 decreased by IKKi deficiency ( Figure 2E).  Echocardiographic analyses were also utilized to evaluate cardiac structures and functions, including the chamber diameter, wall thicknesses and function of the left ventricle. The KO and WT mice that underwent sham surgery did not differ echocardiographically. However, the echocardiographic measurements of LVEDD, LVESD, interventricular septal thickness at end-diastole (IVSD), left ventricular posterior wall thickness at end-diastole (LVPWD), and fractional shortening (FS) indicated deteriorated cardiac hypertrophy and dysfunction in the KO mice compared with the WT mice (Figure 2A). The LV hemodynamic parameters of the anesthetized mice that were obtained during the acquisition of the pressure-volume (PV) loop further confirmed this significantly deteriorated hemodynamic dysfunction (volume and systolic and diastolic function) of the LV in the IKKi-KO mice as shown in Table 2. Under basal conditions, pressure-overloaded KO mice showed significantly increased ratios of HW/BW, HW/TL and LW/BW and cardiomyocyte cross-sectional area (CSA) compared with the WT mice. No significant differences were observed between the sham-operated groups ( Figure 2B). The gross hearts and H&E and WGA staining results were also consistent with the role of IKKi as an inhibitor of cardiac hypertrophy ( Figure 2C). Furthermore, real-time PCR was performed to analyze the mRNA expression of hypertrophic markers ( Figure 2D). As expected, we found significantly higher expression levels of the cardiac fetal genes atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and b-myosin heavy chain (b-MHC) in the KO mice in response to AB. However, the mRNA expression levels of amyosin heavy chain (a-MHC) and sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA2a) were reduced. These results indicate that IKKi deficiency is responsible for the progressive hypertrophic effects and the impairment of cardiac function that is induced by pressure overload.

Forced IKKi expression attenuates the hypertrophic growth of myocytes in vitro
To specifically examine the role of IKKi in cardiomyocytes, we performed gain-of-function studies using cultured H9c2 rat cardiomyocytes. The H9c2 cells were serum-starved for 24 h in 0.5% FBS after infection with Ad-IKKi and then treated with 1 mM Ang II for the indicated times. Ad-IKKi infection led to a substantial increase in the level of IKKi protein in H9c2 rat cardiomyocytes. Further studies showed that the IKKi overexpression induced by Ad-IKKi infection attenuated Ang IImediated cardiomyocyte hypertrophy, as measured by the cell surface area ( Figure 3A). Moreover, RT-PCR showed that IKKi overexpression markedly decreased the mRNA levels of ANP and BNP induced by Ang II (Figure 3B). These in vitro data suggest the inhibitory effect of IKKi on cardiomyocyte hypertrophy.

IKKi deficiency significantly activates AKT/GSK3b/mTOR/ FOXO and NFkB signaling
To elucidate the molecular mechanism by which IKKi deficiency mediates the hypertrophic response, we examined the activation state of AKT and the expression of its downstream targets, including GSK3b, mTOR, forkhead box O3A (FOXO3A), forkhead box O1 (FOXO1) and NFkB using western blotting. The levels of phosphorylated AKT, GSK3b, mTOR, FOXO3A, FOXO1 and NF-kB were significantly increased in the hearts of the KO mice following pressure overload (Figures 4C,  D). We then exposed cultured H9c2 cardiomyocytes infected with Ad-IKKi or Ad-GFP to 1 mMAngII. As shown in Figures 4E and  F,AngII-stimulated AKT/GSK3b/mTOR/FOXO/NF-kB phosphorylation was attenuated by infection with Ad-IKKi.We further investigated the role of MAPK, another important signaling molecule that regulates the development of hypertrophy. However, the levels of phosphorylated MEK-ERK1/2, JNK, p38 and PI3K were similar between the AB groups ( Figures 4A, B).

Effects of IKKi deficiency on cardiac fibrosis induced by pressure overload
Fibrosis contributes to the phenotypic changes associated with the development of pathological cardiac hypertrophy. To detect the extent of fibrosis, we stained heart sections with PSR and found markedly increased perivascular and interstitial fibrosis and LV collagen volumes in the AB-induced KO and WT hearts compared with the sham-operated controls after 4 weeks. However, it should be noted that considerably increased cardiac fibrosis was also present in the KO mice ( Figures 5A, B).
The observed interstitial fibrosis could be attributed to decreased collagen degradation or increased collagen synthesis. To evaluate the effect of IKKi on collagen synthesis, the mRNA expression levels of connective tissue growth factor (CTGF), transforming growth factor (TGF)-b1, TGF-b2, collagen I, collagen III, and fibronectin, which are known mediators of fibrosis, were analyzed. Increased expression levels of CTGF, TGF-b1, TGF-b2, collagen I, collagen III, and fibronectin were detected in the AB-induced KO mice ( Figure 5C). These data suggest that IKKi deficiency promotes cardiac fibrosis.

IKKi deficiency enhances cardiac apoptosis induced by press-ure overload
To further explore the role of IKKi in hypertrophy, we assessed the apoptosis of myocytes after 4 weeks of AB using TUNEL assays. Apoptotic cells were detected in both KO and WT mice, and the fraction of apoptotic versus total cells was significantly higher in the pressure-overloaded hearts of the KO mice ( Figures 6A,B). Furthermore, the levels of cleaved caspase 3, Bax (proapoptotic) and Bcl2 (antiapoptotic) proteins were assessed. The Bax protein level was increased and the Bcl2 protein level was decreased in the pressure-overloaded hearts of the KO mice, indicating a reduced Bcl2/Bax ratio. The level of cleaved caspase-3 was higher in the KO mice in response to AB (Figures 6C-E). These findings indicate that IKKi is involved in apoptosis in hypertrophic hearts subjected to pressure overload.

Discussion
Cardiac hypertrophy is characterized by the reactivation of fetal cardiac genes, increased cross-sectional areas of adult cardiomyocytes, and contractile dysfunction. In this study, we investigated the role of IKKi and its related molecular mechanisms in cardiac hypertrophy. The present study demonstrated that IKKi deficiency deteriorated cardiac hypertrophy and fibrosis. The important novel findings of our study are as follows: (1) IKKi expression is increased in AB-induced hypertrophic heart tissue; (2) IKKi deficiency provokes spontaneous hypertrophy; (3) IKKi deficiency promotes pathological hypertrophy and fibrosis; (4) IKKi overexpression markedly attenuates the hypertrophy of H9c2 rat cardiomyocytes induced by Ang II in vitro; (5) IKKi deficiency exacerbates cardiac remodeling by activating AKT and NF-kB signaling; and (6)IKKi deficiency is involved in apoptosis in hypertrophic hearts subjected to pressure overload As a member of the IKK family, IKKi (inducible IkB kinase), which is also known as IKKe, is involved in the activation of transcription factors [25]. Although IKKi is constitutively expressed in T cells, its expression is mainly regulated by NF-kB in other cell types [26]. Our data demonstrate that IKKi expression is significantly elevated in AB-induced hypertrophic heart tissues, which suggests that it is involved in promoting the development of cardiac hypertrophy and remodeling. This hypothesis is consistent with some studies involving other IKK family members, such as IKKb [18].
The molecular mechanism by which IKKi affects cardiac remodeling remains unclear. To address this issue, we analyzed the activation of hypertrophic signaling pathways in AB mice. Pivotal signaling pathways that functioned in the pathogenesis of cardiac hypertrophy, including the mitogen-activated protein kinases (MAPKs) and AKT pathways, were assessed [10,27,29,30,31]. The downstream targets of AKT include GSK3b, mTOR,FOXO transcription factors and NFkB, all of which are involved in cardiac hypertrophy [10,27,29,30,31,32,33]. In this study,AKT phosphorylation was significantly enhanced in response to hypertrophic stimuli in the KO mice compared with WT mice. Consistent with the observed increase in AKT activity, hypertrophic stimuli caused increased levels of phosphorylated the GSK3b Ser9 and FOXO transcription factors at AKT phosphorylation sites (reducing their anti-hypertrophic effects) and increased activation of mTOR in the IKKi-deficient mice compared with WT mice. However, IKKi did not influence the phosphorylation of ERK1/2, JNK1/2, p38, MAPK or PI3K. Therefore, our study demonstrates that the AKT and NF-kB signalling is a critical pathway by which IKKi influences cardiomyocyte growth. Furthermore, we demonstrated that IKKi overexpression markedly inhibited AKT and NF-kB signaling in cultured cardiomyocytes stimulated by Ang II.However, the mechanism by which IKKi specifically activates AKT signaling remains unknown. In accordance with our findings, TRAF-associated NFkB activator-binding kinase1 (TBK1), another IkB kinase-related kinase, which exhibits 49% identity and 65% similarity to IKKi, another member of the IKK family, has been shown to control the activation of AKT [34,35]. As a ligand for integrins, the absence of IKKi may be compensated for by TBK1,thus modulating integrin signaling or a specific integrin complex in a manner that specifically regulates AKT. Further