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

Abstract We previously observed that disruption of FK506‐binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)‐induced cardiac hypertrophy in mice, whereas the adenovirus‐mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)‐induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6−/−) mice and cardiac‐specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini‐pump. The results showed that FKBP12.6 deficiency aggravated AngII‐induced cardiac hypertrophy, while cardiac‐specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII‐induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII‐induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca2+ ([Ca2+]i), in which the protein significantly inhibited the key Ca2+/calmodulin‐dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF‐2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII‐induced cardiac hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways.


| INTRODUCTION
Cardiac hypertrophy is characterized with myocardial cell hypertrophy, extracellular matrix increase and myocardial interstitial cell proliferation, and it is a compensatory response to variety of cardiovascular diseases such as high blood pressure, heart valve disease and myocarditis. Many factors including mechanical stress and neurohumoural stimulation induce cardiac hypertrophy. 1 Among them, angiotensin II (AngII), a degraded peptide from the renin-angiotensin system, is one of the most powerful stimuli in inducing cardiac hypertrophy and fibrosis. 2 In patients with an excessive activated RAS (rennin-angiotensin system) or enhanced responsiveness to AngII, the inappropriate remodelling and vascular diseases were initiated. 3 The roles of AngII in cardiovascular system are mainly mediated by AT1R activation via complex interacting signalling pathways involving primary stimulation of phospholipase C (PLC), Ca 2+ mobilization, secondary activation of non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (AKT) and mitogenactivated protein kinase. [3][4][5] It has been reported that AngII-activated Ca 2+ signalling pathway initiated the progress of cardiomyocyte hypertrophy. 4,6 AngII induces Ca 2+ influx via G protein-mediated activation of PLC that leads to phosphatidylinositol hydrolysis and formation of inositol trisphosphate (IP3) and diacylglycerol (DAG), in which IP3 binds to its receptor on sarcoplasmic reticulum (SR), opening the channel that allows calcium efflux into the cytoplasm. 3,5 FK506-binding protein 12.6 (FKBP12.6), a member of the FKBPs family, has 85% amino acid sequence identity with FKBP12 7 and is selectively associated with cardiac ryanodine receptor (RyR2) which is predominantly expressed in heart and plays a crucial role in the regulation of Ca 2+ release from cardiac SR. [8][9][10] It has been reported that cyclic ADP Ribose (cADPR) induces a sustained Ca 2+ increase by dissociating FKBP12.6 from RyR2 of the cardiac SR via calciuminduced calcium release (CICR), 4 which further activate Ca 2+ -dependent signalling pathways such as the calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4) and the calmodulin kinaseII (CaMKII)/MEF-2 pathways to lead to cardiac hypertrophy. 3,5,11 NFATc4 transcriptional factor, a key molecule in cardiac hypertrophy induction, serves as a down target of calcineurin and Glycogen synthase kinase 3b (GSK3b), in which its transcriptional activity was regulated by its nucleus-cytoplasmic shuttling. 12 We have previously showed that disruption of the FKBP12.6 gene resulted in cardiac hypertrophy in male mice, 13 and cardiacspecific overexpression of FKBP12.6 that was driven by the a-MHC promoter was able to rescue the cardiac hypertrophy and abnormal calcium release in FKBP12.6 knockout (FKBP12.6 À/À ) mice. 14 Stud-ies showed that cardiac-specific overexpressing FKBP12.6 in mice blunted thoracic aortic constriction (TAC)-induced maladaptive left ventricular remodelling and protected against catecholamine-induced ventricular tachycardia after TAC. 15,16 The cardiac FKBP12.6 overexpression also reduced the molecular signature of left ventricular hypertrophy (LVH) and the mortality in TAC male mice. 17 These results suggest that FKBP12.6 may protect heart from cardiac hypertrophy. However, a study showed that the adenovirusmediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, 18 indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial.
Several lines of evidence suggest that the renin-angiotensin system and AngII may contribute to cardiac hypertrophy in response to pressure overload. 19,20 However, it has been observed that there were disparate effects of ACE inhibitors or disruption of AT1 receptor on the development of LV hypertrophy after pressure overload.
These results indicated that it is not clear whether AngII plays a critical role in the development of cardiac hypertrophy induced by pressure overload. [21][22][23][24][25] Therefore, it is still necessary to clarify the role of FKBP12.6 in AngII-induced cardiac hypertrophy.
This study aimed to clarify whether FKBP12.6 protects hearts from the AngII-induced cardiac hypertrophy and to elucidate the mechanisms of FKBP12.6 in pathological cardiac hypertrophy.
FKBP12.6 À/À mice and the cardiac-specific FKBP12.6 overexpressing mice were used for producing the mouse models of the pathological cardiac hypertrophy with AngII stimulation. In addition, an H9c2 cell line with stable expressing FKBP12.6 exogenously was constructed for elucidating the mechanisms of the protection of FKBP12.6 in AngII-induced cellular hypertrophy.

| Histology and immunohistochemistry
Before mice were killed by cervical dislocation, they were exposed to 5% isoflurane until they lost their righting reflex, and the hearts were carefully excised. Then, heart tissues were kept at À80°C immediately or fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5-lm intervals. The cross-sections were stained with haematoxylin and eosin (H&E) for histological analysis. Picrosirius red staining was used for the histological assessment of collagen types I and III accumulation by polarization microscopy following the instruction of the manufacturer. Wheat germ agglutinin staining was used to evaluate myocyte cross-sectional area. Briefly, heart sections were de-paraffinized and incubated with 100 lg/mL FITC-labelled

| Cell culture and treatment
The overexpressing FKBP12.6 stable H9c2 cells were maintained with 400 lg/mL G418 in DMEM supplemented with 10% heat-inactivated foetal bovine serum. The cells were passaged every 2-3 days when the cells were reaching to 70%-80% confluence. Cultured H9c2 cells of passage 6-15 were used for our experiments. In different experiments, different cell numbers were used. The cells used for RNA, total protein detection and NFATc4 translocation detection were seeded at 1 9 10 4 cells/cm 2 for 24-hours AngII treatment. The cells used for Phalloidin staining were seeded at 2 9 10 3 cells/cm 2 for 24-hours AngII treatment. The cells used for calcium assay, ROS assay and cell protein phosphorylation were seeded at 2 9 10 4 cells/ cm 2 for 20-minutes AngII treatment. The cells were transferred to the DMEM containing 2% FBS for 24 hours at 37°C after overnight culture, and then the cells were stimulated with or without 200 nmol/L AngII (containing 2% FBS) for 20 minutes or 24 hours.

| Western blot analysis
Hearts were dissected and homogenized in RIPA-buffer (10 lL/mg) containing proteinase inhibitors cocktail (Roche Diagnostics, Mannheim, Germany), the cells were also lysed with RIPA-buffer. The cytoplasmic and nuclear proteins were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermofisher Waltham, MA, USA) according to the manufacturer's instructions. The proteins were separated by electrophoresis on 10% to 15% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 hour and incubated at 4°C overnight with the primary antibodies, and then incubated with appropriate secondary antibodies (Thermo fisher, #31460 for rabbit, #31430 for mouse, #A27014 for goat, dilution: 1:5000) at room temperature

| Data analysis
Independent two-sample t-test and two-way ANOVA were applied to determine statistical significance. Differences were considered to be statistically significant when P < .05.

| FKBP12.6 deficiency aggravates AngII-induced cardiac hypertrophy in vivo
To investigate the effects of FKBP12.6 on cardiac hypertrophy, we first examined the expressions of FKPB12.6 in the heart of WT mice in response to AngII stimulation. As showed in Figure 1A, the mRNA levels of FKBP12.6 were down-regulated by 29% in hearts of WT mice after AngII infusion for 14 days, suggesting that FKBP12.6 might play a critical role in AngII-induced cardiac hypertrophy. Given that the expression of FKBP12.6 was decreased in the hearts of WT mice after AngII stimulation, the effects of FKBP12.6 deficiency on AngII-induced cardiac hypertrophy were further investigated using FKBP12.6 À/À mice. As showed in Figure 1B, AngII infusion for 14 days significantly increased the HW/BW ratio in FKBP12.6 À/À mice by 37.9% compared with the 27.2% augment in WT hearts, due to the heart weights of FKBP12.6 À/À mice were significantly increased compared with the WT hearts, while the body weights of both mice were not influenced by AngII stimulation (Table S1), indicating that FKBP12.6 might protect heart from AngII-induced cardiac hypertrophy. Therefore, we further checked the size of cardiomyocytes and the expressions of hypertrophic genes after AngII stimulation. As showed in Figure 1C,D, the cross-sectional areas of cardiomyocytes in FKBP12.6 À/À mice were significantly augmented compared to WT mice after Ang II infusion. Consistent with the above result, disruption of FKBP12.6 significantly increased AngIIinduced expressions of ANF and BNP which served as the hallmarks of cardiac hypertrophy ( Figure 1E,F). In addition, although we observed that AngII stimulation markedly promoted the fibrosis of cardiac tissues in WT mice, there was no significant aggravation of the fibrosis in the null mice ( Figure S1). To examine the effects of FKBP12.6 deficiency on cardiac functions after AngII infusion, echocardiography was measured for each mouse. As analysed in   of WT hearts. However, FKBP12.6 overexpression seems no effect on AngII-induced fibrosis ( Figure S2). Consistent with the above studies, the results of echocardiography (Table S2) also showed that FKBP12.6 TG mice protected heart from AngII-induced hypertrophy, seen as the decreased heart weight, thickness of IVSD, IVSS and LV mass. All the results indicated that overexpression of FKBP12.6 protected heart from AngII-induced cardiac hypertrophy in mice. F I G U R E 1 FKBP12.6 deficiency aggravates AngII-induced cardiac hypertrophy in vivo. FKBP12.6 À/À 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, 9400, 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 AE SEM, n = 5 mice, $ P < .05, $$ P < .01 for FKBP12.6 À/À vs WT, and # P < .05, ## P < .01, ### P < .001 for saline vs AngII infusion groups

cells. And as we expect, Flag-control H9c2 cells (Flag) treated with
AngII were much larger than those of the untreated cells, whereas the overexpression of FKBP12.6 greatly alleviated the AngIIinduced myocyte hypertrophy in H9c2 cells (61.0% vs 25.8%, *P < .05). In addition, we also observed that overexpression of FKBP12.6 was able to prevent the increases of BNP protein expression ( Figure 4F,G), the transcription level of ANF (Figure 4H) F I G U R E 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 FITCconjugated wheat germ agglutinin staining (D, upper, 9400) and quantitative analysis (C, n = 200 cells per section), and the histological examination was analysed by HE staining (D, lower, 940). 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 AE SEM, n = 5, *P < .05 for FKBP12.6 TG vs WT mice, and ## P < .01, ### P < .001 for saline vs AngII infusion groups and b-MHC ( Figure 4I), and the reduced a-MHC expression after Ang II stimulation ( Figure 4J).  on AngII-induced cardiac hypertrophy, the effects of overexpressing F I G U R E 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 AE 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

AngII-induced myocardial apoptosis in H9c2 cells
It has been reported that AngII promotes ROS generation and calcium overload. Severe oxidative stress could lead to the apoptosis of myocardial cells and subsequently activate the TGFb-Smad pro-fibrosis signalling pathway. 28,29 As showed in Figure 7A

| DISCUSSION
In vivo studies showed that the cardiac-specific overexpression of FKBP12.6 protected heart from cardiac hypertrophy. [15][16][17] In contrast, F I G U R E 5 Overexpression of FKBP12.6 significantly reduces the elevation of the intracellular Ca 2+ concentrations ([Ca 2+ ]i) and inhibits the activities of Ca 2+ /calcineurin signalling pathways after AngII stimulation in H9c2 cells. The AngII-induced elevation of the intracellular Ca 2+ was detected by flow cytometric analysis in FKBP12.6 overexpression (F12.6) and Flag-control (Flag) H9c2 cells with Fluo3-AM staining after 20min 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. The data represent the mean AE SEM from three independent experiments, *P < .05 for FKBP12.6-overexpression vs Flag-control and # P < .05, ## P < .01 for PBS vs AngII groups a few studies showed that the increased FKBP12.6 expression might be associated with some cardiovascular diseases, suggesting that the heart may employ FKBP12.6 up-regulation as an adaptive response to ameliorate cardiac dysfunction. 30 However, an in vitro study showed that the adenovirus-mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes due to the activation of ERK1/2, 18 indicating that the role of FKBP12.6 in cardiac hypertrophy is still unclear. In the present study, using both overexpression and knockout mouse and overexpression cell models, we first time demonstrated that FKBP12.6 protects hearts from AngII-induced hypertrophy in vivo and in vitro, seen as lack of FKBP12.6 gene markedly aggravate AngII-induced cardiac hypertrophy, whereas cardiac-specific overexpression of FKBP12.6 prevents the hypertrophic response to AngII. Moreover, we observed that there was no change in the cardiomyocyte sizes between the Flag-control (Flag) and overexpression of FKBP12.6 (F12.6) H9c2 cells. We also found FKBP12.6 overexpression did not promote the activation of ERK1/2 as reported in adenovirusmediated overexpression of FKBP12.6, 18 and it did not prevent the activation of ERK1/2 induced by AngII neither ( Figure S4). Therefore, the different results may be due to using different sources of cardiomyocytes or different approaches to explore the role of the FKBP12.6 overexpression. It has been reported that H9c2 cells can accurately mimic the hypertrophic responses of primary rat neonatal cardiomyocytes in vitro following stimulation with hypertrophic factors such as angiotensin II or endothelin-1, seen as an increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes. 31 H9c2 cells are rat heart embryonic myoblasts, with skeletal muscle properties, which terminally differentiate by fusing and forming multinucleated myotubes. Cardiomyocytes isolated from embryonic or neonatal rat hearts spread quite readily on the culture plate in 1% horse serum. 32 We cultured H9c2 cells in 2% serum medium for a short time (no more than 48 hours) and at the end of our experiments, we did not observe that the cells differentiate into myocytes/myotubes. We showed that the protection of FKBP12.6 on the AngII-induced cell hypertrophy was associated with inhibitions of the Ca 2+ -mediated calcineurin/NFATc4, CaMKII/MEF-2, and AKT/mTOR, AKT/GSK3b/NFATc4 signalling pathways.
Many pieces of evidences suggest that AngII elevates blood pressure, which in turn promotes haemodynamic overload. The adaptation of the heart to this overload is to alter cardiac protein expression and substantial cardiac remodeling. 2,3 In addition to vasoconstrictor effects, AngII also exerts direct growth-promoting effects on cardiac tissues, resulting in myocardial hypertrophy and mechanical dysfunction independently on blood pressure elevation. 3,[33][34][35][36] Both physiological and pathological cardiac hypertrophies are associated with elevation of cardiomyocyte Ca 2+ levels, 37 whereas the F I G U R E 6 Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII-induced hypertrophy through inhibiting the Ca 2+ / CaMKII, AKT/GSK3b 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, GSK3b and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3b protein were performed by density analysis, and the protein expression of p-CaMKII (B), p-AKT (C), p-GSK3b (E) and p-mTOR (F) was normalized to total CaMKII, AKT, GSK3b and mTOR. GAPDH was used as the loading controls. The data represent the mean AE 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 alternations of intracellular Ca 2+ modulate the activities of various Ca 2+ -dependent signalling pathways including calcineurin/NFATc4 and CaMKII/MEF-2 signalling pathways. 38,39 Activated calcineurin is both necessary and sufficient to induce cardiac hypertrophy in vitro and in vivo, cardiac-specific overexpression of calcineurin in mice showed a dramatic increase in heart sizes.
Increased activity of calcineurin results in dephosphorylation of NFATc4, and subsequently induces transcription of foetal cardiac genes via a combinatorial mechanism involving its direct interaction with GATA4. 40 Our results showed that overexpression of FKBP12.6 significantly inhibited AngII-induced calcineurin activities and and the Bax/Bcl2 ratios were quantitatively determined by density analysis (F). GAPDH was used as an internal control. The data represent the mean AE SEM from three independent experiments, *P < .05 for FKBP12.6-overexpression vs Flagcontrol and # P < .05 for PBS vs AngII group, NS represents no significance pathway. CaMKII induces phosphorylation of the class II histone deacetylase 4 (HDAC4), which in turn dissociates from the MEF-2 transcription factor, and translocates to the cytoplasm from the nucleus, leading to the activation of MEF-2, which is sufficient to promote pathological hypertrophy. 38 In our study, overexpression of FKBP12.6 inhibited the AngII-induced phosphorylation of CaMKII (indicative of CaMKII activation) in heart or in H9c2 cells, suggesting that overexpression of FKBP12.6 prevents hearts or H9c2 cells from AngII-induced hypertrophy by suppressing the activation of CaMKII.
Recent studies showed that the mTOR signalling pathway plays a major role in modulating structural and functional adaptation of the heart to both haemodynamic stress and non-haemodynamic factors. 41 Rapamycin acts by irreversibly binding FKBP12 and FKBP12.6, which inhibits mTORC1 assembly, diminished hypertrophic responses to the Gq-protein-coupled receptor agonists AngII. 42 AKT, a member of the AKT/PKG/PKC family of serine/threonine kinases, is known to function both upstream as an activator of mTOR and downstream as a phosphorylation target of mTOR at S473. 43 Our results showed that overexpression of FKBP12.6 appears to be able to prevent AngII-induced the increases of the phosphorylation-AKT and phosphorylation-mTOR and then inhibited the progress of cardiac hypertrophy induced by AngII.
GSK3b, as an inhibitor of hypertrophic signalling in the intact myocardium, is a ubiquitous serine-threonine protein kinase which phosphorylates NFAT proteins and antagonizes the action of calcineurin by promoting NFAT nuclear export, and the activity of GSK3b is regulated by the phosphorylation status of serine-9. AKT has been shown to phosphorylate serine-9 of GSK3b, thereby inactivating the enzyme. 12 As we expected, the phosphorylations of AKT and GSK3b were increased in response to stimulation of AngII Evidences have showed that AngII-induced oxidative stress preserve an important role in pathological hypertrophy. AngII activates membrane NAD(P)H oxidases, such as superoxide and hydrogen peroxide (H 2 O 2 ) to produce ROS, which is involved in the pleiotropic effects of AngII. 3 ROS generation may further induce cell apoptosis, which is involved in antiapoptotic Bcl2 and the pro-apoptotic Bax. 44 The apoptotic cells induced by AngII are then replaced by excessive collagen fibrosis, and reduced capillary density increased oxygen diffusion distances leading to myocardial ischaemia, and finally contribute to the transition from hypertrophy to failure. 28 As showed in our study, overexpression of FKBP12.6 significantly reduced the apoptosis cells and inhibited intracellular Bax/Bcl-2 ratio, but almost has no effects on AngII-induced ROS generation, suggesting that overexpression of FKBP12.6 protects heart from hypertrophy partially through inhibiting apoptosis induced by AngII.
In addition, AngII-induced oxidative stress and cell injury were reported to promote cardiac remodelling through TGFb1 signalling pathway. 45 AngII and TGFb1 do not act independently from one another but rather act as part of a network that promotes cardiac remodeling. 46 TGFb, a potent stimulator of collagen production by cardiac fibroblasts, acts downstream of AngII and promotes myocyte growth and fibrosis. 46 In the canonical pathway, Smad2/3 is phosphorylated by TGFb receptor type 1. There is growing evidence in the literature to suggest that Smad2 is not a mediator of cardiac fibrosis, whereas Smad3 regulates of the secretory phenotype of myofibroblasts and has a detrimental effect on myocardial fibrosis. 46,47 We showed that FKBP12.6 overexpression did minor things in inhibiting the increased level of TGFb1 expression, and phosphorylation-Smad3 induced by AngII. Additionally, it is reported that the TGFb/Smad pathway activates the transcription of several key fibrotic genes through the ERK1/2 pathway. 29 ERK1/2 has been implicated as the regulators of cardiac hypertrophy in both cell culture and genetically modified mouse models. However, genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo, 48 and ERK1/2 pathway is reported to activate the transcription of several key fibrotic genes through TGFb-Smad pathway, 29 indicating that ERK1/2 may not be necessary for cardiac hypertrophy but play an important role in promoting fibrosis. Our results showed that although FKBP12.6 overexpression did not promote the activation of ERK1/2 as reported in adenovirus-mediated overexpression of FKBP12.6, 18 it did not prevent the activation of ERK1/2 induced by AngII neither. Therefore, we speculated that FKBP12.6 overexpression is not sufficient to inhibit the ERK1/2-TGFb1-Smad pathway for protecting hearts from AngII-induced fibrosis.

| CONCLUSION
Our results demonstrated that FKBP12.6 protects heart from AngIIinduced cardiac hypertrophy through preventing the activations of the Ca 2+ /calmodulin-dependent signalling pathways such as calcineurin/NFATc4, CaMKII/MEF-2, AKT/GSK3b/NFATc4 and AKT/ mTOR pathways. Obviously, our findings strongly support that FKBP12.6 plays a protective role in the development of cardiac hypertrophy.

CONF LICT OF I NTEREST
The authors declare no competing financial interests.