PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of f1000resSubmitAuthor GuidelinesAboutAdvisory PanelF1000ResearchView this article
 
Version 2. F1000Res. 2016; 5: 416.
Published online 2016 June 1. doi:  10.12688/f1000research.8154.2
PMCID: PMC4893938
Other versions

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle

Abstract

Higher vertebrates (mammals and birds) express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (α-SMA) was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986) . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

Keywords: Actin isoforms, monoclonal antibodies, epitope, muscle repair

Introduction

Expression of actin isoforms in skeletal muscle at the mRNA and protein levels have been described since the 1980s using tissue extracts ( Gunning et al., 1983; Hayward & Schwartz, 1986; Hayward et al., 1988; Minty et al., 1982; Ordahl, 1986; Paterson & Eldridge, 1984; Sassoon et al., 1988; Vandekerckhove et al., 1986). These studies have indicated that sarcomeric actins (α-CAA and α-SKA) are expressed in fetal and regenerating skeletal muscle, whereas α-SKA becomes the predominant actin isoform in mature skeletal muscle. Noteworthy, during fetal life, another actin isoform, α-SMA, is highly expressed and precedes the sarcomeric isoforms ( Bochaton-Piallat et al., 1992; Hayward & Schwartz, 1986; McHugh et al., 1991; Woodcock-Mitchell et al., 1988).

Nevertheless, very little is known concerning the precise localization of these isoforms due to the lack of specific α-SKA and α-CAA antibodies. The first mAb against α-CAA has allowed the identification, by immunohistochemistry, of a α-CAA transient expression in human skeletal muscle satellite cells during skeletal regeneration induced by muscle injury, while normal skeletal muscle was negative ( Franke et al., 1996). Ten years later, the same group, in a more extensive study, has further analyzed the expression and localization of α-CAA in normal, regenerating, diseased and neoplastic human muscle tissues ( Moll et al., 2006). In human fetal skeletal muscle, a uniform strong α-CAA staining was observed while in normal adult skeletal muscle, α-CAA was identified only in few thin fibers. α-CAA staining of thin fibers became stronger in human regenerating skeletal muscle after traumatic injury, as well as in Duchenne muscular dystrophy (DMD). Whether the persistence of α-CAA in DMD myofibers is due to a lack of differentiation or to a regenerative process deserves further examination. Interestingly, resting satellite cells in healthy adult muscle lack this isoform, whereas, when satellite cells are activated during the regeneration process, α-CAA is up-regulated. Unfortunately, in these two studies, a comparative α-SKA staining was not performed.

In a more recent study, the switch of α-CAA to α-SKA during the differentiation of skeletal muscle from mouse embryonic stem cells has been examined ( Mizuno et al., 2009). In this in vitro system, α-CAA appeared first in myoblasts, with no staining for α-SKA. During cell fusion, α-SKA appeared. When myotubes began to form sarcomeres, α-SKA expression increased while α-CAA began to decrease. Finally, mature skeletal muscle fibers were mainly composed of α-SKA. Although, this in vitro system seems to recapitulate the in vivo skeletal muscle differentiation at the level of sarcomeric actins switching, the in vivo origin of progenitor cells during in vivo muscle differentiation/repair remains elusive.

The sequential expression of the two striated actins during: i) heart development (high expression of α-SKA at birth, predominant expression of α-CAA in differentiated cardiac muscle at adult life) and ii) skeletal muscle development (high expression of α-CAA at birth, predominant of α-SKA in differentiated skeletal muscle at adult life) has been known from mRNA studies for decades, but little is known about the distribution and/or the localization of the two α-sarcomeric muscle actins, because of the lack of double immunostainings availability. Nevertheless, our laboratory, in collaboration with others, has studied α-SKA expression and distribution, in particular in developing and pathological hearts, using affinity polyclonal antibodies ( Clément et al., 1999), again without double immunostaining.

As mentioned above, α-CAA distribution has been studied during muscle development and repair ( Moll et al., 2006), but a comparative study with α-SKA was still missing. Our mAbs were raised using the acetylated N-terminus decapeptide of each isoform (see Table 1). The two different mAb subtypes (anti-α-SKA, IgG2b and anti-α-CAA, IgG1), allowed a clear analysis of the expression and distribution of the two actin isoforms in mature skeletal muscle and during regeneration after micro-lesions by means of highly specific anti- mouse subtype secondary antibodies.

Table 1.

List of monoclonal antibodies against the 6-actin isoforms raised in our laboratory.

The epitope recognized by each antibody is enlightened in bold.

Actin isoformImmunogenMAb nameCloneSubtypeOriginal ReferenceDistributorsCatalog Number
α-SMA AcEEEDSTALVCAnti-α-SM11A4IgG2a Skalli et al., 1986 Abcam
AbD Serotec
Cell Marque Corporation
Dako
eBioscience
EMD Millipore
Genemed Biotechnologies
Nordic-MUbio
R§D Systems
Santa Cruz Biotechnology
Sigma-Aldrich
Spring Bioscience
Zeta Corporation
ab-7817
MCA5781GA
202M
M0851
14–9760
113200
61–0001
MUB0107S
MAB1420
sc-32251
A5228
E14344
Z2066
γ-SMA AcEEETTALVCAnti-γ-SMA20D2IgG1 Arnoldi et al., 2013 NANA
β-CYA AcDDDIAALVCAnti-β-CYA4C2IgG1 Dugina et al., 2009 AbD Serotec
EMD Millipore
Nordic-MUbio
MCA5775GA
MABT825
MUB0110S
γ-CYA AcEEEIAALVCAnti-γ-CYA2A3IgG2b Dugina et al., 2009 AbD Serotec
EMD Millipore
Nordic-MUbio
MCA5776GA
MABT824
MUB0111S
α-SKA and α-CAA
Sarcomeric actins
Rabbit skeletal actinAnti-α-SR15C5IgM Skalli et al., 1988 Dako
Santa Cruz Biotechnology
Sigma-Aldrich
Spring Bioscience
M0874
sc-58670
A 2172
E16654
α-SKA AcDEDETTALVCAnti-α-SKA3B3IgG1 Driesen et al., 2009 Nordic-MUbioMUB0108S
α-SKA AcDEDETTALVCAnti-α-SKA10D2IgG2a Driesen et al., 2009 NANA
α-CAA AcDDEETTALVCAnti-α-CAA22D3IgG1 Driesen et al., 2009 EMD Millipore
Nordic-MUbio
MABT823
MUB0109S

Materials and methods

Reagents details

Details of all reagents with reference to the immunoblot and immunostaining procedure can be found in Table 2 and Table 3. Crucial are the conditions of fixation and permeabilization for relevant immunostaining. With cells in culture as well as with tissues, we have tested a large number of conditions such as MeOH, EtOH, PFA-TX100. By far, the use of PFA, followed by MeOH, as described in Table 3 and previously defined ( Dugina et al., 2009), allowed the best detection of every actin isoform, likely because of availability of the actin molecule N-terminus.

Table 2.

Western blot protocol for the identification of the epitopes recognized by anti-α-SKA (clone 10D2) and anti-α-CAA (clone 22D3).
Protocol stepsReagentConcentration/
dilution
Time
Migration of Purified α-SKA and α-CAA on
SDS-PAGE
1μg/laneovernight
   • Transfer on Nitrocellulose membranes.
   • Membranes cut in strips for Ab-peptides
      incubation.
2 h
Preincubation of antibodies with peptidesMAb:
   • anti-α-SKA (10D2)
   • anti-α-CAA (22D3)
For details, see Table 1

Blocking Peptides (see Figure 1)

1:5000
1:5000


100 μg/ml
1 h
Membranes pretreatment5% dried skimmed milk in TBS5%1 h
Membranes Incubation with antibody-peptidesIn 0.1% TX-100/TBS2 h
Membranes Incubation with 2 nd antibodyGoat Anti-Mouse IgG (H+L) HRP
Conjugate (BioRad) #1706516
In 0.1% TX-100/TBS
1:10’0001 h
HRP activity developmentECL protocol according to (RPN
2109; GE Healthcare),
5–60 sec

Table 3.

Details of immunofluorescence staining protocol.
Protocol stepsReagentConcentration
/dilution
TimeCatalogue
Number
   • Cryopreserved Rat muscles
   • Cryostat sections (3μm)
Fixation   • Paraformaldehyde
   • Methanol
1%
100%
30 min at RT
5 min at –20°C
StainingPrimary Abs:
MAbs:
   • anti-α-SKA (10D2)
   • anti-α-CAA (22D3)
   • anti-α-SMA (1A4)
For details, see Table 1

   • anti-vimentin (clone V9)
Immunogen: Purified vimentin from porcine eye lens
Specificity: the antibody cross-reacts with
vimentin in man, cow, dog, hamster, horse, rhesus
and African green monkey, rabbit, rat and rat
kangaroo. No cross-reaction with mouse vimentin
could be demonstrated, and results on chicken
specimens are contradictory.

Rabbit polyclonal Ab:
   • anti-α-CAA ( Clément et al., 2003)


1:50
1:50
1:50


1:300








1:100
2 h

Home made
Home made
Home made


Dako #M 0725








Home made
Secondary Abs (Jackson Immmunoresearch):
   • AffiniPure Goat Anti-Mouse IgG1, Alexa 488
   • AffiniPure Goat Anti-Mouse IgG1, Alexa 594
   • AffiniPure Goat Anti-Mouse IgG2a, Alexa 488
   • AffiniPure Goat Anti-Mouse IgG2a, Alexa 594
   • AffiniPure Goat Anti-Rabbit-Alexa 594

1:200
1:200
1:200
1:200
1:100
1 h
115-545-205
115-585-205
115-545-206
115-585-206
111-585-144

Animals

All animal experiments (production of antibody in mice, rat wounds, tissue samplings) were approved by and performed in accordance with the cantonal and federal veterinary authorities.

Tissue details

Tibialis muscle specimens from female Wistar rats older than 10 weeks were rapidly embedded in OCT compound (Tissue-Tek), snap-frozen in a beaker containing precooled liquid isopentane, immerged in liquid nitrogen, and stored at -80°C. For muscle repair studies, rat tibialis muscles were injured with a liquid nitrogen cooled needle, using a well-set simple rat model ( Rocheteau et al., 2012). Muscles were taken, OCT embedded and frozen at different days after injury.

Antibody production and details

MAbs against α-SKA and α-CAA were prepared following the Repetitive Immunizations Multiple Sites (RIMMS) strategy ( Kilpatrick et al., 1997). This strategy uses lymphocytes from regional draining lymph nodes and an immunization schedule significantly shorter than conventional techniques: two weeks instead of several months. Along with the use of less antigen, this approach allows to obtain hybridomas in a month.

Mice were immunized with the acetylated N-terminal decapeptide of α-SKA (Ac-DEDETTALVC-COOH) or α-CAA (Ac-DDEETTALVC-COOH) conjugated with keyhole limpet haemocyanin through the cysteine peptide C-terminus (KLH, Imject Maleimide Activated carrier proteins, Pierce) according to the instructions of the manufacturer. Briefly, over a period of 10 days, 5 injections of 5 μg of protein (α-SKA or α-CAA peptide x KLH) emulsified in complete Freund’s adjuvant (first injection) or with incomplete Freund’s adjuvant (for the remaining injections) and RIBI adjuvants (Sigma-Aldrich) were given at six subcutaneous sites proximal to draining peripheral lymph nodes (PLNs) in three anesthetized six-week-old female BALB/c mice (200μl/mice). Two days after the final boosts, animals were sacrificed. PLNs were harvested from popliteal, superficial inguinal, axillary, and brachial lymph nodes and dissociated. Up to 1 × 10 8 lymphocytes per 3 mice, were used for fusions with 2.5 × 10 7 with NSO myeloma cells using 50% polyethylene glycol (PEG 1500, Sigma-Aldrich). Fused cells were plated in 24-well tissue culture plates (4000 cells/well). Hybridomas were grown in DMEM+ pyruvate, 5% FCS, 10% NCTC 109 (Sigma-Aldrich), 1× MEM Non-Essential Amino Acids (Gibco), 1% Hybridoma Fusion and Cloning Supplement (HFCS, Roche), penicillin/streptomycin, and 1× selective media hypoxanthine/aminopterin/thymidine (HAT). Supernatants were harvested after 2 weeks and screened for antibody specificity by ELISA and immunofluorescence staining (see below). Hybridomas from wells of interest were distributed into 96-well plates (1-2-5-10 cells/well) for the first limited dilution using 1× hypoxanthine and thymidine (HT) in place of HAT. After about 10 days of culture, wells with single clones were identified by microscope and their supernatants were harvested for screening. Positive clones were re-plated using another limited dilution for a further 10 days in DMEM containing 10% FCS and antibiotics. Clones secreting the specific mAb were expanded and frozen. Hybridoma were cultivated at post confluence in the same medium for mAb production. Sodium azide (0.1%) was added to the supernatants for storage. See Table 2 and Table 3 for working dilution.

Supernatants of hybridoma cells secreting anti-α-SKA or anti-α-CAA were screened by: i) triple ELISA, using 96 well plates coated with α-SMA, α-SKA and α-CAA BSA-conjugated peptides, using Maleimide Activated BSA (Pierce) according to the instructions of the manufacturer; ii) Western blotting using platelets, heart, aorta, skeletal muscle, gizzard extracts ( Driesen et al., 2009); iii) immunofluorescence using rat lip, skeletal and heart sections. Selected hybridomas were cloned twice by limited dilution, as described above, and the final mAB characterization was performed by immunoblotting after transfer of one-dimensional gels containing tissue and cell extracts and finally by blocking assays ( Chaponnier et al., 1995) using the N-terminal peptides of α-SKA and α-CAA.

Electrophoresis and immunoblot analysis

Purified α-SKA ( Spudich & Watt, 1971) and α-CAA ( Zot & Potter, 1981) were run on 10% SDS-PAGE ( Laemmli, 1970) and electroblotted to nitrocellulose according to Towbin et al. ( Towbin et al., 1979). After preincubation with the respective different length peptides (listed in Figure 1), the antibodies, diluted in Tris-buffered saline (TBS) solution containing 3% BSA and 0.1% Triton X-100, were incubated on membranes strips for two hours at room temperature. After 3 washes with TBS, a second incubation was performed with peroxidase-conjugated affinity purified goat anti-rabbit IgG (Biorad) at a dilution of 1:10,000 in TBS containing 0.1% BSA and 0.1% Triton X-100. Peroxidase activity was developed using the ECL Western blotting system (GE Healthcare), exposed to AX Konica Minolta films for 5–60 sec, and processed with Curix-60 developing machine (Agfa). Blots were scanned and quantified using densitometric analysis ImageJ v1.49 software (NIH, http://rsb.info.nih.gov/ij/).

Figure 1.
Identification, by immunoblots, of the epitope recognized by anti-α-SKA ( A) and anti-α-CAA ( B). Purified α-SKA ( A) or α-CAA ( B) was run on 10% SDS-PAGE and transferred on nitrocellulose membrane. Membrane strips were ...

Immunofluorescence microscopy

Cryopreserved tissues were sliced (3 μm) with a cryostat microtome (Microm). Sections were positionned on glass slides, fixed with 1% PFA for 30 min at room temperature, followed by three washes with PBS and a 3 min treatment with methanol at -20°C. After three 5 min washes with PBS at RT, tissue sections on glass slides were incubated with primary (2h) and secondary antibodies (1h) at appropriate dilutions (see Table 3). Normal rat serum (1:50) was used to block non-specific sites, and DAPI for nuclear staining. After washing in PBS, sections on slides were mounted in polyvinyl alcohol (PVA: 50 mM Tris-phosphate pH 9.0, 0.1% chlorobutanol, 20% polyvinyl alcohol, 0.5‰ phenol red, 20% glycerol) ( Lennette, 1978). Images were acquired using a Zeiss Axiophot microscope (Carl Zeiss), equipped with plan apochromatic 10x, 20x, or 40x objectives and a high sensibility color camera (Axiocam, Zeiss).

After a careful selection of highly specific secondary antibodies against mouse subtypes, testing a large panel of these antibodies, we selected those commercialized by Jackson Immunoresearch and Southern Biotechnology.

Results

Raw data for Figure 1a,b

Copyright : © 2016 Chaponnier C and Gabbiani G
Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Identification of the epitopes recognized by the actin isoform antibodies

Acetylated N-terminal peptide of different length (4–10 amino acids) and non-acetylated N-terminal decapeptide of α-SKA and α-CAA were tested for their blocking ability of the respective mAb ( Figure 1). We identified the epitope of α-SKA (AcDEDETTA) and of α-CAA (AcDDEET). The epitope of the other actin isoforms were previously identified and are listed in Table 1 (in bold letters). Noteworthy, the acetyl group is a critical element of the epitope of each isoform.

Comparison of distribution of α-SKA and α-CAA, in mature skeletal muscle

The normal myonuclear turnover in rodents is estimated at 1–2% per week ( Schmalbruch & Lewis, 2000). As a first investigation, we have compared the distribution of α-CAA with α-SKA, α-SMA and vimentin on cryostat sections of adult rat tibialis muscle. Although a minority of myocytes expressed α-CAA, we have observed two types of α-CAA positive cells in skeletal fibers: one was mainly located in interstitial connective tissue ( Figure 2A, a–c, d–f) and the other was localized in the muscle mass ( Figure 2A, g–i, arrowhead). The first type corresponded most likely the “muscle spindles”, described by Moll et al. ( Moll et al., 2006), as “modified muscle fibers of neuromuscular spindles, believed to act as sensors of muscle tension”. The “spindle” cells expressed exclusively α-CAA, whereas the “classical muscle fibers”, in addition to a high expression of α-CAA, displayed α-SKA at various levels ( Figure 2A, g–i, j–l), probably according to the state of differentiation during self-regeneration of myocytes.

Figure 2.
Rat adult skeletal muscle co-stained with isoform specific antibodies.

It is well known that during skeletal muscle development, α-SMA is the first muscle actin to be expressed in myocytes during fetal life. Therefore, we also investigated the expression of α-SMA during muscle self-regeneration. We observed that a few regenerating α-CAA positive fibers displayed α-SMA ( Figure 2B, d–f).

As vimentin is expressed in the capsule of muscle spindle cells ( Cizková et al., 2009), we investigated further whether the α-CAA positive fibers located in interstitial spaces could be identified as component of muscle spindles. After co-staining of muscle sections for α-CAA and vimentin, we confirmed that a capsule containing vimentin-positive cells surrounded isolated α-CAA positive spindle fibers ( Figure 2C, a–c). Noteworthy, regenerating fibers displayed contacts with vimentin positive cells ( Figure 2C, d–f).

Distribution of α-SMA, α-SKA and α-CAA, in mature skeletal muscle during the repair process

The use of specific mAbs against actin isoforms allows the tracking and follow-up of myocytes regeneration during the muscle repair process. In particular, complete and fast repair can be observed after muscle micro-lesions obtained after light injury induced by nitrogen-cooled needle application. At early stage (4 day post-injury), an important population of α-CAA positive myofibers was observed ( Figure 3A) at injury sites. These cells, being α-SKA negative or marginally positive, are probably in an early stage of differentiation. Few of them co-expressed α-SMA ( Figure 3C, a–c, arrowhead). Only rarely, α-SMA positive myofibroblasts, the hallmark of fibrotic process ( Tomasek et al., 2002) were detected ( Figure 3C, a–c, arrow). At 5d post-injury, fibers started to express more importantly α-SKA in addition to α-CAA in the regenerating location ( Figure 3B, a–f). In our model, muscle regeneration was very rapid, as expression of α-SMA was not any longer detectable 5d after injury ( Figure 3C, d–f). At 9d post-injury, co-expression of α-SKA and α-CAA became rare ( Figure 3B, g–i). These results suggest that skeletal muscle has a high capacity of regeneration after micro-injury and that actin isoform specific mAbs represent an important tool for muscle regeneration tracking.

Figure 3.
Regenerating rat adult skeletal muscle co-stained with isoform specific antibodies.

Conclusion

In conclusion, α-CAA, in conjunction with the expression of α-SMA and α-SKA, appears to represent a valuable marker for the identification of myofibers regeneration in skeletal muscle and for the analysis of the degree of fiber differentiation. For this purpose, it is important to use well-characterized and specific antibodies. Furthermore, high quality anti-mouse subtype secondary antibodies allow double immunostaining necessary for this type of investigation.

Data availability

The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Chaponnier C and Gabbiani G

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). http://creativecommons.org/publicdomain/zero/1.0/

F1000Research: Dataset 1. Raw data for Figures 1a, b., 10.5256/f1000research.8154.d117164 ( Chaponnier & Gabbiani, 2016).

Acknowledgments

We thank Anita Hiltbrunner and Aman Ahmed Mohamed, for their high quality technical assistance, Dr. Marie-Luce Bochaton-Piallat for advice and help with Photoshop and Dr. Stéphane König, Department of Basic Neurosciences, for advice and discussion about the muscle micro-lesion model.

Notes

[version 2; referees: 3 approved]

Funding Statement

This work was supported by the Swiss National Science Foundation (grant number 310030_125320) to C.C.

Notes

Revised. Amendments from Version 1

We like to thank all reviewers for their helpful comments.  

  • A few words have been changed in the new version following the suggestions given by the referees.  
  • A new figure (Supplemental figure 1) showing the specificity of the antibodies is provided, with raw data of Western blots on different tissue extracts.  
  • A new figure (Supplemental Figure 2) showing αCAA positive myofibers with centrally located nuclei during muscle regeneration is provided (Enlargement of Figure 2Af and 3Ai).

We believe that future studies will benefit from our effort to generate these highly specific mAbs against actin isoforms.

Supplemental Figures

Supplemental Figure 1.
Immunodetection of α-CAA and α-SKA in total tissue extracts by Western blots.
Supplemental Figure 2.
Enlargement of Figure 2Af ( a) and Figure 3Ai ( b), showing that, either in normal adult muscle ( a), or at 4d post-injury during the healing process ( b), regenerating αCAA (green) positive myofibers with centrally located nuclei (blue) are detectable. ...

References

  • Arnoldi R, Hiltbrunner A, Dugina V, et al. : Smooth muscle actin isoforms: a tug of war between contraction and compliance. Eur J Cell Biol. 2013;92(6–7):187–200. 10.1016/j.ejcb.2013.06.002 [PubMed] [Cross Ref]
  • Bochaton-Piallat M, Ropraz P, Gabbiani G, et al. : Actin isoform and intermediate filament protein expression in human developing skeletal muscle. Basic Appl Myol. 1992;2(2):83–87. Reference Source
  • Chaponnier C, Goethals M, Janmey PA, et al. : The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo. J Cell Biol. 1995;130(4):887–95. 10.1083/jcb.130.4.887 [PMC free article] [PubMed] [Cross Ref]
  • Chaponnier C, Gabbiani G.: Dataset 1 in: Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle. F1000Research. 2016. Data Source [PMC free article] [PubMed]
  • Cizková D, Soukup T, Mokrý J.: Expression of nestin, desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles. Histochem Cell Biol. 2009;131(2):197–206. 10.1007/s00418-008-0523-7 [PubMed] [Cross Ref]
  • Clément S, Chaponnier C, Gabbiani G.: A subpopulation of cardiomyocytes expressing alpha-skeletal actin is identified by a specific polyclonal antibody. Circ Res. 1999;85(10):e51–8. 10.1161/01.RES.85.10.e51 [PubMed] [Cross Ref]
  • Clément S, Orlandi A, Bocchi L, et al. : Actin isoform pattern expression: a tool for the diagnosis and biological characterization of human rhabdomyosarcoma. Virchows Arch. 2003;442(1):31–8. 10.1007/s00428-002-0728-4 [PubMed] [Cross Ref]
  • Driesen RB, Verheyen FK, Debie W, et al. : Re-expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies. J Cell Mol Med. 2009;13(5):896–908. 10.1111/j.1582-4934.2008.00523.x [PMC free article] [PubMed] [Cross Ref]
  • Dugina V, Zwaenepoel I, Gabbiani G, et al. : Beta and gamma-cytoplasmic actins display distinct distribution and functional diversity. J Cell Sci. 2009;122(Pt 16):2980–8. 10.1242/jcs.041970 [PubMed] [Cross Ref]
  • Franke WW, Stehr S, Stumpp S, et al. : Specific immunohistochemical detection of cardiac/fetal alpha-actin in human cardiomyocytes and regenerating skeletal muscle cells. Differentiation. 1996;60(4):245–50. 10.1046/j.1432-0436.1996.6040245.x [PubMed] [Cross Ref]
  • Gunning P, Ponte P, Blau H, et al. : alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol. 1983;3(11):1985–95. 10.1128/MCB.3.11.1985 [PMC free article] [PubMed] [Cross Ref]
  • Hayward LJ, Schwartz RJ.: Sequential expression of chicken actin genes during myogenesis. J Cell Biol. 1986;102(4):1485–93. [PMC free article] [PubMed]
  • Hayward LJ, Zhu YY, Schwartz RJ.: Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression. J Cell Biol. 1988;106(6):2077–86. 10.1083/jcb.106.6.2077 [PMC free article] [PubMed] [Cross Ref]
  • Kilpatrick KE, Wring SA, Walker DH, et al. : Rapid development of affinity matured monoclonal antibodies using RIMMS. Hybridoma. 1997;16(4):381–389. 10.1089/hyb.1997.16.381 [PubMed] [Cross Ref]
  • Laemmli UK.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–685. 10.1038/227680a0 [PubMed] [Cross Ref]
  • Lennette DA.: An improved mounting medium for immunofluorescence microscopy. Am J Clin Pathol. 1978;69(6):647–8. [PubMed]
  • McHugh KM, Crawford K, Lessard JL.: A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat. Dev Biol. 1991;148(2):442–58. 10.1016/0012-1606(91)90263-3 [PubMed] [Cross Ref]
  • Minty AJ, Alonso S, Caravatti M, et al. : A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA. Cell. 1982;30(1):185–92. 10.1016/0092-8674(82)90024-1 [PubMed] [Cross Ref]
  • Mizuno Y, Suzuki M, Nakagawa H, et al. : Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells. Histochem Cell Biol. 2009;132(6):669–72. 10.1007/s00418-009-0650-9 [PubMed] [Cross Ref]
  • Moll R, Holzhausen HJ, Mennel HD, et al. : The cardiac isoform of alpha-actin in regenerating and atrophic skeletal muscle, myopathies and rhabdomyomatous tumors: an immunohistochemical study using monoclonal antibodies. Virchows Arch. 2006;449(2):175–91. 10.1007/s00428-006-0220-7 [PubMed] [Cross Ref]
  • Ordahl CP.: The skeletal and cardiac alpha-actin genes are coexpressed in early embryonic striated muscle. Dev Biol. 1986;117(2):488–92. 10.1016/0012-1606(86)90315-5 [PubMed] [Cross Ref]
  • Paterson BM, Eldridge JD.: alpha-Cardiac actin is the major sarcomeric isoform expressed in embryonic avian skeletal muscle. Science. 1984;224(4656):1436–8. 10.1126/science.6729461 [PubMed] [Cross Ref]
  • Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, et al. : A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. Cell. 2012;148(1–2):112–25. 10.1016/j.cell.2011.11.049 [PubMed] [Cross Ref]
  • Sassoon DA, Garner I, Buckingham M.: Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development. 1988;104(1):155–64. [PubMed]
  • Schmalbruch H, Lewis DM.: Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles. Muscle Nerve. 2000;23(4):617–26. 10.1002/(SICI)1097-4598(200004)23:4<617::AID-MUS22>3.0.CO;2-Y [PubMed] [Cross Ref]
  • Skalli O, Gabbiani G, Babaï F, et al. : Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. II. Rhabdomyosarcomas. Am J Pathol. 1988;130(3):515–31. [PubMed]
  • Skalli O, Ropraz P, Trzeciak A, et al. : A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103(6 Pt 2):2787–96. 10.1083/jcb.103.6.2787 [PMC free article] [PubMed] [Cross Ref]
  • Spudich JA, Watt S.: The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971;246(15):4866–71. [PubMed]
  • Tomasek JJ, Gabbiani G, Hinz B, et al. : Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3(5):349–63. 10.1038/nrm809 [PubMed] [Cross Ref]
  • Towbin H, Staehelin T, Gordon J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76(9):4350–4354. 10.1073/pnas.76.9.4350 [PubMed] [Cross Ref]
  • Vandekerckhove J, Bugaisky G, Buckingham M.: Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms. J Biol Chem. 1986;261(4):1838–43. [PubMed]
  • Woodcock-Mitchell J, Mitchell JJ, Low RB, et al. : Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation. 1988;39(3):161–6. 10.1111/j.1432-0436.1988.tb00091.x [PubMed] [Cross Ref]
  • Zot HG, Potter JD.: Purification of actin from cardiac muscle. Prep Biochem. 1981;11(4):381–95. 10.1080/00327488108065530 [PubMed] [Cross Ref]

Review Summary Section

Review dateReviewer name(s)Version reviewedReview status
2016 June 2Naohiro HashimotoVersion 2Approved
2016 April 12Shoichiro OnoVersion 1Approved
2016 April 11Omar SkalliVersion 1Approved
2016 April 8Naohiro HashimotoVersion 1Approved with Reservations

Approved

1Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Ōbu, Japan
Competing interests: No competing interests were disclosed.
Review date: 2016 June 2. Status: Approved

I understand the purpose of this report is the validation of the actin isoforms antibodies. Muscle biologists would like to hear answers for questions derived from this study. 

For example:

  1. What is a role of  myofibroblasts appear in regenerating muscles and disease muscles?
  2. Does each actin isoform play a distinct role during  muscle regeneration?

Particularly, a role of aCAA in newly formed myofibers is interesting.

I expect the panel of antibodies against actin isoforms will help future studies to reveal distinct role(s) of actin isoforms in muscle regeneration.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Approved

Shoichiro Ono, Referee1
1Department of Pathology and Department of Cell Biology, Emory University, Atlanta, GA, USA
Competing interests: No competing interests were disclosed.
Review date: 2016 April 12. Status: Approved

Drs. Chaponnier and Gabbiani report production and validation of monoclonal antibodies that specifically recognize alpha-skeletal muscle actin (aSKA) or alpha-cardiac alpha actin (aCAA), which are different only in a few amino acids at the N-terminus. The new antibody against aSKA (10D2) is IgG2a, while anti-aCAA (22D3) is IgG1, which allows researchers to perform double staining in immunohistochemistry to distinguish these closely related actin isoforms. The validation in immunohistochemistry using rat tissues clearly demonstrates specific labeling of different subsets of cells by the isoform-specific antibodies. These antibodies should be outstanding resources to characterize actin isoform expression and subcellular localization in muscle development, regeneration, and diseases, as well as to investigate functions of actin isoforms. I have a few suggestions for a revision mostly for clarification purposes.

  1. In abstract and introduction, “higher vertebrates” should be more clearly defined. Classification of six actin isoforms in 3 subgroups applies to mammals and birds. It should be useful for researchers who use non-mammalian or non-avian species to state whether this classification applies to other vertebrates. A recent analysis by Gunning et al. 1 indicates that zebrafish has 10 muscle actins, suggesting some gene duplications and isoform divergence in fish.
  2. Page 3, left column, 3 rd paragraph, “sarcomers” should be “sarcomeres”.
  3. In Materials and Methods “Antibody production and details”, this section should also include description of how the monoclonal antibodies were prepared after the clonal selection has been completed, which should include whether the antibodies were finally prepared in culture media with or without serum or in ascites, and whether protein concentrations were determined.
  4. Also In Materials and Methods “Antibody production and details”, the selection process includes screening using Western blotting using extracts from several different tissues. To improve transparency of the validation process, representative images of the Western blots preferably showing a wide molecular weight range should be included to demonstrate specificity.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

References

1. Gunning PW, Ghoshdastider U, Whitaker S, Popp D, Robinson RC.: The evolution of compositionally and functionally distinct actin filaments. J Cell Sci.2015;128(11) : 10.1242/jcs.165563 2009-19 10.1242/jcs.165563 [PubMed] [Cross Ref]

Approved

Omar Skalli, Referee1
1Department of Biological Sciences, Integrated Microscopy Center(IMC), University of Memphis, Memphis, TN, USA
Competing interests: No competing interests were disclosed.
Review date: 2016 April 11. Status: Approved

The paper by Chaponnier and Gabbiani reports the production of two monoclonal antibodies, one specific for skeletal actin and the other for cardiac actin. Being able to obtain these antibodies is a momentous accomplishment given that these two actin isoforms differ from each other by only a few amino acids. The authors rigorously demonstrate the specificity of each antibodies for their respective isoform by Western blotting and competitive assay with the immunogen. Because the antibodies are from different subtypes, they can be used by double immunofluorescence to investigate the regulation of skeletal and cardiac actin in different conditions. The authors provide stunningly beautiful immunofluorescence images that demonstrate the utility of their antibodies to pinpoint different cellular populations during skeletal muscle repair. It is to be expected that these antibodies will be extremely useful to studies questions related to cardiac and skeletal muscle physiology and pathology.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Approved with Reservations

1Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Ōbu, Japan
Competing interests: No competing interests were disclosed.
Review date: 2016 April 8. Status: Approved with Reservations

Higher vertebrates express six different highly conserved actin isoforms.  The authors determined the precise localization of alpha-skeletal actin (aSKA) and alpha-cardiac actin (aCAA) in normal and regenerating rat skeletal muscles. The distinct distribution of the two alpha actin isoforms seems very interesting. However, the authors should describe the results of their immunofluorescent analysis on skeletal muscle more precisely and carefully. 

  1. The authors often used terms “renewing muscle (fibers)” and “regenerating fibers”.  However, there is no definition of them. The authors should show the presence of centrally located nuclei in those myofibers because those are found in newly formed myofibers.
  2. The authors identified myofibroblasts in regenerating skeletal muscles as the alpha-SMA (aSMA) positive mononuclear cells. However, myoblasts also express aSMA.  Thus, the authors should determine the absence of myogenic lineage markers such as MyoD in those cells.
  3. Intracellular localization of alpha actin isoforms should be shown in pictures with higher magnification in addition to the present Figure 2 and 3. Those data will provide important information suggesting different function of those actin isoforms.
  4. The term “self-renewal” is used incorrectly. For examples, “self-renewal in muscle cells” and “during muscle self-renewal” are difficult to understand.  I wonder whether “muscle cells” mean muscle satellite cells, myoblasts (muscle progenitor cells), or myofibers.°  “Muscle  self-renewal” means muscle regeneration?

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Articles from F1000Research are provided here courtesy of F1000 Research Ltd