S100a8/9 (S100 Calcium Binding Protein a8/9) Promotes Cardiac Hypertrophy Via Upregulation of FGF23 (Fibroblast Growth Factor 23) in Mice

Abstract

Background: S100a8/9 (S100 calcium binding protein a8/9) belongs to the S100 family and has gained a lot of interest as a critical regulator of inflammatory response. Our previous study found that S100a8/9 homolog promoted aortic valve sclerosis in mice with chronic kidney disease. However, the role of S100a8/9 in pressure overload-induced cardiac hypertrophy remains unclear. The present study was to explore the role of S100a8/9 in cardiac hypertrophy.

Methods and results: Cardiomyocyte-specific S100a9 loss or gain of function was achieved using an adeno-associated virus system, and the model of cardiac hypertrophy was established by aortic banding-induced pressure overload. The results indicate that S100a8/9 expression was increased in response to pressure overload. S100a9 deficiency alleviated pressure overload-induced hypertrophic response, whereas S100a9 overexpression accelerated cardiac hypertrophy. S100a9-overexpressed mice showed increased FGF23 (fibroblast growth factor 23) expression in the hearts after exposure to pressure overload, which activated calcineurin/NFAT (nuclear factor of activated T cells) signaling in cardiac myocytes and thus promoted hypertrophic response. A specific antibody that blocks FGFR4 (FGF receptor 4) largely abolished the prohypertrophic response of S100a9 in mice.

Conclusions: In conclusion, S100a8/9 promoted the development of cardiac hypertrophy in mice. Targeting S100a8/9 may be a promising therapeutic approach to treat cardiac hypertrophy.

Keywords: S100a8/9 protein; cardiac hypertrophy; fibroblast growth factor 23; fibroblast growth factor receptor 4; therapeutic target.

Figure 1. S100a8/9 was increased during the hypertrophic response.

A and B, The mRNA levels of S100a8 and S100a9 in Sham and aortic banding hearts (n=5). C, Six weeks after AB surgery, Western blot analysis, and quantitative results of S100a8 and S100a9 (n=6). Values represent the mean±SEM. *P<0.05 vs Sham group. AB indicates aortic banding; and S100a8/9, S100 calcium binding protein a8/9.

Figure 2. S100a8/9 deficiency attenuated pressure overload‐induced cardiac hypertrophy and fibrosis in mice.

A, Six weeks after AB surgery, hematoxylin–eosin and picrosirius red staining of hearts with or without the infection of AAV9‐shS100a9 (n=6). B and C, Statistical results of the HW/BW and HW/TL of 4 groups (n=12). D, Cross‐sectional areas of myocytes (n=6). E, Statistical results of the fibrosis areas (n=6). F through H, LVIDd, IVSd, and EF of mice after AB surgery (n=12). I–L, mRNA levels of hypertrophy‐related genes (n=6). M through P, The mRNA levels of fibrosis‐related genes (n=6). Values represent the mean±SEM. *P<0.05 vs Sham+shRNA, ^# P<0.05 vs AB+shRNA. AB indicates aortic banding; α‐MHC, α‐myosin heavy chain; α‐SMA, α‐smooth muscle actin; ANP, atrial natriuretic peptide; β‐MHC, β‐myosin heavy chain; BNP, B‐type natriuretic peptide; BW, body weight; EF, ejection fraction; HW, heart weight; IVSd, interventricular septal thickness at diastole; LVIDd, left ventricular internal diastolic diameter; S100a8/9, S100 calcium binding protein a8/9; TGF‐β, transforming growth factor β; and TL, tibia length.

Figure 3. Overexpression of S100a9 in the hearts promoted pressure overload‐induced cardiac hypertrophy.

A, Six weeks after AB surgery, hematoxylin–eosin and picrosirius red staining of hearts with or without the infection of AAV9‐S100a9 (n=6). B and C, Statistical results of the HW/BW and HW/TL of 4 groups (n=12). D, The cross‐sectional areas of myocytes (n=6). E, Statistical results of the fibrosis areas (n=6). F through H, LVIDd, IVSd, and EF of mice after AB surgery (n=12). I through L, mRNA levels of hypertrophy‐related genes (n=6). M through P, mRNA levels of fibrosis‐related genes (n=6). Values represent the mean±SEM. *P<0.05 vs Sham+GFP, ^# P<0.05 vs AB+GFP. AAV9 indicates adeno‐associated virus 9; AB, aortic banding; α‐MHC, α‐myosin heavy chain; α‐SMA, α‐smooth muscle actin; ANP, atrial natriuretic peptide; β‐MHC, β‐myosin heavy chain; BNP, B‐type natriuretic peptide; BW, body weight; EF, ejection fraction; GFP, green fluorescent protein; HW, heart weight; IVSd, interventricular septal thickness at diastole; LVIDd, left ventricular internal diastolic diameter; S100a8/9, S100 calcium binding protein a8/9; TGF‐β, transforming growth factor β; and TL, tibia length.

Figure 4. S100a8/9 promoted phenylephrine‐induced hypertrophy of cardiomyocytes in vitro.

A, Representative α‐actinin staining and cell area in neonatal rat cardiomyocytes (NRCMs) stimulated by recombinant S100a8/9 or not (n=6). B, mRNA levels of hypertrophic markers in NRCMs of indicated groups (n=6). C, Representative α‐actinin staining and cell area in NRCMs infected with siS100a9 or not (n=6). D, mRNA levels of hypertrophic markers in NRCMs of indicated groups (n=6). Values represent the mean±SEM. For A and B, *P<0.05 vs PBS+Vehicle, ^# P<0.05 vs phenylephrine+S100a8/9. For C and D, *P<0.05 vs PBS+siRNA, ^# P<0.05 vs phenylephrine+siS100a9. ANP indicates atrial natriuretic peptide; β‐MHC, β‐myosin heavy chain; GFP, green fluorescent protein; and S100a8/9, S100 calcium binding protein a8/9.

Figure 5. S100a8/9 activated calcineurin/NFAT signaling pathway during phenylephrine‐induced hypertrophy of cardiomyocytes in vitro.

A, Cell areas of NRCMs treated with several inhibitors of hypertrophy‐related kinases (n=5). cyclosporin A (an inhibitor of calcineurin), VIVIT (an inhibitor of NFAT), CCT129957 (an inhibitor of PLCγ), PD169316 (an inhibitor of p38), SP600125 (an inhibitor of JNK), U0126 (an inhibitor of ERK), BMS‐345541 (an inhibitor of NF‐κB), and AKTi (an inhibitor of AKT) were used. B, mRNA levels of atrial natriuretic peptide (ANP) in indicated groups (n=5). C and D, Calcineurin activity in the hearts following AB with overexpression or knockdown of S100a9 (n=6). E and F, NFAT transcriptional activity in the hearts following AB with overexpression or knockdown of S100a9 (n=6). G and H, mRNA level of calcineurin 1 (RCAN1) in the hearts following AB with overexpression or knockdown of S100a9 (n=6). I, Representative Western blots and the statistical results of the protein levels (n=6). Values represent the mean±SEM. *P<0.05 vs the matched control group. AB indicates aortic banding; ANP, atrial natriuretic peptide; NF‐κB, nuclear factor‐κB; NFAT, nuclear factor of activated T cells; NRCM, neonatal rat cardiomyocyte; PCNA, proliferating cell nuclear antigen; PLCγ, phospholipase C‐γ; P‐PLCγ, phospho‐PLCγ; RCAN1, regulator of calcineurin 1; and S100a8/9, S100 calcium binding protein a8/9.

Figure 6. Mice with S100a9 overexpression exhibited increased cardiac expression of FGF23.

A, FGF23 and FGF2 mRNA levels in S100a9‐overexpressed and control groups after AB surgery (n=5). B and C, Representative Western blots and quantitative results of FGF23 in S100a9‐overexpression or knockdown mice after AB surgery (n=6). D through E, FGF23 concentration in phenylephrine‐treated NRCMs after simulated by recombinant S100a8/9 or siS100a9 (n=6). F and G, Representative α‐actinin staining and cell area in NRCMs treated with anti‐FGFR4 or not (n=5). H, mRNA levels of ANP in NRCMs of indicated groups (n=5). I, NFAT transcriptional activity in NRCMs of indicated groups (n=5). Values represent the mean±SEM. *P<0.05 vs the matched control group. AB indicates aortic banding; ANP, atrial natriuretic peptide; FGF23, fibroblast growth factor 23; FGFR4, FGF receptor 4; IgG, immunoglobulin G; NFAT‐luc, nuclear factor of activated T cells‐luciferase; NRCM, neonatal rat cardiomyocyte; and S100a8/9, S100 calcium binding protein a8/9.

Figure 7. FGFR4 blocking abolished the prohypertrophic effect of S100a8/9 overexpression in response to pressure overload.

A, Statistical results of the HW/TL of mice injected with an FGFR4 blocking antibody (anti‐FGFR4) (n=8–10). B, Hematoxylin–eosin and picrosirius red staining of hearts with anti‐FGFR4 or not (n=4–5). C, The cross‐sectional areas of myocytes (n=4–5). D, Statistical results of the fibrosis areas (n=4–5). E and F, LVIDd and EF of mice with anti‐FGFR4 or not (n=8–10). G through I, Calcineurin activity, NFAT transcriptional activity, and RCAN1 mRNA level in hearts with anti‐FGFR4 or not (n=4–5). Values represent the mean±SEM. *P<0.05 vs the matched control group. AB indicates aortic banding; EF, ejection fraction; FGFR4, fibroblast growth factor receptor 4; HW, heart weight; IgG, immunoglobulin G; LVIDd, left ventricular internal diastolic diameter; NFAT‐luc, nuclear factor of activated T cells‐luciferase; RCAN1, regulator of calcineurin 1; S100a8/9, S100 calcium binding protein a8/9; and TL, tibia length.