Nebulin regulates the assembly and lengths of the thin filaments in striated muscle.

Cell culture, siRNA, and Lat B treatment Rat fetal cardiac myocytes were isolated by J. Bahl and Y. Wu (University of Arizona, Tucson, AZ) and maintained as described previously ( Bang et al., 2001 ; Kazmierski et al., 2003 ). 2 nM dsRNAs were incubated with 6 μl of Cytofectene (Bio-Rad Laboratories) in 100 μl OptiMEM for 15 min at 37°C and added to myocytes on day 3. Transfection efficiency was >80%, and >50 cultures were analyzed. Four dsRNAs to the rat nebulin sequence (19 mers + 5′ AA overhangs) were synthesized, using Ambion's website and Silencer Construction kit, and blasted to ensure no significant homology to nebulette or other known sequences in the National Center for Biotechnology Information database. Three of the four dsRNAs significantly knocked down nebulin in cardiac myocytes (target cDNA: 5′-GGAGACGGTGTACGAGGAG-3′; 5′-CCACAACTACAAGGACATC-3′; and 5′-CAAGGACATCTGACTATGA-3′), using concentrations titrated from 0.5–10 nM, with 2 nM being the lowest effective dosage. Control siRNAs included GAPDH (Ambion), a scrambled nebulin siRNA, and the three siRNAs to nebulin containing 6-bp changes in their centers. Nebulin reduction was confirmed by immunofluorescence with three independent antibodies ( Millevoi et al., 1998 ; McElhinny et al., 2001 ; Kazmierski et al., 2003 ). For live cell imaging, cells were cotransfected with GFP-tropomyosin (a gift from J.-C. Perriard, Swiss Federal Institute of Technology, Zurich, Switzerland) and imaged as described previously ( Mudry et al., 2003 ). To depolymerize thin filaments, we added 20 μM Lat B (Invitrogen) to cells for 4 h (8 h after siRNA treatment), and then washed them out and let them recover for 4 h. For skeletal myotube cultures, myoblasts were isolated from rat hind limbs. The deboned tissue was suspended in 10 ml trypsin/EDTA (Life Technologies) and incubated for 10 min at 37°C. After centrifugation, the pellets were resuspended in MEM + 15% chick embryo extract + 10% horse serum, and cells were preplated at 37°C for 1 h on 100-mm dishes. Collected cells were plated at 4 × 10 5 cells/ml in 35-mm culture dishes containing 12-mm round coverslips coated with rat-tail collagen (Sigma-Aldrich). Cells were transfected with siRNA 24 h later, except this time using 10 nM siRNA. After 3 d, cells were treated with 1 μg/ml 1-[β-D-arabinofuranosyl]-cytosine (Ara-; Sigma-Aldrich) in MEM with 2.5% chick embryo extract and 10% horse serum and fixed on days 5 and 6. RT-PCR Total RNA was harvested from cardiac myocytes within 24 h of siRNA treatment, or from skeletal myotubes within 48 h, using the RNeasy Mini kit (QIAGEN), or with Trizol (Sigma-Aldrich), and quantified using a Biophotometer (Eppendorf). cDNA was synthesized from 1–3 μg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen). 0.5–3 μl of template were used per 25 μl PCR reaction. Reagents were from the TripleMaster Taq kit (Eppendorf). For nebulin amplification, at least 28–30 cycles were performed (with the threshold of detection being ∼30 cycles in cDNA from cardiac myocytes and ∼27 from skeletal myotubes), and for other transcripts, 26–30 cycles were used. Primers used included rat nebulin (forward, 5′-ACTGTCTTCCATCCCGTCAC-3′, and reverse, 5′-GCCATACATCCAGCCTTCAT-3', to amplify a 202-bp product; rat nebulette (forward, 5′-ATTGGGAAGGGCTACAGCTT-3′, and reverse, 5′-GAAGCCTCTTCCCTTCGTCT-3′ to amplify a 196-bp product); rat GAPDH (forward, 5′-CCAGTATGATTCTACCCACGGC-3′, and reverse, 5′-CGGAGATGATGACCCTTTTGGC-3′, to amplify a 227-bp product); and Tmod1 (forward, 5′-ACTGTAAGGCCATGGACAGC-3′, and reverse, 5′-GCTGCAGTTGTGTTTCAAGG-3′, to amplify a 141-bp product). All PCR products were sequenced.

Western blotting

Myotube lysates were solubilized in SDS sample buffer, sonicated, and incubated at 70°C for 5 min before loading onto a 4–20% gradient SDS-PAGE gel. After transfer to nitrocellulose, strips were probed with anti–COOH- and anti–NH 2 -terminal nebulin antibodies (∼1 μg/ml), followed by anti–rabbit IgG-conjugated HRP (1:25,000; Jackson ImmunoResearch Laboratories). After incubation in SuperSignal chemiluminescent substrate (Pierce Chemical Co.), the strips were exposed to BioMax MR film (Eastman Kodak Co.), and band intensity was quantified using National Institutes of Health image.

Immunofluorescence microscopy

Cells were stained as described previously ( Gregorio and Fowler, 1995 ). All cultures were double or triple stained to distinguish myocytes from fibroblasts and/or to evaluate the intensity of nebulin staining. Myocytes were treated with relaxing buffer (150 mM KCl, 5 mM MgCl 2 , 10 mM MOPS, pH 7.4, 1 mM EGTA, and 4 mM ATP) and fixed in 3% PFA for 15 min. Cells were incubated with affinity purified rabbit anti–NH 2 and anti–COOH-terminal nebulin antibodies (∼5 μg/ml), monoclonal antimyosin F59 antibodies (1:10 of culture supernatant; provided by F. Stockdale, Stanford University, Stanford, CA), rabbit antititin antibodies (A168-170 at 1:500; Z1-Z1 at 1:100; Centner et al., 2000 ), monoclonal antisarcomeric α-actinin antibodies (1:1,500; EA-53; Sigma-Aldrich), affinity-purified rabbit anti–human Tmod1 antibodies (10 μg/ml), monoclonal anticardiac actin antibodies (1:10; Ac1-20.4.2; American Research Products, Inc.), and monoclonal antitropomyosin CH1 antibodies (1 μg/ml; Developmental Studies Hybridoma Bank). The following secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and Invitrogen: goat anti–mouse AlexaFluor 488 (1:1,000), goat anti–mouse Texas red (1:600), donkey anti–rabbit Texas red (1:600), goat anti–rabbit AlexaFluor 350 (1:300), and goat anti–mouse AlexaFluor 350 (1:200) IgG. AlexaFluor phalloidin 488 (Invitrogen) or Texas red phalloidin (Sigma-Aldrich) labeled thin filaments, and DAPI (5 μg/ml; Sigma-Aldrich) labeled myotube nuclei. Coverslips were mounted using Aqua Poly/Mount (Polysciences, Inc.) and analyzed on a microscope (Axiovert; Carl Zeiss MicroImaging, Inc.) using a 63 (NA 1.4) or 100× (NA 1.25) objective, and micrographs were collected as digital images on a camera (Orca-ER; Hamamatsu) using OpenLab software (Improvision). Imaging was also performed using a microscope (DeltaVision Deconvolution model D-OL; Olympus) with a 100× objective (1.3 NA) using a charge-coupled device camera (series 300; Photometrics), and on a multiphoton microscope (model 510; Carl Zeiss MicroImaging, Inc.) using a 100× objective (1.4 NA). For pixel intensity plots, images of phalloidin-stained thin filaments were oriented parallel to the long axis in Adobe Photoshop, and intensity levels along the lengths of two adjacent sarcomeres were quantified using boxes drawn to encompass 69 × 6 total pixels (SoftWorx Data Inspector). Images were processed using Adobe Photoshop 7.0, and statistical analyses were performed using Microsoft Excel.