Ea 53

Cultured cardiac myocytes were fixed using 2% paraformaldehyde in 5 mg/ml sucrose for 5 min and permeabilized in 0.3% Triton-X in PBS for 10 min. NRVM were stained for sarcomeric α-actinin (EA-53; Sigma-Aldrich; 1:1000). Cy3-conjugated goat-anti-mouse (Jackson Immuno Research) was used as secondary antibody at 1:300. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; 400 ng/ml in PBS for 10 min). Immunofluorescence images were taken on a Leica TCS-SP5 confocal microscope with a 63x oil objective. Myocyte cross-sectional area was measured based on α-actinin-positive staining using Leica AF6000 fluorescence software.

Rabbit polyclonal antisera were raised against minispryn as follows. A PCR product encoding amino acids 1 to 227 of minispryn was produced by PCR using the primers, 5′-TGAATTCATGGAGGAGGAAGCAGAG and 5′-TGTCGACTCAATTGTAATGGCACTCAAAGTTTT. This region of minispryn has no sequence similarity to myospryn minimising any potential cross-reactivity between antibodies raised against the two proteins. The resulting PCR product was cloned downstream of thioredoxin in pET32a (Novagen) to produce a chimeric fusion protein that was used to immunize New Zealand white rabbits. Anti-thioredoxin antibodies were removed by affinity purification on a Sulfolink (Pierce) thioredoxin column and the resulting anti-minispryn antibody (819) was affinity-purified against the cognate antigen coupled to Sulfolink. The des122 anti-myospryn antibody was raised against amino acids 2791 to 2913 of murine myospryn in rabbits as described previously ^{9 (link)}. The anti-α-actinin monoclonal antibody, clone EA-53, was purchased from Sigma. The 34C (anti-ryanodine receptor) and XIIC4 (anti-sarcalumenin) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (Iowa)^{37 (link)}. The anti-SERCA2 antibody (MA3-910) from was obtained from Affinity Bioreagents (Thermo Fisher Scientific).

Neonatal rat ventricular myocytes were isolated from Sprague–Dawley rats by Percoll gradient centrifugation and cultured as previously described (Böckmann et al., 2019 (link)). After starvation in 20% M199 overnight, NRVM were stimulated for 48 h with either vehicle, 20 μM phenylephrine (PE), 20 ng/mL recombinant oncostatin M (OSM; Ala24-Arg206) in the absence or presence of 100 ng/mL recombinant soluble αKlotho [sKL; 6 His-tagged Ala35-Lys982 (Arg948Lys)] (all R&D Systems), or 2% serum from Ctrl and AAV-Fgf23 mice, respectively. To analyze hypertrophic cell growth, NRVM were fixed in 4% paraformaldehyde, permeabilized in Triton X-100, and incubated with mouse sarcomeric α-actinin antibody (EA-53, Sigma Aldrich) followed by incubation with goat anti-mouse secondary antibody. 4′,6-Diamidino-2-phenylindole (DAPI, Sigma Aldrich) was used to visualize nuclei. Immunofluorescence images were taken on a Zeiss Axio Observer Z1 microscope (Carl Zeiss) with a 20× objective. Cardiac myocyte cross-sectional area was quantified in at least 100 cells per group using Carl Zeiss ZEN software.

For EdU detection, freshly isolated muscle satellite cells were cultured for 3–4 days in growth medium (GM) (DMEM-HG containing 20% FCS (Trace Biosciences, N.S.W., Australia), 2.5 ng/ml basic fibroblast growth factor (bFGF) (FGF2:PeproTech, London, UK), and penicillin (100 U/ml)-streptomycin (100 μg/ml) (Gibco BRL, Gaithersburg, MD, USA)) on culture dishes coated with Matrigel (BD Biosciences). Thirty-six hours before fixation, EdU was added to the GM medium. The cultured cells were fixed with 4% PFA for 10 minutes and then permeabilized with 0.25% Triton X-100 in PBS for 20 minutes. The Click chemical reaction was performed according to the manufacturer’s instructions using a Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA, USA). Coverslips were mounted using Vectashield (Vector Laboratories).
For fusion index analyses, freshly isolated muscle satellite cells were cultured for 3 days in GM on culture dishes coated with Matrigel (BD Biosciences). Then the medium was changed to differentiation medium containing DMEM-HG, 5% horse serum, and penicillin-streptomycin for 3 days. The cultured cells were fixed with 4% PFA as described above. After blocking with 5% skimmed milk, the cells were stained with anti-sarcomeric α-actinin antibody (Sigma-Aldrich, clone EA-53).