells and rat L6 skeletal muscle myoblasts[Sharp et al., 1992; Shimizu et al., 1995]. However, thepresence of SMA in myotubes derived from differenti-ated myoblasts in tissue culture may not be fully repre-sentative of what occurs in the animal.

In this study, we have sought to ascertain the extentand timing of SMA appearance and disappearance inmouse primary myoblasts in culture, and have askedwhether these dynamics are reflected in cultured myo-blasts after implantation into adult skeletal muscle. Weshow here that mouse primary myoblasts express SMAtransiently during differentiation in culture and lose theirSMA with the onset of contractility and the appearanceof sarcomeric actin isoforms, suggesting that SMA mayplay a role in the morphological changes that accompanyskeletal myogenesis. Likewise, the cells express SMAshortly after implantation into adult skeletal muscle andlack SMA after fusion into pre-existing host musclefibers. Primary myoblasts are, therefore, capable of re-prising their embryonic SMA expression program both inculture and in adult muscle.

MATERIALS AND METHODS

Cells and Culture Conditions

Two isolates of C57BL/6 primary myoblasts wereused for this study. The isolate used for in vitro studieswas derived from neonatal mice as described previously[Rando and Blau, 1994]. The myoblasts used for in vivocell implantation were from a pre-existing isolate ex-pressing the lacZ gene from an MMLV retroviral pro-moter [Rando and Blau, 1994]. Primary myoblasts werecultured on collagen-coated tissue culture dishes at 5%CO2 as described previously [Springer et al., 1997].C2C12 myoblasts were grown on uncoated dishes inDMEM supplemented with 15% calf serum and 5% fetalbovine serum at 10% CO2. Differentiation medium con-

sisted of DMEM containing 5% horse serum for primarymyoblasts or 2% horse serum for C2C12 myoblasts.Differentiation of both cell types took place at 10% CO2.

Immunofluorescent Staining of Cells

For immunofluorescent visualization of actin, cellswere fixed in their dishes on appropriate days for 15 minin 1.5% formaldehyde in PBS, rinsed in PBS, blockedand permeablized with 0.5% purified casein (blockingreagent, NEN Life Science Products, Boston, MA) in astaining buffer consisting of 2% normal goat serum and0.3% Triton X-100 in PBS for 45 min, rinsed in PBS, andstored at 4°C until all time points were collected. Regionsof cells were isolated in their dishes with a hydrophobicmarking pen (PAP pen, Research Products International,Mount Park, IL) and immunostained in the dish withdroplets of a mouse IgG antibody against �-smooth mus-cle actin (1A4, ICN Biomedicals, Aurora, OH) at 1:400dilution for 1 h at room temperature followed by anAlexa488-conjugated goat-anti-mouse IgG secondaryantibody (Molecular Probes, Eugene, OR) at 1:200 dilu-tion for 1 h at room temperature. In some cases, cellswere also stained with a mouse IgM antibody against�-sarcomeric actin (5C5, Sigma, St. Louis, MO) at 1:200dilution followed by a Texas Red-conjugated goat-anti-mouse IgM secondary antibody (Molecular Probes) at1:200 dilution. Hoechst 33258 was sometimes includedwith the secondary antibody. Negative controls were alsoincluded in which the primary antibodies were omitted.

Immunoblotting

Cultured cells were trypsinized, centrifuged, andlysed in a buffer containing 1% Triton X-100, 20 mMTris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mMEDTA pH 8.0, 1 mM PMSF, and complete proteaseinhibitor cocktail (Roche Mannheim, Germany). Twentymicrograms total protein was loaded on a 10–20% Tris-HCl polyacrylamide gel (Bio-Rad, Hercules, CA) andtransferred to Immobilon-P membrane (Millipore, Bed-ford, MA). Equal loading of protein was assessed byPonceau S (Sigma) staining of the blot. The membranewas pre-blocked in 5% powdered milk in Tris-bufferedsaline containing 0.1% Tween-20 (TBS-T). The blot wasthen incubated with a 1:800 dilution of anti-�-smoothmuscle actin antibody (the same antibody that was usedfor immunofluorescence) in blocking solution for 1 h,washed three times with TBS-T, and incubated for 1 hwith a 1:2,500 solution of a horseradish peroxidase-linked anti-mouse Ig antibody (Amersham, Buckingham,UK) in TBS-T. The blot was then washed three timeswith TBS-T and developed using the ECL Western Blotdetection system (Amersham).

178 Springer et al.

Implantation of Myoblasts Into Mouse Muscle

Nude mice were obtained from the Stanford Uni-versity Department of Comparative Medicine and treatedin accordance with the guidelines of the Stanford Uni-versity Administrative Panel on Laboratory Animal Care.Myoblasts were trypsinized and injected once into eachtibialis anterior of Metofane-anesthetized mice as previ-

ously described [Rando and Blau, 1994; Springer et al.,1997]. Each injection consisted of 5 � 105 cells in 5 �lof PBS with 0.5% BSA.

Tissue Section Histology

On the designated days, mice were sacrificed andtheir muscle tissue was isolated, frozen in freezing iso-

Fig. 1. Transient production of SMA in primary myoblasts differentiating in culture. SMA is stainedfluorescent green and nuclei are fluorescent blue; fluorescence images are paired with corresponding phasecontrast images. SMA is absent from the vast majority of undifferentiated cells, and is present in mostcells after 1 day in differentiation conditions. Mitotic cells (arrows in 1-day panel) still lack SMA. Afterbeing present for several days during fusion into myotubes, SMA has begun to disappear from maturemyotubes by 7 days under differentiation conditions (arrows in 7-day panels). Bar � 50 �m.

SMA in Skeletal Muscle Myoblasts 179

Figure 2.

Figure 3.

pentane, cryosectioned, and stained histologically withX-gal (20-�m sections) or hematoxylin and eosin (H&E;10-�m sections) as described previously [Rando andBlau, 1994; Springer et al, 1997]. X-gal-stained sectionswere viewed using Nomarski optics. For immunofluores-cent staining of neighboring 10-�m sections, the sectionswere fixed and stained using the procedures describedabove for cell staining.

RESULTS

Primary Myoblasts Transiently Produce�-Smooth Muscle Actin in Culture

Primary myoblasts were induced to differentiate inculture by switching to serum-poor differentiation medium(DM). At different times after induction of differentiation,cells were fixed and accumulated until all samples could beimmunostained for �-smooth muscle actin (SMA) simulta-neously (Fig. 1). The first time point examined consisted ofcells that were never exposed to DM and were fixed imme-diately after removal of growth medium. These undifferen-tiated cells did not stain positive for SMA except for anoccasional cell that displayed faint staining. However, afterone day in DM, the myoblasts had elongated and the vastmajority stained brightly positive for SMA. A few cells stilldid not stain; these cells usually were undergoing mitosisand, therefore, clearly had not entered the differentiationpathway. After 3 days in DM, cells had fused and manylong multinucleate myotubes were apparent, but they werestill flat, nuclei were disorganized, and contractility had notyet begun. At this point, bright SMA staining was observedin all myotubes. After 7 days, when many myotubes hadchanged from flat to cylindrical morphology and had be-come contractile, a substantial number of myotubes nolonger stained positive for SMA, exhibiting no staining atall even when neighboring cells were still brightly stained.

Disappearance of �-Smooth Muscle ActinCoincides and Overlaps With Appearance of �-Sarcomeric Actin and the Onset of Contractility

Because the disappearance of SMA occurredaround the time at which contractility began, we double-

stained myotubes at the 7-day time point to visualizeboth SMA and sarcomeric actin, using an antibody thatrecognizes the skeletal and cardiac sarcomeric isoformsof �-actin but not the smooth muscle isoform (Fig. 2). Inall cases, myotubes that did not possess detectable SMAdid possess sarcomeric actin. The converse was alsogenerally true, although some myotubes and an occa-sional unfused cell possessed both isoforms. Some un-fused cells did not show evidence of either isoform.

SMA staining could occasionally be observed in astriated pattern in contractile myotubes (Fig. 3). Thispattern was not observed in the earlier 3-day myotubes,before the onset of contractility. Double staining for bothactin isoforms showed co-localization of striation pat-terns that were not present when primary antibody wasomitted, showing that the staining was specific and notdue to binding of the secondary antibody. This striatedSMA staining pattern was not due to cross-reactivity ofthe antibody with sarcomeric actin, as there were manyexamples of sarcomeric actin staining in which there wasno trace of SMA staining.

�-Smooth Muscle Actin Is Produced DuringDifferentiation of C2C12 Myoblasts

In order to determine if SMA also appears duringdifferentiation of an established myoblast cell line, myo-blasts from the C2C12 cell line were examined both byimmunoblotting and by immunostaining in culture.C2C12 cells are essentially immortal, grow significantlyfaster than primary myoblasts, and do not require acollagen substrate for growth, but they retain their myo-blast identity in terms of characteristic myogenic geneexpression and the ability to fuse in culture [Blau et al.,1983; Yaffe and Saxel, 1977]. At appropriate times afterinduction of differentiation, C2C12 cultures were dividedand processed either for protein extraction and immuno-blotting or for immunofluorescent staining, using thesame antibody against SMA (Fig. 4). SMA was notdetectable by either approach in undifferentiated cells,although the protein samples analyzed by immunoblot-ting consisted of equal amounts of protein, and roughlyequal loading was confirmed by Ponceau S staining ofthe blot. These cells did not elongate as early as theprimary myoblasts; thus, the next time point examinedwas after 2 days in DM, at which time SMA was visibleby immunofluorescence in myotubes and in very fewunfused cells but was not detectable in most unfusedcells. At this time, immunoblotting showed a single spe-cific band corresponding to the expected size of SMA (42kDa). SMA was similarly detected by both analyticalapproaches performed after 4 days. Unlike the primarymyoblasts, C2C12 myoblasts did not exhibit an observ-able decrease in SMA even at 9 days after the change to

Fig. 2. Actin isoform distribution in a population of myotubes de-rived from primary myoblasts. Myotubes that no longer contain SMA(green stain) now stain positive for �-sarcomeric actin (red stain).Some unfused cells are stained with both antibodies (yellow; arrow inc), as are occasional fused myotubes (arrow in a). Bar � 50 �m.

Fig. 3. Co-localization of SMA and sarcomeric actin in striatedpatterns in myotubes derived from primary myoblasts. SMA (green;a,d) and sarcomeric actin (red; b,e) overlap in a striated pattern insome myotubes (c,f). Arrows point to regions of striated immunostain-ing pattern. Bar � 50 �m.

SMA in Skeletal Muscle Myoblasts 181

DM (not shown). However, because both culture condi-tions and differentiation conditions for C2C12 myoblastsare different from those for primary myoblasts, it isunknown whether this reflects a fundamental differencebetween cell types or simply a different response todifferent conditions. Nonetheless, both by cell-specificimmunostaining and population-wide immunoblotting,C2C12 myoblasts were observed to produce SMA as aspecific response to differentiation.

�-Smooth Muscle Actin Appears Transiently AfterImplantation of Primary Myoblasts Into Muscle

Because all of the studies described above weredone in culture, we also examined the behavior of SMAin primary myoblasts upon implantation into mouse skel-etal muscle. Upon implantation into skeletal muscle,myoblasts fuse into pre-existing muscle fibers, reprisingtheir role in muscle healing [Dhawan et al., 1991; Watt etal., 1982]. Primary myoblasts expressing the lacZ genewere implanted into the tibialis anterior muscle of eachleg of 6 mice, resulting in 12 injected muscles. Two mice

per time point were sacrificed after 1, 9, and 57 days, andthe injected muscles were isolated, cryosectioned, andstained with H&E to visualize tissue morphology and withX-gal to identify myoblasts and fibers into which they hadfused. For the legs that showed detectable X-gal staining,neighboring sections were immunostained for SMA.

As shown in Figure 5, myoblasts were observed in the1-day tissue sections as regions of mononuclear cells visibleby H&E staining and stainable by X-gal. Smaller regions ofmononuclear cells that were not stained by X-gal mostlikely corresponded to the inflammatory response to theinjection. The X-gal stained regions also stained positive forSMA in neighboring sections. Sometimes muscle fibersalong the needle track were also fluorescent, but this fluo-rescence was not due to SMA staining, as those fibers werealso fluorescent in negative controls lacking primary anti-body. The X-gal stained regions of myoblasts were notfluorescent in those controls, indicating that the SMA stain-ing seen in the unfused myoblasts was real.

In the 9-day samples, myoblast fusion into pre-existing fibers was complete and there was no indication

Fig. 4. Appearance of SMA in C2C12 differentiating cultures. Pro-tein extracts prepared from cultures of differentiating C2C12 myo-blasts are shown in the immunoblot (top left). A single specific bandat approximately 42 kDa is evident after 2 and 4 days in DM, but isundetectable in the sample from undifferentiated cells. Ponceau Sstaining of the blot (shown below) confirmed approximately equal

loading of protein in all lanes. SMA immunofluorescence and corre-sponding phase contrast images depict the appearance of SMA inthe same cultures within 2 days of the switch to differentiation me-dium. Arrow points to a region of striated immunostaining pattern.Bar � 50 �m.

182 Springer et al.

of unfused cells in H&E-stained sections. X-gal stainingshowed many blue muscle fibers, identifying them as thefibers into which the myoblasts had fused. However,these fibers did not stain positive for SMA, nor did anyother part of the muscle other than the blood vessels,which were expected to stain positive due to their smoothmuscle component. The same result was observed for theday-57 samples (not shown).

DISCUSSION

The appearance of SMA during skeletal muscledifferentiation appears to be a universal phenomenon ofskeletal muscle differentiation, occurring during embry-onic development, differentiation of satellite cells juxta-

posed to isolated muscle fibers, and myoblast differenti-ation in culture and after implantation into muscle. Alongwith the appearance of cardiac actin, it may be indicativeof either a developmental decision or a regulatory mech-anism, as all three isoforms are expressed with the ap-propriate isoform ultimately emerging as the dominantone. However, because SMA appears first and is replacedby sarcomeric isoforms, the SMA is likely to play anactive role in the preparation of the differentiating myo-blasts for the appearance of sarcomeric actins. In ourexperiments, the appearance of SMA during primarymyoblast differentiation in culture occurred rapidly uponthe shift to differentiation conditions and coincided withthe elongation of the myoblasts in preparation for fusion.The sarcomeric isoform did not appear until after fusion

Fig. 5. Transient production of SMA in primary myoblasts afterintramuscular implantation. a–d, e–g: Sets of adjacent sections takenfrom two different muscles 2 days after implantation of primarymyoblasts expressing the lacZ gene. a,e: hematoxylin/eosin staining ofmyoblasts and inflammatory cells that respond to the needle injury. b,f:X-gal staining, which stains the myoblasts blue but leaves the inflam-matory cells unstained. c,g: SMA staining at these regions, showingSMA� myoblasts (e.g., large arrow in c) and SMA� blood vessels(e.g., small arrow in c). Non-specific fluorescence is occasionallyvisible in certain muscle fibers along the needle track (arrowhead in c).

That this fluorescence is non-specific and does not correspond to SMAis confirmed by negative control immunostaining of an adjacent tissuesection without the primary antibody (d); the non-specific fluorescenceis still visible while the specific SMA staining of myoblasts and bloodvessels is absent in this control. h–j: adjacent tissue sections frommuscle 9 days after myoblast implantation. Hematoxylin/eosin andX-gal staining (h,i) confirm that the myoblasts have fully fused intopre-existing muscle fibers, but SMA staining is not evident in thesefibers, only in blood vessels. Arrows in h–j point to correspondinglocations. Bar � 200 �m.

SMA in Skeletal Muscle Myoblasts 183

into myotubes was complete throughout most of theculture. Thus, it is possible that SMA plays a role in themorphological changes that lead to cell fusion, a role thatsarcomeric isoforms may be ill-equipped to fulfill be-cause of their inclination towards myofibril assembly.Supporting this hypothesis is the observation that preco-cious expression of skeletal actin in C2C12 myoblastsprevents them from spreading during differentiation[Gunning et al., 1997]. However, SMA does not appearto be critical to this process because mice deficient inSMA are still able to produce skeletal muscle [Schildm-eyer et al., 2000]. Unlike skeletal actin, precocious ex-pression of cardiac actin does not interfere with skeletalmuscle differentiation [Gunning et al., 1997]; therefore,in SMA null mice, it is possible that cardiac actin com-pensates for the lack of SMA in this process. SMA andcardiac actin may be used by the myoblasts to establishmyotube morphology locking it into an appropriate shapeprior to the polymerization of skeletal actin with myosin.

SMA staining appeared in a striated pattern inmany mature myotubes that had become contractile, co-localizing with sarcomeric actin. This suggests that theSMA is capable of inserting into nascent sarcomeresduring skeletal myogenesis before being replaced bysarcomeric isoforms. A similar phenomenon has beenpreviously observed in cultured cardiac myocytes, inwhich SMA can localize to sarcomeres to a limited extent[Perriard et al., 1992; von Arx et al., 1995]. There arealso precedents for �-actin isoforms replacing each otherunder circumstances of insufficient expression of oneisoform. For example, SMA null mice show surprisingviability, being able to grow to adulthood and reproduce;this is likely due to compensation by the skeletal isoformthat is expressed inappropriately in vascular tissue ofthese mice [Schildmeyer et al., 2000]. Likewise, micedeficient in cardiac actin, while unable to grow to adult-hood, show an abnormal increase in both skeletal actinand SMA [Kumar et al., 1997], and mechanical load-induced cardiac hypertrophy leads to re-activation ofSMA gene expression in the heart [Black et al., 1991].Feedback mechanisms are likely to exist that allow �-ac-tin isoform deficiencies to be sensed, resulting in abnor-mal expression of the other �-actin genes in a compen-satory manner. Not surprisingly, regulatory elements thatrespond to basic helix-loop-helix factors have been iden-tified in the SMA promoter that guide expression insmooth vs. skeletal muscle [Johnson and Owens, 1999].The correct regulation of this promoter, therefore, islikely to be critical to successful differentiation. How-ever, prior reports showing a severalfold increase inactive transcription of the SMA gene during differentia-tion of L6 myoblasts, and the presence of SMA mRNA incultured C2C12 myoblasts as well as in myotubes, sug-

gest that both transcriptional and post-transcriptionalmechanisms may play a role in this regulation [Sharp etal., 1992; Shimizu et al., 1995].

Whether SMA plays a regulatory role in myoblastdifferentiation or is merely regulated itself, it is likelythat the different �-actin isoforms are an important partof the balance of regulatory factors that control cell fateduring myogenesis. Previous studies from this laboratoryshowed that human �-skeletal and �-cardiac actins wereboth expressed in cultured human hepatocytes and fibro-blasts when those cells were fused to mouse myoblasts instable heterokaryons [Blau et al., 1985; Hardeman et al.,1986]. Moreover, the human �-cardiac actin gene wasexpressed only at low levels after being transfected intomouse fibroblasts, but fusion of those cells to myoblastscaused the gene to be expressed at increased levels[Hardeman et al., 1988]. Conversely, forced expressionof �-skeletal actin in undifferentiated C2C12 myoblastshas been shown to result in precocious accumulation ofseveral differentiated muscle-specific structural proteins,as well as the regulatory protein MyoD [Gunning et al.,2001]. Clearly, the balance of pro- and anti-myogeniccytoplasmic regulatory factors greatly influences the ex-pression of the skeletal and cardiac �-actin genes. It isinteresting to note that some but not all rhabdomyosar-comas (skeletal muscle tumors) express SMA in additionto the expected skeletal muscle proteins [Babai et al.,1988; Cintorino et al., 1989; Skalli et al., 1988]. Thetransient production of SMA in the several models ofskeletal muscle differentiation discussed here suggeststhat all three �-actin isoforms are involved in thisbalance.

One potential caveat to consider regarding the in-tramuscular implantation of myoblasts is that Darby et al.[1990] reported that myofibroblasts that appear duringwound healing can also transiently express SMA. Sincethe injection of myoblasts into muscle constitutes awound of the muscle, the possibility that myofibroblastshave accumulated at the wound and are expressing SMAshould not be discounted. However, the SMA expressionattributed to the implanted myoblasts occurred very fast,within one day of implantation, which matches the timecourse of SMA induction during differentiation in cul-ture, and is much faster than the 6-day time course ofmyofibroblast accumulation described by Darby et al.[1990]. In addition, Figure 5a–d shows both the im-planted mass of X-gal-staining myoblasts and the un-stained wound of the needle track; only myoblasts stainpositive for SMA while the wound does not. Therefore,SMA expression by myofibroblasts is unlikely to be aconfusing factor in this experiment.

The transient appearance of SMA after myoblastimplantation into adult muscle should be heeded when

184 Springer et al.

using such myoblast implantation as a method of genedelivery [Blau and Springer, 1995]. The implantation ofgenetically altered myoblasts that produce recombinantproteins relevant to vascular biology can be evaluated byassaying for proteins of the vasculature, including SMA[Springer et al., 1998, 2000]. The spurious appearance ofSMA during differentiation and fusion of these myo-blasts may be misinterpreted as a vascular response ifappropriate controls are not performed.

In summary, we have shown that mouse primarymyoblasts transiently produce SMA during differentia-tion in culture and after implantation into adult skeletalmuscle, reprising their embryonic program of develop-ment. SMA then frequently disappears, and is replacedby sarcomeric actin isoforms. SMA and the other �-actinisoforms appear to be coordinately regulated during skel-etal muscle differentiation and are likely to be involvedin the balance of myogenic regulators within the cell, andmay play distinct roles in the morphological changesassociated with myogenesis as well.

ACKNOWLEDGMENTS

We thank Peggy Kraft for invaluable technical as-sistance, and Andrea Banfi, Barbara Munz, George Sen,and Tom Wehrman for insightful discussion during theresearch and helpful comments on the manuscript. Thiswork was supported by an H.H.M.I pre-doctoral fellow-ship to C.R.O., NIH grant HL65572 to H.M.B. andM.L.S., and a grant from the Muscular Dystrophy Asso-ciation of America as well as NIH grants HD18179,AG09521, and CA59717 to H.M.B.

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