Calcineurin-NFAT dynamics correspond to cardiac remodeling during aortic banding and debanding, mimicking aortic valve replacement

Figures

• 

  FIGURE 1. Increased activation of calcineurin-NFAT signaling in the myocardium of aortic stenosis patients. Transmural left ventricular biopsies were collected per-operatively from patients with aortic stenosis (AS) and controls. For patient details, see Table 1. Expression of regulator of calcineurin 1-4 (RCAN1-4) is directly regulated by calcineurin-NFATc activation. (A) Relative level of RCAN1-4 mRNA (n = 9). Ribosomal protein L32 (RPL32) was used for normalization. (B) Representative immunoblot and quantitative data of RCAN1-4 (n = 8). GAPDH was used for loading control. Data shown as single data points and mean ± SEM. Statistical differences were tested using a t-test vs. controls. (C) Pearson correlation of RCAN1-4 mRNA vs. NPPB mRNA (the transcript encoding brain natriuretic peptide (BNP)), normalized to RPL32, in AS biopsies (n = 9).

• 

  FIGURE 2. Increased abundance of NFATc1-c4 proteins in the myocardium of aortic stenosis patients. Transmural left ventricular biopsies were collected per-operatively from patients with aortic stenosis (AS) and controls. For patient details, see Table 1. (A) Representative immunoblots of nuclear factor of activated T-cell (NFAT) c1, NFATc2, NFATc3, and NFATc4 proteins from NFATc1-c4-transfected human embryonic kidney (HEK) 293 cells. DNA constructs consisted of NFATc1-c4 genes cloned in frame with enhanced green fluorescent protein (EGFP) and were used for antibody validation (n = 3). Actin was used for loading control. (B) Representative immunoblots of protein and phosphorylation levels of NFATc1-c4 in biopsies from patients AS and controls (n = 3). GAPDH was used for loading control.

• 

  FIGURE 3. Sustained elevation of cardiac calcineurin-NFATc activation after aortic banding in mice. Wild-type (WT) C57BL/6JBomTac mice were subjected to aortic banding (AB) 24 h–18 weeks as part of a previously published study (Melleby et al., 2016). Controls consisted of sham-operated mice for each time-point. Expression of regulator of calcineurin 1-4 (RCAN1-4) is directly regulated by calcineurin-NFATc activation. (A) Representative immunoblots and (B) quantitative data of RCAN1-4 in left ventricles (LV) at indicated time-points following AB (n = 6). Vinculin was used for loading control. (C) Relative mRNA abundance of RCAN1-4 (n = 3–10). Ribosomal protein L32 (Rpl32) was used for normalization. Data shown as single data points and mean ± SEM. Statistical differences were tested using t-test vs. respective sham-operated controls. (D) Pearson correlation of RCAN1-4 mRNA vs. NPPB mRNA (the transcript encoding brain natriuretic peptide (BNP)), normalized to RPL32, in mouse hearts (n = 87).

• 

  FIGURE 4. Increased cardiac levels of NFATc4 after aortic banding in mice. Wild-type (WT) C57BL/6JBomTac mice were subjected to aortic banding (AB) 24 h–18 weeks as part of a previously published study (Melleby et al., 2016). (A–D) Relative mRNA abundance of nuclear factor of activated T-cell (NFAT) isoforms c1 (A), c2 (B), c3 (C), and c4 (D) at indicated time-points following AB, compared to respective sham-operated controls (n = 3–10). Ribosomal protein L32 (Rpl32) was used for normalization. Data shown as single data points and mean ± SEM. Statistical differences were tested using t-test vs. respective sham-operated controls. (E) Representative immunoblot of NFATc4 after AB for 1 week (AB1w) (n = 9–10).

• 

  FIGURE 5. Cardiac calcineurin-NFAT activation induced by aortic banding was reversed by relief of pressure overload in mice. Wild-type (WT) C57BL/6JBomTac mice were subjected to aortic banding (AB) or sham operation for 1 week (AB1w and Sham AB1w, respectively), or 1 week of aortic de-banding subsequent to 1 week of aortic banding (DB; AB1wDB1w) and of sham-operated controls (Sham AB1wDB1w). For animal characteristics, see Table 2. Expression of regulator of calcineurin 1-4 (RCAN1-4) is directly regulated by calcineurin-NFATc activation. Relative mRNA ((A); n = 6–11) and protein ((B,C); n = 6–10) levels of RCAN1-4. Expression of ribosomal protein L32 (Rpl32) was used for mRNA normalization. (D,E) Relative protein levels of calcineurin (CnA subunit). (E,F) Relative protein levels of glycogen synthase kinase 3β (GSK-3β). D-F n = 3–6. Vinculin was used for protein loading control. Data shown as single data points and mean ± SEM (A,C,D,F). Statistical differences were tested using a one-way ANOVA with Dunnett’s multiple comparison test vs. sham AB1w or AB1w.

• 

  FIGURE 6. Prolonged pressure overload prior to its relief attenuated reversal of cardiac calcineurin-NFATc activation in mice. NFAT-luciferase reporter mice were subjected to 1 (AB1w) or 4 (AB4w) weeks of aortic banding (AB) or sham-operation, or 1 or 4 weeks of AB followed by 1 week of aortic de-banding (DB; AB1wDB1w or AB4wDB1w). For animal characteristics, see Table 3. Data shown as single data points and mean ± SEM. (A) NFAT-luciferase activity was measured in entire left ventricles (LVs). Statistical differences were tested using a one-way ANOVA with Dunnett’s multiple comparison test vs. sham AB1w and vs. AB1w. A t-test was used to compare AB4wDB1 vs. AB4w; ns, non-significant. No statistical testing was performed vs. Sham AB4w due to n = 2. N = 8–11 for 1w dataset; n = 2–4 for 4w dataset. (B) NFAT-luciferase activity in cardiomyocytes from neonatal NFAT-luciferase mice following 24 h of mechanical stress, with or without 24 h of recovery. Cells from n = 3 separate cell isolations were used, with n = 6 technical replicates for each round (total n = 18). Statistical differences were tested using one-way ANOVA with Dunnett’s multiple comparison test vs. control cells, or vs. stress.