Cardiomyocyte-specific overexpression of syndecan-4 in mice results in activation of calcineurin-NFAT signalling and exacerbated cardiac hypertrophy

Fig. 1. Generation of mice with cardiomyocyte-specific overexpression of syndecan-4. A Mice with cardiomyocyte-specific overexpression of syndecan-4 (Sdc4-Tg) were generated by inserting mouse Sdc4 cDNA upstream of the mouse Myh6 promoter, followed by the simian virus (SV40) splice acceptor site and polyadenylation signal sequence (schematic shown). A C57BL/6J mouse line with stable, germline transmission of the Sdc4-Tg allele was established. See Table 1 for baseline characteristics. B Representative genotyping gel of DNA from mice at weaning age, showing a 110 bp SV40 fragment used to identify Sdc4-Tg mice. A control fragment was amplified in all mice. C Relative mRNA expression of Sdc4 in the left ventricle (LV), right ventricle (RV), atria, fat, intestine, lung, liver, kidney, spleen, brain and skeletal muscle (gastrocnemius) of Sdc4-Tg and WT mice (n = 3), normalized to Gapdh. D Relative mRNA expression of Sdc1-3 in the LV, normalized to Gapdh. Data are mean ± SEM. Statistical differences (C, D) were tested using t-test vs. respective control tissue; **p < 0.01, ***p < 0.001. E, F Representative immunoblots of syndecan-4 in LV protein lysates (E; vinculin was used for loading…

Fig. 2. Sdc4-Tg mice show exacerbated cardiac remodelling upon pressure overload. A Representative magnetic resonance imaging (MRI), mid-ventricular, short-axis view, of Sdc4-Tg and wild-type (WT) mice subjected to aortic banding (AB) or sham operation for 20 weeks (20w). See Tables 2 and SI for characteristics post-AB or sham at 2, 6, 12 and 20w. Relative mRNA levels of signature molecules of heart failure, Nppa and Nppb [B, C; encoding atrial and brain natriuretic peptides (ANP and BNP, respectively)] in left ventricle (LV), right ventricle (RV) and atria of Sdc4-Tg and WT at 20w post-AB or sham (n = 7–9). Relative mRNA levels of markers of pathological cardiomyocyte remodelling, Myh6 and Myh7 [D, E; encoding α- and β-myosin heavy chain (MHC), respectively] in the LV of Sdc4-Tg and WT mice after 20w of AB or sham. Gene expression was normalized to Gapdh. Data are mean ± SEM. Statistical differences were tested using one-way ANOVA with Dunnett’s post-testing vs. respective WT sham, *p < 0.05, **p < 0.01, ***p < 0.001; or vs. WT AB20w, #p < 0.05

Fig. 3. Sdc4-Tg mice show increased NFAT activation upon pressure overload. A Relative NFAT-luciferase (luc) activity in whole left ventricle (LV) of Sdc4-Tg -NFAT-luc and NFAT-luc mice at baseline (6–8 weeks of age, n = 8), and after two weeks of aortic banding (AB) or sham operation (n = 6–15). See Table SII for mouse characteristics. Relative mRNA levels of calcineurin (encoded by Ppp3cb; B) and nuclear factor of activated T-cells c4 (NFATc4, encoded by Nfatc4; C) in the LV of Sdc4-Tg and WT mice after 20 weeks of AB. Gene expression was normalized to Gapdh. Data are mean ± SEM. Statistical differences were tested using t-test (baseline), or one-way ANOVA with Dunnett´s post-testing vs. NFAT-luc sham, *p < 0.05; and vs. NFAT-luc AB2w, #p < 0.05 (A), or using one-way ANOVA with Dunnett´s post-testing vs. respective WT sham, ***p < 0.001; and vs. WT AB20w. D Immunoprecipitation of full-length (FL) syndecan-4 from LVs of Sdc4-Tg mice, using an antibody detecting an extracellular epitope (see Fig. S1 for syndecan-4 antibody validation), or rat IgG as control. Representative immunoblots of syndecan-4 and calcineurin (n = 3). As positive controls for the calcineurin antibody, mouse brain…

Fig. 4. Cardiomyocytes from adult Sdc4-Tg mice show increased diastolic Ca2+ levels. A Representative fluorescence confocal microscopy images of cardiomyocytes isolated from adult Sdc4-Tg and wild-type (WT) mice, loaded with the fluorescence-labelled Ca2+ indicator Fluo 4-AM. Representative tracings of cells paced at 0.5 Hz (baseline condition; B) and 2 Hz (stressed condition; C). D, E Ca2+ transient characteristics of n = 16–19 cells from n = 3 WT and Sdc4-Tg mice, paced at 0.5 Hz and 2 Hz. Data are mean ± SEM. Statistical differences were tested using an unpaired t-test vs. respective WT control, *p < 0.05, **p < 0.01

Fig. 5. Cultured neonatal cardiomyocytes overexpressing Sdc4 show increased calcineurin-dependent hypertrophic growth and NFAT activation. A Relative mRNA level of Sdc4 in neonatal cardiomyocytes (NCM) from rats transduced with an adenovirus encoding Sdc4 (AdSdc4) or control (AdNull), n = 11–12. B Representative immunoblot (n = 3) showing increased full-length (FL) syndecan-4 protein and shedding, i.e., increased levels of the cellular fragment (CF) remaining in cells after shedding of the ectodomain, in NCM protein lysates. Vinculin was used for loading control. C Radioactive H3-leucine incorporation, used to estimate protein synthesis and hypertrophic growth in vitro, in NCM transduced with AdSDC4 or AdNull (n = 8), or co-treated with Cyclosporine A (CsA, n = 5–6), an inhibitor of calcineurin. Serum-treated cells served as positive control of growth (n = 3). Relative expression of signature molecules of heart failure, Nppa and Nppb [D, E; encoding atrial and brain natriuretic peptides (ANP and BNP, respectively)], and the nuclear factor of activated T-cells (NFAT)-responsive gene Rcan1-4 (F) in NCM transduced with AdSdc4 or AdNull, n = 11–12. Data are mean ± SEM. Gene expression was…