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

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.

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 × 10⁸ lymphocytes per 3 mice, were used for fusions with 2.5 × 10⁷ 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. Clones secreting the specific mAb were expanded and frozen.

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 incubated with the mAb anti-α-SKA (A) or anti-α-CAA (B) alone (control, lane 1) or mixed with the listed peptides (2–9). The epitope recognized by anti-α-SKA (A) includes the acetyl group and the first 7 amino acids of the α-SKA sequence. The epitope recognized by anti-α-CAA (B) includes the acetyl group and the first 5 amino acids of the α-CAA sequence.

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 (1h) and secondary antibodies (30 min) 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.

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-renewal of myocytes.

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

A) Co-staining with anti-α-SKA (red) and anti-α-CAA (green) allows the detection of isolated thin α-CAA positive fibers in interstitial connective tissue (transversal sections, a–c: longitudinal section, d–f) and of renewing muscle fibers expressing both isoforms (longitudinal section, g–l, arrowhead), or only α-CAA (arrow). Merged images are shown on right column. Bars = 50 μm. B) Co-staining with anti-α-CAA (green) and anti-α-SMA (red) shows that α-CAA positive spindle cells are in close connection with α-SMA positive vessel (a–c), that early muscle fiber self-renewal is characterized by co-expression of both isoform (d–f, arrowhead), although more advanced regenerated fibers are only α-CAA positive (g–i). Merged images are shown on right column. Bar = 50 μm. C) Co-staining with anti-α-CAA (red) and anti-vimentin (green) allows the detection of α-CAA positive muscle spindle cells surrounded by a capsule containing vimentin positive cells (transversal section, a–c) and of renewing α-CAA positive fibers in contact with vimentin positive cells (longitudinal section, d–f). Merged images are shown on right column. Bar = 50 μm.

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-renewal. 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 (Cizkova 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, renovating 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 renewal 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.

A) At 4d post-injury, co-staining with anti-α-SKA (red) and anti-α-CAA (green) shows the presence of α-CAA positive fibers in the injured muscle area (a–i). At this stage of regeneration, only a few fibers co-express both isoforms (d–i), with a low α-SKA level (g–i). Merged images are shown on right column. Bars = 50 μm. B) At 5d–9d post-injury, co-staining with anti-α-SKA (red) and anti-α-CAA (green) shows that after 5d, most fibers co-express both isoforms (a–f), whereas at 9d (g–i), α-SKA positive fibers become predominant. Merged images are shown on right column. Bars = 50 μm. C) At 4d–5d post-injury, co-staining with anti-α-SMA (red) and anti-α-CAA (green) shows that during the healing process, only a few fibers co-express both isoforms after 4d (a–c, arrowhead), whereas after 5d, only α-CAA positive fibers are detected (d–f). After 4d, myofibroblasts might participate to the repair process and are detected by using the anti-α- SMA mAb (a–c, arrow). Merged images are shown on right column. Bars = 50 μm.