Direct Actions of Dapagliflozin and Interactions with LCZ696 and Spironolactone on Cardiac Fibroblasts of Patients with Heart Failure and Reduced Ejection Fraction

Figure 1. Migration rate of cardiac fibroblasts: role of heart failure and SGLT2 inhibition. Representative image of migratory rate at 24 h following scratch (A) and time course of mean migratory rate (% healing) in cardiac fibroblasts of healthy controls and patients with heart failure with reduced ejection fraction (HFrEF) (B). Migration rate was analysed in cells of HFrEF (C, n = 5) or control (D, n = 4) groups treated with vehicle and increasing concentrations (100 nM and 1 μM) of SGLT2 inhibitor (SGLT2i). The effects of SGLT2i interaction with other frequently used drugs in heart failure patients, LCZ696 (E, n = 5), and spironolactone (F, n = 5), on the migration rate of cardiac fibroblasts, were determined in cells isolated from HFrEF patients. The extra sum‐of‐squares F determined differences in the fit of time‐course curves. P values and comparisons are expressed next to each time‐course curve. Significance is considered when P < 0.05.

Figure 2. Expression of biomarkers of heart failure in cardiac fibroblasts. Volcano plots show the values of fold change (Log2, x‐axis) by the FDR‐adjusted P‐value (−Log10 of P‐value, y‐axis) in the concentration of the heart failure biomarkers atrial natriuretic polypeptide (ANP); fibronectin (FN1); galectin 3 (GAL3); collagen type I‐alpha‐1 (COL1A1); matrix metalloproteinase‐2 (MMP‐2), ‐3 (MMP‐3), and ‐9 (MMP‐9); interleukins (IL) IL‐6 and IL‐1β; procollagen I C‐terminal propeptide (PICP) and N‐terminal procollagen III propeptide (PIIINP). Highlighted are biomarkers that were at least 1.5‐fold down‐regulated (red) or up‐regulated (green), with P < 0.05. Changes in heart failure biomarkers were analysed in untreated fibroblasts from patients with HFrEF (n = 5) relative to the levels found in untreated cells of healthy controls (n = 4) (A). The effects of SGLT2 inhibition in the expression of heart failure biomarkers were determined in cells from HFrEF (B, n = 5) or healthy controls (C, n = 4) treated with 100 nM of SGLT2i expressed as fold change relative to their respective untreated cells. Differences were analysed by multiple t‐tests with false discovery rate (FDR) correction using the…

Figure 3. Association of SGLT2i with LZC696 or spironolactone on heart failure biomarkers. Box and whiskers plot shows the distribution and the mean of levels (pg/mL) of the heart failure biomarkers fibronectin (FN1); galectin 3 (GAL3); matrix metalloproteinase‐3 (MMP‐3) and ‐9 (MMP‐9); and interleukins (IL) IL‐6 and IL‐1β in cardiac fibroblasts of HFrEF patients, as indicated. The levels of biomarkers were determined in cells treated with vehicle (NT), 100 nM SGLT2 inhibitor (SGLT2i), the combination of 100 nM SGLT2i + 100 nM LCZ696 or the combination of 100 nM SGLT2i + 100 nM Spironolactone. Brown Forsythe and Welch ANOVA analysed the dependence of data on the type of treatment with Dunnett's T3 post hoc analysis for multiple comparisons. P‐values and comparisons are expressed on top of box plots. Significance is considered when P < 0.05.

Figure 4. Phosphorylation array. Volcano plot (A) shows the values of fold change (Log2, x‐axis) by the FDR‐adjusted P‐value (−Log10 of P‐value, y‐axis) in phosphorylation signal of cells treated with SGLT2i relative to the densitometric signal of untreated samples. Highlighted in red are phosphorylation signals that were significantly down‐regulated (P < 0.05). Box and whiskers plots show the distribution and the mean fold change (Log2) in the phosphorylation signals of AKT kinase at Threonine 308 and Serine473 site (B), glycogen synthase kinase‐3 (GSK3) at subunits alpha and beta (C), protein‐tyrosine kinase 2 (PYK2) (D), signal transducer and activator of transcription 3 (STAT3) (E), and components of the mitogen‐activated protein kinase cascade, MEK1/2 (F) and ERK1/2 (G). Phosphorylation signals were determined in lysate of cardiac fibroblasts from HFrEF treated with vehicle (NT), 100 nM SGLT2 inhibitor (SGLT2i), the combination of 100 nM SGLT2i + 100 nM LCZ696 or the combination of 100 nM SGLT2i + 100 nM Spironolactone. Brown Forsythe and Welch ANOVA analysed the dependence of data on the type of treatment with Dunnett's T3 post hoc analysis for multiple comparisons. P values and…