Mbers, p50, p52, p65 (RelA), c-Rel, and RelB. These members share an N-terminal Rel homology domain (RHD),which is responsible for DNA binding and homo- and heterodimerization [11,12]. In the absence of a stimulus, NF-kB dimers normally combine with one of three typical IkB proteins, IkBa, IkBb or IkBe, or the precursor protein p100. Stimulation with cytokines or other agonists results in the phosphorylation of IkB by the inhibitory-kB kinase (IKK) complex, which includes IKKa, IKKb and IKKc, triggering the degradation of IkB. Then, the freed NF-kB translocates to the nucleus, where it binds to and activates the promoters of the NFkB responsive genes [11,12]. Recent DprE1-IN-2 custom synthesis studies have shown that NF-kB directly exerts its role or alternatively involved in G protein-coupled receptor agonist- or tumour necrosis factor a(TNFa)-induced cardiac hypertrophy and pathological remodeling and fibrosis and NF-kB inhibition attenuates cardiac hypertrophy [13,14,15,16,17]. Furthermore, IKKb-deficient mice exhibit cardiac dilation and dysfunction 
and lung congestion [18]. The inducible IkB kinase (IKKi/IKKe) a constitutively active serine-threonine IKK-related kinase shares 31 amino acid identity with IKKb in the highly conserved Nterminal kinase domain but differs from IKKb in severalIKKi Deficiency Promotes Cardiac Hypertrophyimportant aspects [19]. For example, IKKi is expressed in the cells and tissues of the immune system [19]. Recent studies have shown that human IKKi has two novel splice variants, IKKe-sv1 and IKKe-sv2, which have cell type- and stimulus-specific protein expression [20]. Some groups have described a role for IKKi in infectious diseases and cancer [21,22,23,24,25,26]. However, it has not been shown to be involved in cardiovascular disease. In this study, for the first time, we used IKKi-knockout (KO) mice to investigate the role of IKKi in cardiac hypertrophy induced by pressure overload. We demonstrate that IKKi deficiency in mice leads to cardiac hypertrophy, fibrosis, and cardiac dysfunction, indicating a crucial role for IKKi in regulating cardiac hypertrophy.recorded continuously using a Millar Pressure-Volume System (MPVS-400, Millar Instruments, Houston, TX, USA), and the recordings were further analyzed using PVAN data analysis software.Histological analysis and apoptotic cell assayThe hearts were excised, washed with PBS, arrested in diastole with 10 potassium chloride solution, weighed, placed in 10 formalin, and embedded in paraffin. They were then cut transversely and close to the apex to visualize the left and right ventricles. Several sections of each heart (4? mm thick) were prepared, stained with hematoxylin and eosin (H E) for histopathology or picrosirius red (PSR) for collagen deposition by standard procedures and then visualized by light microscopy. For the myocyte cross-sectional area, sections were stained for membranes with FITC-conjugated WGA (Invitrogen) and for nuclei with DAPI. Single myocytes were measured using a quantitative digital image analysis system (Image 57773-65-6 custom synthesis Pro-Plus, version 6.0). The outlines of 100 myocytes were traced for each group. Cell death by apoptosis was evaluated using a TUNEL assay, which was performed on sections with In Situ Apoptosis Detection Kit (Roche, 11684817910) according to the manufacturer’s recommendations.Materials and Methods Animals and animal modelsAll animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, which was publ.Mbers, p50, p52, p65 (RelA), c-Rel, and RelB. These members share an N-terminal Rel homology domain (RHD),which is responsible for DNA binding and homo- and heterodimerization [11,12]. In the absence of a stimulus, NF-kB dimers normally combine with one of three typical IkB proteins, IkBa, IkBb or IkBe, or the precursor protein p100. Stimulation with cytokines or other agonists results in the phosphorylation of IkB by the inhibitory-kB kinase (IKK) complex, which includes IKKa, IKKb and IKKc, triggering the degradation of IkB. Then, the freed NF-kB translocates to the nucleus, where it binds to and activates the promoters of the NFkB responsive genes [11,12]. Recent studies have shown that NF-kB directly exerts its role or alternatively involved in G protein-coupled receptor agonist- or tumour necrosis factor a(TNFa)-induced cardiac hypertrophy and pathological remodeling and fibrosis and NF-kB inhibition attenuates cardiac hypertrophy [13,14,15,16,17]. Furthermore, IKKb-deficient mice exhibit cardiac dilation and dysfunction and lung congestion [18]. The inducible IkB kinase (IKKi/IKKe) a constitutively active serine-threonine IKK-related kinase shares 31 amino acid identity with IKKb in the highly conserved Nterminal kinase domain but differs from IKKb in severalIKKi Deficiency Promotes Cardiac Hypertrophyimportant aspects [19]. For example, IKKi is expressed in the cells and tissues of the immune system [19]. Recent studies have shown that human IKKi has two novel splice variants, IKKe-sv1 and IKKe-sv2, which have cell type- and stimulus-specific protein expression [20]. Some groups have described a role for IKKi in infectious diseases and cancer [21,22,23,24,25,26]. However, it has not been shown to be involved in cardiovascular disease. In this study, for the first time, we used IKKi-knockout (KO) mice to investigate the role of IKKi in cardiac hypertrophy induced by pressure overload. We demonstrate that IKKi deficiency in mice leads to cardiac hypertrophy, fibrosis, and cardiac dysfunction, indicating a crucial role for IKKi in regulating cardiac hypertrophy.recorded continuously using a Millar Pressure-Volume System (MPVS-400, Millar Instruments, Houston, TX, USA), and the recordings were further analyzed using PVAN data analysis software.Histological analysis and apoptotic cell assayThe hearts were excised, washed with PBS, arrested in diastole with 10 potassium chloride solution, weighed, placed in 10 formalin, and 
embedded in paraffin. They were then cut transversely and close to the apex to visualize the left and right ventricles. Several sections of each heart (4? mm thick) were prepared, stained with hematoxylin and eosin (H E) for histopathology or picrosirius red (PSR) for collagen deposition by standard procedures and then visualized by light microscopy. For the myocyte cross-sectional area, sections were stained for membranes with FITC-conjugated WGA (Invitrogen) and for nuclei with DAPI. Single myocytes were measured using a quantitative digital image analysis system (Image Pro-Plus, version 6.0). The outlines of 100 myocytes were traced for each group. Cell death by apoptosis was evaluated using a TUNEL assay, which was performed on sections with In Situ Apoptosis Detection Kit (Roche, 11684817910) according to the manufacturer’s recommendations.Materials and Methods Animals and animal modelsAll animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals, which was publ.