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Published as doi: 10.1096/fj.09-129783.
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(The FASEB Journal. 2009;23:2205-2214.)
© 2009 FASEB

Context-dependent functional substitution of {alpha}-skeletal actin by {gamma}-cytoplasmic actin

Michele A. Jaeger*, Kevin J. Sonnemann*, Daniel P. Fitzsimons{dagger}, Kurt W. Prins* and James M. Ervasti*,1

* Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA; and

{dagger} Department of Physiology, University of Wisconsin, Madison, Wisconsin, USA

1 Correspondence: Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. E-mail: jervasti{at}umn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We generated transgenic mice that overexpressed {gamma}-cyto actin 2000-fold above wild-type levels in skeletal muscle. {gamma}-cyto actin comprised 40% of total actin in transgenic skeletal muscle, with a concomitant 40% decrease in {alpha}-actin. Surprisingly, transgenic muscle was histologically and ultrastructurally identical to wild-type muscle despite near-stoichiometric incorporation of {gamma}-cyto actin into sarcomeric thin filaments. Furthermore, several parameters of muscle physiological performance in the transgenic animals were not different from wild type. Given these surprising results, we tested whether overexpression of {gamma}-cyto actin could rescue the early postnatal lethality in {alpha}-sk actin-null mice (Acta1–/–). By quantitative Western blot analysis, we found total actin levels were decreased by 35% in Acta1–/– muscle. Although transgenic overexpression of {gamma}-cyto actin on the Acta1–/– background restored total actin levels to wild type, resulting in thin filaments composed of 60% {gamma}-cyto actin and a 40% mixture of cardiac and vascular actin, the life span of transgenic Acta1–/– mice was not extended. These results indicate that sarcomeric thin filaments can accommodate substantial incorporation of {gamma}-cyto actin without functional consequences, yet {gamma}-cyto actin cannot fully substitute for {alpha}-sk actin.—Jaeger, M. A., Sonnemann, K. J., Fitzsimons, D. P., Prins, K. W., Ervasti, J. M. Context-dependent functional substitution of {alpha}-skeletal actin by {gamma}-cytoplasmic actin.


Key Words: isoforms • isoform substitution • myofibril • sarcomere • thin filament • nemaline myopathy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACTIN IS ONE OF THE MOST abundant proteins in nature and is involved in a multitude of diverse cellular processes. Six unique actin isoforms exist within higher eukaryotes, each encoded by separate genes (1) . Based on their predominant tissue-specific location, the actins are categorized into 4 muscle-specific isoforms [{alpha}-skeletal ({alpha}-sk), {alpha}-cardiac ({alpha}-ca), {alpha}-smooth ({alpha}-sm), and {gamma}-smooth ({gamma}-sm)] and 2 ubiquitously expressed isoforms [β-cytoplasmic (β-cyto) and {gamma}-cytoplasmic ({gamma}-cyto)]. The biological significance of multiple actin isoforms is not well understood, particularly since all 6 isoforms exhibit an extraordinary degree of amino acid sequence identity (>93%). However, several lines of evidence suggest that each isoform serves a unique purpose. First, all 6 actin isoforms are conserved from birds to mammals (2) and expressed in a tissue-specific and developmentally regulated manner (3 , 4) . Multiple actin isoforms also exist within an individual cell at specific ratios (4 5 6 7) . In addition, actins are sorted into different subcellular compartments (4 , 8 9 10) , suggesting that cells can distinguish between isoforms.

Dramatic developmental shifts in actin isoform expression make skeletal muscle an ideal system for studying the role of multiple actins. Proliferating skeletal myoblasts almost exclusively express β- and {gamma}-cyto actin, but on differentiation these cytoplasmic isoforms are drastically down-regulated and sequentially replaced by {alpha}-ca and {alpha}-sm, and ultimately {alpha}-sk actin (11 , 12) . Although {alpha}-sk actin predominates in mature skeletal muscle, small amounts of other muscle and cytoplasmic actins remain. These actin populations sort to discrete areas of the muscle: sarcomeric actin ({alpha}-ca and {alpha}-sk) to the thin filament and {gamma}-cyto actin to the cortical actin cytoskeleton (13 , 14) . Although {gamma}-cyto actin comprises only 1/4000 of the actin population (15) , muscle-specific ablation of {gamma}-cyto actin results in muscle that develops normally, but leads to a progressive myopathy (16) .

Studies performed on cultured muscle cells have provided further evidence that cytoplasmic actin isoforms have distinct roles in muscle cells. Alteration of the normal 2:1 β- to {gamma}-cyto actin ratio in myoblasts via overexpression of either cytoplasmic actin perturbed cellular morphology and cytoarchitecture (4) . Moreover, transfection of cytoplasmic actins into adult cardiomyocytes resulted in deconstruction of the thin filament and cessation of spontaneous contractions, while transfection of muscle actins had no effect (17) . Thus, in vitro studies suggest that perturbation of the native muscle actin population with cytoplasmic actins is detrimental to muscle cells.

To examine the effects of altering myocyte cytoplasmic actin populations in vivo, we generated transgenic mice that overexpressed {gamma}-cyto actin specifically in skeletal muscle. Contrary to in vitro based predictions, >2000-fold overexpression of {gamma}-cyto actin did not adversely affect skeletal muscle. Furthermore, {gamma}-cyto actin incorporated into thin filaments without altering the functional performance of skeletal muscle. Given these surprising results, we addressed whether gross overexpression of {gamma}-cyto actin rescued the postnatal lethality of {alpha}-sk actin-null mice (18) . Despite a restoration of total actin to wild-type levels, expression of the {gamma}-cyto actin transgene on the {alpha}-sk actin-null background was not able to rescue postnatal lethality, suggesting that {gamma}-cyto actin cannot form functional thin filaments in skeletal muscle independent of {alpha}-sk actin. Our results demonstrate a context-dependent functional substitution of {alpha}-sk actin by {gamma}-cyto actin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of transgenic mice
The full coding sequence of the {gamma}-cyto actin cDNA was reverse-transcribed from wild-type murine kidney RNA using the SuperScript OneStep RT-PCR with Platinum Taq kit (Invitrogen, Carlsbad, CA, USA) and primers KJS41 (5'-gatcgcaATGgaagaagaaatcgc-3') and KJS42 (5'tgcctggcacctgctcagtc-3'). The Actg1 3'UTR was subsequently amplified using RT-PCR primers KJS45 (5'-cgaaatgcttctagatggactgag-3') and KJS46 (5'-ttctttacacaatgacgtgttgctgg-3') and cloned in frame with the coding sequence. This coding sequence-3'UTR construct was inserted downstream of the human skeletal {alpha}-actin (HSA) promoter and between the vp1 intron and tandem SV40 polyadenylation sequences of the HSAvpA expression cassette (kindly provided by Dr. Jeffrey Chamberlain, University of Washington, Seattle, WA, USA) to generate HSAcgaTg. The HSA promoter through the polyadenylation sequence fragment was excised from the plasmid backbone with ClaI/PvuI digestion and microinjected into fertilized C57Bl/6 zygotes, which were then implanted into pseudopregnant female ICR mice. Resultant offspring were screened with transgene-specific primers KJS49 (5'-gtcacccacgtatgagtctttctgg-3') and KJS51 (5'gaagcagacagtattcagcaagtaactg-3') to identify mice carrying the transgene. Six founder mice were positively identified, and 4 of the 5 that survived to breeding age showed germline transmission and muscle-specific overexpression of {gamma}-cyto actin. Transgenic line 3 mice showed the highest {gamma}-cyto actin protein expression level and were used for all experiments. Acta1+/– male breeder mice were kindly provided by Dr. Nigel Laing and Dr. Kristen Nowak (University of Western Australia, Perth, Australia). Animals were housed and treated in accordance with the standards set by the institutional animal care and use committees at the University of Minnesota and University of Wisconsin.

Purification of bovine {gamma}-cyto actin
The purification of {gamma}-cyto actin from bovine brain was modified from Renley et al. (19) . 125 g of unstripped mature bovine brain (Pel-Freeze Biologicals, Rogers, AR, USA) was cut into small pieces and placed in a cooled blender cup with 7 vol of G-buffer + protease inhibitors (2 mM Tris-HCl, 0.2 mM CaCl2, 1 mM NaN3, 0.1 mM ATP, 0.5 mM DTT, 0.1 mM benzamidine, 0.2 mM PMSF, 1.1 µM leupeptin, and 76 nM aprotinin, pH 7.5, at 4°C). The brain was homogenized on high speed (30 s on/ 30 s off, 3 times) and spun at 9000 g for 15 min. The resulting supernatant was spun at 125,000 g and filtered through 8 layers of cheesecloth. The supernatant was run over a column of 25 ml of DNase-I-Affi-Gel 10 and washed sequentially with 75 ml of G-buffer + protease inhibitors, 75 ml of 0.2 M NH4Cl in G-buffer, and 75 ml of G-buffer. Actin was eluted off of the DNase-I-Affi-Gel 10 column and onto a 2-ml DEAE-Sepharose column with 60 ml of 30% deionized formamide in G-buffer. The DEAE-Sepharose column was then washed with 10 ml of G-buffer, and the actin was eluted with 18 1-ml fractions of 0.3 M KCl in G-buffer. Fractions containing actin (as determined by Coomassie gels and A280 readings) were pooled and stored at –80°C.

Determination of actin concentration in skeletal muscle
Known amounts of purified bovine brain actin or purified {alpha}-sk actin (purchased from Cytoskeleton, Denver, CO, USA) and known amounts of SDS extracts from Actg1-TG gastrocnemius or quadriceps femoris muscles were run side by side on a 3–12% SDS-polyacrylamide gel and transferred to nitrocellulose. Two {gamma}-cyto actin antibodies (monoclonal mouse 2–4 and polyclonal affinity-purified rabbit 7577) were used for Western blotting, each at 1:1000 dilution. Two {alpha}-sarc actin antibodies were also used [monoclonal mouse 5c5 (Sigma, St. Louis, MO, USA) and monoclonal mouse Alpha-Sr-1 (Dako, Glostrup, Denmark)] at 1:1000 dilution. For blots comparing {alpha}-sarc actin levels between wild-type and Actg1-TG mice, fast skeletal myosin heavy chain (monoclonal mouse MY-32; Sigma) was used at a dilution of 1:1000 to adjust for equal loading between wild-type and Actg1-TG samples. To determine the concentration of actin (µg) in the SDS extracts, a concentration standard curve was derived from the known values of either purified bovine {gamma}-cyto actin or purified {alpha}-sk actin using the densitometry and concentration standards feature on the Li-Cor software program (Li-Cor Biosciences, Lincoln, NE, USA).

Isolation of myofibrils
Strips of skeletal muscle were tied to small wooden dowels and soaked overnight at 4°C in 50% glycerol/50% wash solution (150 mM NaCl, 2 mM MgCl2, 10 mM imidazole, 2 mM EGTA, 0.5 mM DTT, and 0.1 mM PMSF, pH 7.0). Muscles were then removed from the dowels, diced into small pieces with scissors, and incubated with stirring at 4°C for 30 min in 20 vol of cold wash buffer to remove glycerol. Fresh wash buffer (20 vol) was then added to the muscle, and it was homogenized on ice for 30 s with a Tissue Tearor homogenizer (Biospec Products, Bartlesville, OK, USA) on low setting. After centrifugation at 4000 g for 10 min, the pellet was washed by resuspending 3 times in 20 vol of wash buffer to remove cytoplasmic components, pelleting at 4000 g each time. The pellet was then resuspended and incubated for 20 min at 4°C in wash buffer containing 0.5% Triton X-100. After centrifugation at 4000 g for 10 min, the pellet was washed three more times with wash buffer, pelleting each time at 4000 g for 10 min. After the final wash and spin, the top white fluffy layer containing myofibrils was resuspended in wash buffer.

Confocal microscopy
Mouse skeletal muscle was dissected, frozen in melting isopentane, and mounted in O.C.T. medium (TissueTek, Torrance, CA, USA) for cryosectioning. Transverse sections (10 µm) of skeletal muscle were immediately fixed in 4% paraformaldehyde in PBS for 10 min, washed with PBS 3 x 2 min, and blocked with 5% goat serum in PBS for 30 min. Primary antibody was diluted in PBS and applied to sections overnight at 4°C or at room temperature for 4 h. Sections were washed 3 x 2 min with PBS and secondary antibody (Alexa 488 and Alexa 568; Invitrogen) applied for 30 min at 37°C. After several washes with PBS, slides were coverslipped with a drop of ProLong Gold AntiFade with DAPI (Invitrogen). The following antibody dilutions were used for immunofluorescence experiments performed on transverse sections: 1:75 affinity purified {gamma}-cyto actin rabbit 7577, 1:200 laminin {alpha}-2 rat clone 4H8–2 (Sigma) and 1:100 slow skeletal myosin clone NOQ7.5.4D (Sigma).

For immunofluorescence microscopy of isolated myofibrils, the myofibrils were diluted in ddH2O and spread out to dry on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA). The samples were then fixed, blocked, and stained as described above using the following dilutions: 1:75 affinity purified {gamma}-cyto actin rabbit 7577, 1:100 fast myosin heavy chain clone MY-32 (Sigma), 1:500 {alpha}-actinin clone EA-53 (Sigma), 1:50 {alpha}-actin clone Sr-1 (Dako).

All images were obtained on an Olympus Fluoview 1000 Single Photon confocal microscope equipped with Olympus PlanApoN 1.42 NA x60 oil, UPlanFL N 1.30 NA x40 oil, and UPlan SApo 0.40 NA x10 objective lenses (Olympus, Tokyo, Japan). Images were collected with Olympus Fluoview 1.7b software and assembled and processed with ImageJ (National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop (version 8.0; Adobe Systems, San Jose, CA, USA) software.

Serum creatine kinase analysis
Serum was collected and stored at –80°C until analysis. Serum (10 µl) was applied to a Vitros CK DT slide (Ortho-Clinical Diagnostics, Inc., Rochester, NY, USA) and analyzed with a Kodak Ektachem DT60 (Eastman Kodak Company, Rochester, NY, USA) and DTSE II module (Ortho-Clinical Diagnostics, Inc.).

Transmission electron microscopy
Anesthetized mice were transcardially perfused with 3% buffered glutaraldehyde, and desired muscles were excised and placed in glutaraldehyde for 2 additional hours. After extensive washing with 0.1 M PBS, samples were treated with 2% osmium tetroxide for 1 h, dehydrated in ethanol, and embedded in EMbed-812 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were poststained with 5% uranyl acetate and 1% lead citrate and viewed on a Jeol 100CX transmission electron microscope (Jeol Ltd., Akishima, Japan).

Force measurements
Ex vivo force measurements
The extensor digitorum longus muscle from 4-mo-old mice was dissected; one tendon was attached to a rigid support and the other to a dual-mode servomotor. The muscle was allowed to equilibrate in a Ca2+-Ringer’s solution continuously gassed with 95% O2/5% CO2 to maintain a pH of 7.6 at 30°C. Platinum plate electrodes were positioned on either side of the muscle, and stimulation was induced by a single pulse lasting 200 µs. The muscle was adjusted to the length (L0) at which maximal twitch force was achieved. The muscle was then subjected to an eccentric contraction regimen consisting of 5 maximal tetanic stimulations. Each stimulation was carried out over 700 ms; during the final 200 ms, the muscle was lengthened at a velocity of 0.5 L0/s, resulting in a total stretch of 10% L0.

In vivo physiological performance
Whole-body tension measurements were based on established techniques by Carlson and Makiejus (20) . A mouse was attached by the tail to a horizontally mounted force transducer using suture silk and subsequently placed in an apparatus that allowed only forward movement. The force evoked by gentle tail pinches (~5 pinches/min) was recorded for 5 min, and the top 5 values were identified and normalized to body mass.

Maximal exercise performance was tested on a Columbus Instruments treadmill with an uphill grade of 15°(Columbus Instruments, Columbus, OH, USA). Mice were acclimated to the treadmill by running at a speed of 10 m/min for 5 min, 3 times/wk, for 2 wk. To determine maximal exercise performance mice were run on the treadmill for 5 min, at a speed of 10 m/min, followed by a 1-m/min increase in speed every minute until exhaustion. Mice were considered exhausted when they refused to stay off a shock bar for at least 5 s. Maximal exercise capacity was determined as the average duration of 2 trials separated by 2 d.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Actg1-TG mice overexpress {gamma}-cyto actin >2000-fold in skeletal muscle
We used the HSA promoter to drive overexpression of the Actg1 cDNA in murine skeletal muscle to examine how elevated levels of {gamma}-cyto actin affect skeletal muscle in vivo. SDS lysates from transgenic {gamma}-cyto actin (Actg1-TG) skeletal muscle were analyzed alongside a calibration curve of purified {gamma}-cyto actin and quantified using the Li-Cor Odyssey infrared imaging system (Fig. 1A ). Quantitative Western blotting with 2 different {gamma}-cyto actin antibodies (15) gave a value of 409 ± 43 µM {gamma}-cyto actin in Actg1-TG skeletal muscle (Fig. 1A, C ). In wild-type skeletal muscle, {gamma}-cyto actin was previously measured at 0.197 ± 0.05 µM (15) , but required extraction and enrichment in order to be measured. Thus, Actg1-TG mice minimally expressed {gamma}-cyto actin at levels 2000-fold above wild type. Using the same methods, the abundance of {alpha}-sarcomeric actin ({alpha}-sk and {alpha}-ca) was measured at 941 ± 123 µM for wild-type skeletal muscle (Fig. 1B ), which is very similar to the 893 ± 42 µM {alpha}-sarc actin reported previously for wild-type skeletal muscle (15) . In Actg1-TG skeletal muscle, {alpha}-sarc actin abundance was measured at 571 ± 51 µM (Fig. 1A, C ). Together, {gamma}-cyto actin (409 µM) and {alpha}-sarc actin (571 µM) levels in Actg1-TG skeletal muscle totaled 980 µM, which is consistent with {alpha}-sarc actin abundance in wild-type muscle (941 µM) (Fig. 1C ). Thus, up-regulation of {gamma}-cyto actin in Actg1-TG skeletal muscle led to a concomitant down-regulation of {alpha}-sarc actin to maintain total actin levels similar to {alpha}-sarc actin abundance in wild-type muscle.


Figure 1
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  		Figure 1. Actg1-TG mice express {gamma}-cyto actin 2000-fold above wild-type levels in skeletal muscle. A, B) Quantitative Western blot analysis was performed to determine the concentration of {gamma}-cyto actin (A) and {alpha}-sarc actin (B) in skeletal muscle. C) Actg1-TG mice expressed {gamma}-cyto actin in skeletal muscle at levels that were >2000-fold above wild-type levels. However, total actin levels were not altered due to decreased {alpha}-sarc actin expression in skeletal muscle. *Wild-type skeletal muscle {alpha}-sarc actin levels previously calculated (15) . D) {gamma}-Cyto actin staining was predominately seen in capillaries (arrowhead), blood vessels (double-headed arrow) and nerves (arrow) in wild-type skeletal muscle. In addition, {gamma}-cyto actin brightly stained the internal structure of fast-twitch fibers in Actg1-TG skeletal muscle.

{gamma}-cyto actin incorporates into Actg1-TG thin filaments
To determine the localization of {gamma}-cyto actin in Actg1-TG mice, immunofluorescence microscopy was performed on transverse cryosections of wild-type and Actg1-TG muscles. In wild-type skeletal muscle, {gamma}-cyto actin polyclonal antibodies brightly stained capillaries, blood vessels and nerves (Fig. 1D ). No internal staining was observed in wild-type myofibers. However, the internal space of many Actg1-TG fibers stained intensely, while some smaller fibers were devoid of staining (Fig. 1D ). Staining with slow myosin heavy chain and {gamma}-cyto actin antibody demonstrated that many fibers devoid of staining were slow-twitch fibers, although not exclusively. This staining pattern was consistent with reports that the HSA promoter is preferentially expressed in fast-twitch myofibers (21) . Staining of gastrocnemius, quadriceps femoris, tibialis anterior, extensor digitorum longus, and diaphragm with {gamma}-cyto actin antibody showed that >90% of the fibers within each muscle had medium to high-intensity staining. Fibers that showed little to no {gamma}-cyto actin expression tended to cluster in focal areas of muscle, whereas other larger regions showed homogenous bright staining (see gastrocnemius staining in lower right panel of Fig. 1D ).

The internal space of muscle fibers is comprised of a densely packed network of myofibrils. Myofibrils are composed of repeating thick- and thin-filament bundles that make up the contractile sarcomere. Normally, {alpha}-sk actin is the dominant, if not sole, actin isoform within sarcomeric thin filaments of adult skeletal muscle. To determine whether the intense {gamma}-cyto actin staining present in Actg1-TG muscle resulted from {gamma}-cyto actin incorporation into thin filaments, immunostaining was performed on isolated myofibrils. Bright staining of {gamma}-cyto actin was observed interspersed between thick filaments in Actg1-TG myofibrils (as indicated by fast myosin heavy chain staining, Fig. 2A ), but was absent in wild-type myofibrils. Also consistent with thin-filament incorporation, {gamma}-cyto actin staining flanked {alpha}-actinin Z-disk staining (Fig. 2B ). Myofibrils probed with both {alpha}-sarc actin and {gamma}-cyto actin showed areas of colocalization (Fig. 2C ). However, some areas showed differential enrichment of either {gamma}-cyto actin or {alpha}-sarc actin (Fig. 2C ). Combined, these immunofluorescence analyses demonstrate that {gamma}-cyto actin in Actg1-TG mice localized to areas that are consistent with incorporation into thin filaments.


Figure 2
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  		Figure 2. {gamma}-cyto actin incorporates into Actg1-TG skeletal muscle thin filaments. A) Isolated myofibrils from wild-type or Actg1-TG mice were stained with {gamma}-cyto actin and fast myosin heavy chain. B, C) Actg1-TG isolated myofibrils were stained with {gamma}-cyto actin and {alpha}-actinin or {alpha}-sarc actin. Boxed areas in left panels of C are magnified at right. Scale bars = 5 µm.

Gross overexpression of {gamma}-cyto actin does not adversely affect muscle cell architecture or integrity in vivo
Based on the detrimental effects of overexpressing {gamma}-cyto actin in vitro (4 , 17 , 22) , we examined Actg1-TG mice for evidence of muscle pathology. Transmission electron microscopy of Actg1-TG skeletal muscle revealed well-defined sarcomeres and overall ultrastructure that was indistinguishable from wild-type skeletal muscle (Fig. 3A ). Hematoxylin and eosin (H&E)-stained transverse cryosections of Actg1-TG skeletal muscle also demonstrated that the general fiber architecture (shape and size) was similar to wild-type skeletal muscle (Fig. 3B ). In addition, no fibrosis, fat deposits, or mononuclear cell infiltration were observed. To quantitatively assess whether gross overexpression of {gamma}-cyto actin in muscle resulted in muscle pathology, levels of centrally nucleated fibers (CNFs) in H&E-stained tibialis anterior and gastrocnemius muscle were quantified from wild-type, Actg1-TG, and dystrophin-deficient mdx mice. Muscle nuclei are normally located at the periphery of myofibers; however, the nuclei are located internally in a cell that has been damaged and subsequently regenerated. In Actg1-TG tibialis anterior and gastrocnemius muscles, CNF levels were very low (<2% at all ages) and not significantly different from wild type (Fig. 3C ). In contrast, the mdx mouse exhibited >30% CNF in each muscle at all ages examined (Fig. 3C ). In addition to histological parameters, many mouse models of muscle disease exhibit high serum levels of the muscle enzyme creatine kinase, indicative of sarcolemmal damage. Actg1-TG mice had wild-type serum creatine kinase levels, while mdx mice had significantly higher serum creatine kinase levels (Fig. 3C ). Overall, Actg1-TG skeletal muscle had normal cytoarchitecture with no evidence of increased cell death or membrane damage.


Figure 3
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  		Figure 3. Gross overexpression of {gamma}-cyto actin in skeletal muscle does not result in muscle pathology. A) Transmission electron micrographs of 2-mo-old wild-type and Actg1-TG gastrocnemius muscle. No ultrastructural abnormalities were observed in Actg1-TG samples. Scale bars = 2 µm. B) H&E-stained cryosections from 1-yr-old wild-type and Actg1-TG gastrocnemius muscle. C) CNFs were quantified from entire H&E sections of tibialis anterior and gastrocnemius muscles at various time points. Both wild-type and Actg1-TG muscles had low levels of CNFs, as well as low levels of serum creatine kinase, compared to the myopathic mdx mouse model. *P < 0.05 vs.wild-type and Actg1-TG samples; 1-way ANOVA with Tukey’s multiple comparison post hoc test; n ≥ 3 for each genotype/time point. Error bars = SEM.

Actg1-TG mice exhibit normal ex vivo and in vivo muscle performance
Because perturbation of the actin composition in cardiac muscle thin filaments altered cardiac muscle contractile properties (23) , a series of in vivo and ex vivo force measurements were performed with Actg1-TG mice to determine whether incorporation of {gamma}-cyto actin into sarcomeres affected muscle performance. Ex vivo analyses were conducted on isolated extensor digitorum longus muscles, which expressed high levels of {gamma}-cyto actin (Fig. 4A ). Twitch force—the amount of force elicited by a single action potential—was identical between wild-type and Actg1-TG muscle (Fig. 4A ). Similarly, the maximal force generated during tetanic stimulation was indistinguishable between the 2 groups (Fig. 4A ). Force generation following a series of eccentric (lengthening) contractions was also examined. While eccentric contractions are injurious to even wild-type muscle, several animal models of muscular dystrophy display significantly greater susceptibility to eccentric contraction induced muscle damage (24 25 26 27) . Actg1-TG and wild-type extensor digitorum longus muscles showed a similar decrement in maximal force production after four eccentric contractions (Fig. 4A ). Similarly, in vivo studies examining treadmill endurance and whole-body force generation showed no differences between wild-type and Actg1-TG mice (Fig. 4B ). Overall, thin filaments composed of 40% {gamma}-cyto actin were functionally equivalent to wild-type filaments.


Figure 4
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  		Figure 4. Actg1-TG mice exhibit normal ex vivo and in vivo muscle performance. A) Extensor digitorum longus muscle stained with {gamma}-cyto actin antibody. Scale bar = 100 µm. Ex vivo force measurements: normalized twitch force, tetanic force, and force deficit after 4 eccentric contractions did not differ between wild-type and Actg1-TG extensor digitorum longus muscles (Student’s t test; n=4 mice/genotype). B) In vivo force measurements: time to exhaustion on a treadmill did not differ between wild-type and Actg1-TG mice (t test; n=5 wild-type mice, n=4 Actg1-TG mice). Whole-body tension also did not differ between wild-type and Actg1-TG mice. Average of the top 5 pulling responses is depicted (Student’s t test; n=9 wild-type mice, n=5 Actg1-TG mice). Error bars = SEM.

Gross overexpression of {gamma}-cyto does not rescue {alpha}-sk actin-null mouse lethality
As sarcomeres containing near-stoichiometric amounts of {gamma}-cyto actin were fully functional (Fig. 4 ), we tested the hypothesis that overexpression of {gamma}-cyto actin in skeletal muscle could rescue the postnatal lethality associated with the absence of {alpha}-sk actin. Mice null for {alpha}-sk actin (Acta1–/–) are indistinguishable from their littermates at birth but die by postnatal day 10 (18) . Similarly, humans born without {alpha}-sk actin are severely impaired and often die within the first few months of life (28) . We crossed Acta1+/– with Actg1-TG mice to generate mice that were null for {alpha}-sk actin and overexpressed {gamma}-cyto actin in skeletal muscle (Acta1–/–;TG). Both Acta1–/– and Acta1–/–;TG mice were born at expected Mendelian ratios (Fig. 5A ). Interestingly, in the absence of {alpha}-sk actin, Acta1–/–;TG mice expressed {gamma}-cyto actin at significantly higher levels (631 µM) than Actg1-TG mice (409 µM) (Fig. 5B ), indicating that transgene expression was context dependent. Although Acta1–/– mice are able to form skeletal muscle via the up-regulation of {alpha}-ca and {alpha}-sm actin (18) , quantitative Western blotting of total muscle extracts with a pan-actin antibody revealed that Acta1–/– skeletal muscle contained 35% less actin than wild-type skeletal muscle (Fig. 5C ). Total actin levels were restored when the Actg1 transgene was expressed on the Acta1–/– background (Fig. 5C ). Despite wild-type levels of actin, Acta1–/–;TG mice did not thrive. Similar to Acta1–/– pups, Acta1–/–;TG pups failed to gain weight normally (Fig. 5D ), were smaller in stature than their littermates (Fig. 5E ), and died by 15 d of age (Fig. 5F ). Acta1–/–;TG skeletal muscle was histologically indistinguishable from Acta1–/– skeletal muscle (data not shown), displaying prominent interstitial space between fibers, as described previously (18) . Collectively, these results indicate that, while {gamma}-cyto actin can contribute to functional thin filaments in the presence of {alpha}-sk actin, {gamma}-cyto actin cannot serve as a functional replacement for {alpha}-sk actin.


Figure 5
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  		Figure 5. Transgenic {gamma}-cyto actin overexpression cannot rescue Acta1–/– lethality. A) All pups were born at expected Mendelian ratios as determined by {chi}2 analysis. B) {gamma}-Cyto actin levels were significantly higher in Acta1–/–;TG skeletal muscle compared to adult Actg1-TG skeletal muscle. *P = 0.0007; unpaired t test. C) Total actin abundance was significantly lower in Acta1–/– skeletal muscle compared to wild-type skeletal muscle. However, total actin abundance was restored with transgenic overexpression of {gamma}-cyto actin. *P < 0.0001 vs. Acta1+/+; single-sample 2-tailed t test. D) Acta1–/– and Acta1–/–;TG pups weighed significantly less than their littermates from 7 d of age until their natural death. *P < 0.01, Acta1–/–;TG vs. Acta1+/+; #P < 0.01, Acta1–/– vs. Acta1+/+; 2-way ANOVA. E) Acta1–/– and Acta1–/–;TG mice were smaller in stature compared to littermates. Representative image of an Acta1–/–;TG pup at 11 d of age (arrow). F) Age of death did not differ significantly between Acta1–/– and Acta1–/–;TG mice. P > 0.05; paired t test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We examined whether a cytoplasmic actin can function as a muscle actin in vivo by transgenically overexpressing {gamma}-cyto actin in skeletal muscle and observed that {gamma}-cyto actin can form functional thin filaments in a context-dependent manner. A 2000-fold up-regulation of {gamma}-cyto actin and corresponding decrease in {alpha}-sarc actin resulted in {gamma}-cyto actin representing nearly 50% of the total actin population in transgenic skeletal muscle. The maintenance of total actin at wild-type levels supports previous observations that sarcomeric protein levels are maintained at strict stoichiometries (29 30 31 32) . We also observed that {gamma}-cyto actin incorporated efficiently into myofibrillar thin filaments, which agrees with most in vitro studies (33 , 34) . Although von Arx et al. (17) reported that {gamma}-cyto actin overexpression dramatically perturbed the cytoarchitecture and contractile function of cardiac myocytes, our results demonstrate that {gamma}-cyto actin is capable of near stoichiometric incorporation into skeletal myofibrils in vivo without disrupting skeletal muscle cytoarchitecture or function.

Actg1-TG muscle performance was identical to wild type as measured through several ex vivo and in vivo assays, suggesting that there is considerable functional redundancy between {gamma}-cyto and {alpha}-sk actin isoforms. Therefore, we tested whether {gamma}-cyto actin overexpression could rescue the early postnatal lethality associated with total {alpha}-sk actin deficiency. Acta1–/– mice can breathe, suckle, and walk at birth (18) . Although Acta1–/– skeletal muscle up-regulates alternative actin isoforms ({alpha}-ca, {alpha}-sm, and {gamma}-sm) and exhibits no obvious ultrastructural defects, mice fail to gain weight, have weaker muscles, and die by 10 d of age (18) . We measured the total actin abundance in Acta1–/– skeletal muscle and found that it was significantly lower (35%) than in wild-type mice. We also noted that Acta1+/– skeletal muscle expressed 17% less actin (Fig. 5C ), but heterozygous mice did not present with an overt phenotype (ref. 18 and unpublished results). Although {gamma}-cyto actin overexpression restored total muscle actin to wild-type levels, the Actg1 transgene was not able to significantly prolong survival in Acta1–/– pups. From these data, we conclude that thin-filament actin isoform composition—and not concentration—is critical for normal skeletal muscle function. Similar results were observed in Drosophila melanogaster, whereby complete replacement of flight muscle actin with cytoplasmic actin resulted in disruption of flight muscle structure and function (35) .

In contrast to our data, transgenic overexpression of {alpha}-ca actin fully rescued Acta1–/– mice (36) , supporting the observation that survival beyond birth in ACTA1-null humans correlates with the extent of {alpha}-ca actin up-regulation (28) . Similarly, transgenic overexpression of {gamma}-sm actin rescued {alpha}-ca actin-mice from embryonic and postnatal lethality (37) . In both {alpha}-sk- and {alpha}-ca-null mice, up-regulation of alternative actin isoforms resulted in the formation of sarcomeric structures. However, transgenic overexpression of distinct muscle actin isoforms was necessary to rescue lethality, arguing that a threshold level of muscle actin expression is necessary for viability. In all cases where transgenic or endogenous up-regulation of a muscle actin compensated for a muscle actin deficiency the muscle was functionally distinct from wild type (36 37 38) , arguing that muscle actins are optimally suited for their respective tissue-specific functions.

Our results extend the prior studies by demonstrating that thin filaments composed of 40% {gamma}-cyto actin retain wild-type function, yet thin filaments composed of 60% {gamma}-cyto actin in the absence of {alpha}-sk actin result in nonviable mice. The former result is surprising given that actin isoforms show different efficiencies in activating skeletal muscle myosin ATPase (39 40 41 42) and overexpression of {gamma}-sm actin in wild-type hearts altered contractile performance (23) . We can envision several scenarios that may explain our current results. First, at 40% incorporation of {gamma}-cyto actin into Acta1+/+ sarcomeres, each {gamma}-cyto actin molecule would likely be adjacent to an {alpha}-sk actin molecule, which may allosterically influence {gamma}-cyto actin molecules to behave like {alpha}-sk actin subunits. At a higher molar ratio such as that measured in the Acta1–/–;TG skeletal muscle (60%), {gamma}-cyto actin molecules bounded by identical neighbors may function as cytoskeletal filaments with altered structure or dynamics that no longer properly support skeletal myosin or thin-filament regulatory protein function. Alternatively, the combined up-regulation of {gamma}-cyto, {alpha}-ca, {alpha}-sm, and {gamma}-sm isoforms in Acta1–/–;TG skeletal muscle may yield sarcomeric thin filaments that are simply too heterogeneous to sustain normal muscle function. Yet a third possibility is that there may be an unidentified function for {alpha}-sk actin that is independent of muscle contraction, perhaps outside of the thin-filament compartment.

The functional significance behind the strict conservation of actin coding sequences throughout evolution remains elusive. Our data indicate that partial substitution of one actin isoform for another without functional consequences is insufficient to conclude there is complete functional redundancy. Therefore, the development of new mouse "rescue" models designed to investigate functional redundancy among actin family members in a tissue-specific context is currently the most logical route to answer such questions. In addition, future studies to identify how isoform substitution influences the expression or function of actin regulatory proteins are likely required before we can adequately understand the importance and significance of the 6 highly conserved, yet functionally distinct actin isoforms.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Nigel Laing and Dr. Kristen Nowak (University of Western Australia, Perth, Australia) for generously providing Acta1+/– mice and for sharing their data prior to publication, and Dr. Richard L. Moss for helpful comments and suggestions. This study was supported by American Heart Association Predoctoral Fellowships to M.A.J and K.J.S., and NIH grant AR049899 to J.M.E.

Received for publication January 13, 2009. Accepted for publication February 12, 2009.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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K. J. Nowak, G. Ravenscroft, C. Jackaman, A. Filipovska, S. M. Davies, E. M. Lim, S. E. Squire, A. C. Potter, E. Baker, S. Clement, et al.
Rescue of skeletal muscle {alpha}-actin-null mice by cardiac (fetal) {alpha}-actin
J. Cell Biol., June 1, 2009; 185(5): 903 - 915.
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