Figure 1. mAKAPβ-anchored RSK3 regulates SRF S¹⁰³ phosphorylation, a determinant of myocyte growth in width vs. length.

A, Adult rat ventricular myocytes were infected with adenovirus expressing untagged β-galactosidase (β-gal, control) or HA-tagged RSK3 and maintained in minimal media for 24 hours before immunocytochemistry and measurement of cell width and length (maximum dimension parallel or perpendicular to striations; Bar - 25 μm). Anti-actinin antibody - red; nuclei - blue; anti-HA antibody – green and grayscale panels below. n = 4. Bars and colored symbols indicate average mean and means of independent experiments using different myocyte preparations, respectively. p-values obtained by paired t-test. B, SRF domain structure, showing nuclear localization site (NLS), MADS DNA-binding and heterodimerization domain, and C-terminal transactivation domain.¹⁵ C, Co-immunoprecipitation of endogenous complexes from mouse heart extracts. n = 3. D, HA-tagged RSK3 WT or S218A inactive mutant⁴ and/or myc-mAKAPβ were expressed in COS-7 cells for co-immunoprecipitation assay of ternary complexes. n = 3. E, Neonatal rat ventricular myocytes were transfected with siRNA and treated +/− 10 μmol/L phenylephrine (PE) for 2 days. n = 3-4. * vs. control siRNA + PE. Bar graph below blots shows ratio of phosphorylated to total SRF for PE-treated samples above. F,G, Myocytes were infected with adenovirus expressing Flag-tagged SRF WT, S103D, S103A or control β-galactosidase virus and treated +/− 20 μmol/L phenylephrine (PE) for 1 day. Flag-SRF – green, sarcomeric α-actinin – red, nuclei – blue. * vs. no drug for same protein; ^† vs. β-gal under the same treatment condition; ^‡ vs. SRF WT under the same treatment condition. Bars - 25 μm. F: n = 3-4; G: n = 5. Data in E-G were analyzed by matched, two-way ANOVA with Tukey post-hoc testing. *,^‡ p < 0.05; **,^††,^‡‡ p < 0.01; ***,^†††,^‡‡‡ p < 0.001.