FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca²⁺/calmodulin‐mediated signalling pathways in vivo and in vitro

Figure 1. FKBP12.6 deficiency aggravates AngII‐induced cardiac hypertrophy in vivo. FKBP12.6−/− and WT mice were subjected to 14 d AngII or saline infusion, and then the hearts were isolated from the mice for histological and biochemical analyses. The mRNA expressions of FKBP12.6 in hearts from WT mice after AngII or saline infusion were determined by qRT‐PCR (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) (B). The myocyte cross‐sectional areas were stained with FITC‐conjugated wheat germ agglutinin (C), and quantitatively analysed (D, ×400, n = 200 cells per section). The mRNA expressions of hypertrophic makers ANF (E) and BNP (F) were analysed by qRT‐PCR. The data represent the mean ± SEM, n = 5 mice, $ P < .05, $$ P < .01 for FKBP12.6−/− vs WT, and # P < .05, ## P < .01, ### P < .001 for saline vs AngII infusion groups

Figure 2. Cardiac‐specific overexpression of FKBP12.6 protects hearts from AngII‐induced cardiac hypertrophy in vivo. The expressions of FKBP12.6 protein in the hearts from three lines 2‐month‐old FKBP12.6 transgenic mice (FKBP12.6 TG) and their WT littermates were determined by Western blot (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) in FKBP12.6 TG and WT mice which were subjected to 14 days AngII or saline infusion (B). The myocyte cross‐sectional areas were determined by FITC‐conjugated wheat germ agglutinin staining (D, upper, ×400) and quantitative analysis (C, n = 200 cells per section), and the histological examination was analysed by HE staining (D, lower, ×40). The expressions of BNP protein (E&F) and the mRNA expressions of hypertrophic makers BNP (G) and ANF (H) in hearts from FKBP12.6 TG and WT mice after AngII or saline infusion were analysed by Western blot and qRT‐PCR. The data represent the mean ± SEM, n = 5, *P < .05 for FKBP12.6 TG vs WT mice, and ## P < .01, ### P < .001 for saline vs AngII infusion groups

Figure 3. Cardiac‐specific overexpression of FKBP12.6 markedly inhibited AngII‐induced elevation of calcineurin, CaMKII and AKT activities in mice. The expressions of calcineurin (CaN) and MCIP1.4 (a highly sensitive readout for calcineurin activity) were detected by Western blot (A) and quantitatively determined by density analysis (B,C) in hearts from FKBP12.6 TG and WT mice that were subjected to 14 days of AngII or saline infusion. The expressions of the total or phosphorylated proteins including CaMKII, AKT and mTOR were detected by Western blot analysis (D‐F) and the protein expression of p‐CaMKII (G), p‐AKT (H) and p‐mTOR (I) was normalized to total CaMKII, AKT and mTOR in the hearts of the mice. GAPDH expression was used as loading control. The data represent the mean ± SEM, n = 5, *P < .05 for FKBP12.6 TG vs WT mice and # P < .05 for saline vs AngII infusion groups, NS represents no significance

Figure 4. Overexpression of FKBP12.6 protects H9c2 cells from AngII‐induced hypertrophy. FKBP12.6 overexpressing stable H9c2 cell lines were prepared as described in the methods, and both FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of FKBP12.6 proteins were detected by Western blot (A) and quantitatively determined by density analysis (B). The mRNA expression of FKBP12.6 was also examined by qRT‐PCR (C). The cell sizes of FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells were detected by Phalloidin‐Tetramethylrhodamine staining (D, ×200), and the cell area was calculated by measuring at least 200 cells per slide using Image‐Pro Plus 6 software (E). The expressions of BNP protein (F&G) and the mRNA expression of ANF (H), β‐MHC (I) and α‐MHC (J) were determined by Western blot analysis and qRT‐PCR, respectively. All results were presented from three independent experiments, and the data represent the mean ± SEM, *P < .05, ***P < .001 for FKBP12.6‐overexpression vs Flag‐control groups and # P < .05, ## P < .01 for PBS vs AngII groups

Figure 5. Overexpression of FKBP12.6 significantly reduces the elevation of the intracellular Ca2+ concentrations ([Ca2+]i) and inhibits the activities of Ca2+/calcineurin signalling pathways after AngII stimulation in H9c2 cells. The AngII‐induced elevation of the intracellular Ca2+ was detected by flow cytometric analysis in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells with Fluo3‐AM staining after 20‐min PBS or AngII treatment (A) and quantitatively determined by measuring the mean fluorescence intensity of Fluo‐3 in each group (B). FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of calcineurin (CaN) and MCIP1.4 proteins were detected by Western blot (C) and quantitatively determined by density analysis (D and E), respectively. The NFATc4 protein levels in cytosolic and nuclear fractions were analysed by Western blot (F). TATA‐binding protein (TBP), a nuclear protein marker, and Tubulin, a cytosolic protein marker, were used as an internal control, respectively. The contents of NFATc4 protein in nuclei (G) and cytosole (H) were quantitatively determined by density analysis.…

Figure 6. Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca2+/CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, *P < .05, ***P < .001 for FKBP12.6‐overexpression vs Flag‐control and # P < .05, ## P < .01 for PBS vs AngII groups

Figure 7. Overexpression of FKBP12.6 attenuates AngII‐induced myocardial apoptosis in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or 200 nmol/L AngII for 20 min. The intracellular ROS productions were measured by a flow cytometry stained with CM‐H2DCFDA (A) and the mean DCF‐fluorescence intensity in different group of cells was quantitatively determined (B). Cell apoptosis was detected by a flow cytometry with annexin V‐FITC/propidium iodide (PI) double staining. Representative flow cytometric dot plots (x‐axis: annexin V staining/y‐axis: PI staining) show two‐cell populations: annexin V/PI‐negative cells (normal cells, lower left quadrant) and annexin V‐positive/PI‐negative cells (early apoptotic cells, lower right quadrant) and annexin V‐positive/PI‐negative cells(late apoptotic cells, lower right quadrant) (C). The annexin V‐positive/PI‐negative cells (apoptosis cells) are shown in (D). The expressions of Bax and Bcl2 proteins were detected by Western blot (E) in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells after treated with AngII (200 nmol/L) for 24 h, and the Bax/Bcl2 ratios were quantitatively determined…