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.
Free full text: Click here
