Ing other IKK family members, such as IKKb [18]. The molecular mechanism

Ing other IKK family members, such as IKKb [18]. The molecular mechanism by which IKKi affects cardiac Clavulanate (potassium) site 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 GSK3bSer9 and FOXO transcription factors at AKT phosphorylation sites (reducing their anti-hypertrophic effects) and increased activation of mTOR in the IKKi-deficient 25331948 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 experiments are needed to determine the molecular signaling mechanism by which IKKi regulates AKT. It is worth noting that the AKT pathway is a non-specificIKKi Deficiency Promotes Cardiac HypertrophyTable 2. Anatomic and hemodynamic parameters in IKKi KO and WT mice at 4 weeks after surgery.ParameterSham WT(n = 6) IKKi KO(n = 6) 27.1960.32 4.4360.04 4.9060.10 6.5160.08 462610 117.1564.04 10.2461.28 10.5161.46 24.8561.83 10663.836781.97 28967.336357.21 64.0262.AB WT(n = 6) 27.8960.34 6.4960.08* 5.1960.13 9.9660.08* 47768 150.8562.16* 17.7562.14* 23.5861.93* 34.4461.32* 8171.176326.82* 27658.176346.37* 39.1862.40* IKKi KO(n = 6) 28.0260.40 8.5360.42*# 9.3360.76*# 12.8360.48*# 462618 149.2562.30* 24.3161.84*# 37.2363.46*# 48.3763.29*# 6694.336306.38*# 26578.176416.63*# 25.8363.11*#BW (g) HW/BW(mg/g) LW/BW(mg/g) HW/TL(mg/cm) HR (beats/min) ESP (mmHg) EDP (mmHg) ESV (ml) EDV (ml) dP/dt max (mmHg/s) dP/dt min (mmHg/s) EF( )27.5060.52 4.2960.07 5.2060.05 6.4060.13 48369 105.7461.58 9.8460.19 10.0960.44 26.8260.78 10585.476540.98 29177.346269.63 65.6061.BW,body weight;HW/BW,heart weight/body weight;LW/BW,lung weight/body weight; HW/TL,heart weight/tibial length; HR,heart rate; ESP, end-systolic pressure; EDP, end-diastolic pressure; ESV, endsystolic volume; EDV, end-diastolic volume; EF, ejection fraction; dP/dtmax, 11089-65-9 web maximal rate of pressure development; dP/dtmin, maximal rate of pressure decay. *P,0.05 vs 26001275 WT/sham; # P,0.05 vs WT/AB after AB. doi:10.1371/journal.pone.0053412.tFigure 3. IKKi overexpression attenuates my.Ing 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 GSK3bSer9 and FOXO transcription factors at AKT phosphorylation sites (reducing their anti-hypertrophic effects) and increased activation of mTOR in the IKKi-deficient 25331948 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 experiments are needed to determine the molecular signaling mechanism by which IKKi regulates AKT. It is worth noting that the AKT pathway is a non-specificIKKi Deficiency Promotes Cardiac HypertrophyTable 2. Anatomic and hemodynamic parameters in IKKi KO and WT mice at 4 weeks after surgery.ParameterSham WT(n = 6) IKKi KO(n = 6) 27.1960.32 4.4360.04 4.9060.10 6.5160.08 462610 117.1564.04 10.2461.28 10.5161.46 24.8561.83 10663.836781.97 28967.336357.21 64.0262.AB WT(n = 6) 27.8960.34 6.4960.08* 5.1960.13 9.9660.08* 47768 150.8562.16* 17.7562.14* 23.5861.93* 34.4461.32* 8171.176326.82* 27658.176346.37* 39.1862.40* IKKi KO(n = 6) 28.0260.40 8.5360.42*# 9.3360.76*# 12.8360.48*# 462618 149.2562.30* 24.3161.84*# 37.2363.46*# 48.3763.29*# 6694.336306.38*# 26578.176416.63*# 25.8363.11*#BW (g) HW/BW(mg/g) LW/BW(mg/g) HW/TL(mg/cm) HR (beats/min) ESP (mmHg) EDP (mmHg) ESV (ml) EDV (ml) dP/dt max (mmHg/s) dP/dt min (mmHg/s) EF( )27.5060.52 4.2960.07 5.2060.05 6.4060.13 48369 105.7461.58 9.8460.19 10.0960.44 26.8260.78 10585.476540.98 29177.346269.63 65.6061.BW,body weight;HW/BW,heart weight/body weight;LW/BW,lung weight/body weight; HW/TL,heart weight/tibial length; HR,heart rate; ESP, end-systolic pressure; EDP, end-diastolic pressure; ESV, endsystolic volume; EDV, end-diastolic volume; EF, ejection fraction; dP/dtmax, maximal rate of pressure development; dP/dtmin, maximal rate of pressure decay. *P,0.05 vs 26001275 WT/sham; # P,0.05 vs WT/AB after AB. doi:10.1371/journal.pone.0053412.tFigure 3. IKKi overexpression attenuates my.