Zhongzhou Yang

S6K inhibition renders cardiac protection against myocardial infarction through PDK1 phosphorylation of Akt.

In the present study, we observed a rapid and robust activation of the ribosomal protein S6K (S6 kinase) provoked by MI (myocardial infarction) in mice. As activation of S6K promotes cell growth, we hypothesized that increased S6K activity contributes to pathological cardiac remodelling after MI and that suppression of S6K activation may prevent aberrant cardiac remodelling and improve cardiac function. In mice, administration of rapamycin effectively suppressed S6K activation in the heart and significantly improved cardiac function after MI. The heart weight/body weight ratio and fibrotic area were substantially reduced in rapamycin-treated mice. In rapamycin-treated mice, decreased cardiomyocyte remodelling and cell apoptosis were observed compared with vehicle-treated controls. Consistently, inhibition of S6K with PF-4708671 displayed similar protection against MI as rapamycin. Mechanistically, we observed significantly enhanced Thr308 phosphorylation and activation of Akt in rapamycin- and PF-4708671-treated hearts. Cardiomyocyte-specific deletion of PDK1 (phosphoinositide-dependent kinase 1) and Akt1/3 abolished cardioprotection after MI in the presence of rapamycin administration. These results demonstrate that S6K inhibition rendered beneficial effects on left ventricular function and alleviated adverse remodelling following MI in mice by enhancing Akt signalling, suggesting the therapeutic value of both rapamycin and PF-4708671 in treating patients following an MI.

PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development

ABSTRACT One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.

Genetic and Pharmacological Inhibition of Rheb1-mTORC1 Signaling Exerts Cardioprotection against Adverse Cardiac Remodeling in Mice

A previous study indicated that Rheb1 is required for mammalian target of TOR complex 1 (mTORC1) signaling in the brain. However, the function of Rheb1 in the heart is still elusive. In the present study, we deleted Rheb1 specifically in cardiomyocytes and found that reduced Rheb1 levels conferred cardioprotection against pathologic remodeling in myocardial infarction (MI) and pressure overload (transverse aortic constriction) mouse models. Cardiomyocyte apoptosis was reduced and mTORC1 activity was suppressed in cardiomyocyte Rheb1-deletion mice, suggesting that Rheb1 regulates mTORC1 activation in myocardium. Furthermore, we demonstrated that astragaloside IV (As-IV) could inhibit mTORC1, and As-IV treatment displayed similar protection against MI and transverse aortic constriction as Rheb1 genetic inhibition. This study indicates that Rheb1 is essential for mTORC1 activation in cardiomyocytes and suggests that targeting Rheb1-mTORC1 signaling, such as by As-IV treatment, may be an effective therapeutic method for treating patients with adverse cardiac remodeling after MI and hypertrophy.

Deletion of Akt1 causes heart defects and abnormal cardiomyocyte proliferation.

The PI3K-PDK1-PKB/Akt (PI3K, phosphoinositide-3 kinase; PDK1, phosphoinositide-dependent protein kinase 1; PKB, protein kinase B) signaling pathway plays a critical role in a variety of biological processes including cell survival, growth and proliferation, metabolism and organogenesis. Previously, we generated Akt1-deficient mice and found high neonatal mortality with unknown causes. Here we report that histological analysis of Akt1-deficient embryos and newborns revealed heart defects and decreased cell proliferation. Echocardiographic study of Akt1-deficient mice indicated decreased heart function. Further investigation revealed that Akt1 deficiency caused substantial activation of p38MAPK in the heart. Breeding the Akt1-deficient mice to mice that were heterozygous for a null p38α partially rescued the heart defects, significantly decreased post-natal mortality, and restored normal patterns of cardiomyocyte proliferation. Our study suggests that Akt1 is essential for heart development and function, in part, through suppression of p38MAPK activation.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

Signaling by kinases downstream of the Hippo homolog mediates thymocyte migration. Sending thymocytes into action MST1, the mammalian homolog of Hippo, plays a role in apoptosis and cellular proliferation by activating the kinase LATS, which inhibits the transcriptional coactivator YAP; however, MST1 also functions independently of LATS and YAP in T cell adhesion and migration. Tang et al. generated mice with a T cell–specific deficiency in both isoforms of the LATS-related kinase NDR. These mice had reduced numbers of naïve T cells in the periphery because mature thymocytes were trapped in the thymus. Chemoattractants stimulated actin polymerization and the migration of thymocytes in an MST1- and NDR-dependent manner, suggesting that the NDRs act downstream of MST1 to mediate thymocyte egress. The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell–specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

The alteration of protein prenylation induces cardiomyocyte hypertrophy through Rheb–mTORC1 signalling and leads to chronic heart failure

G protein‐regulated cell function is crucial for cardiomyocytes, and any deregulation of its gene expression or protein modification can lead to pathological cardiac hypertrophy. Herein, we report that protein prenylation, a lipidic modification of G proteins that facilitates their association with the cell membrane, might control the process of cardiomyocyte hypertrophy. We found that geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in protein prenylation, played a critical role in postnatal heart growth by regulating cardiomyocyte size. Cardiac‐specific knockout of GGPPS in mice led to spontaneous cardiac hypertrophy, beginning from week 4, accompanied by the persistent enlargement of cardiomyocytes. This hypertrophic effect occurred by altered prenylation of G proteins. Evaluation of the prenylation, membrane association and hydrophobicity showed that Rheb was hyperactivated and increased mTORC1 signalling pathway after GGPPS deletion. Protein farnesylation or mTORC1 inhibition blocked GGPPS knockdown‐induced mTORC1 activation and suppressed the larger neonatal rat ventricle myocyte size and cardiomyocyte hypertrophy in vivo, demonstrating a central role of the FPP–Rheb–mTORC1 axis for GGPPS deficiency‐induced cardiomyocyte hypertrophy. The sustained cardiomyocyte hypertrophy progressively provoked cardiac decompensation and dysfunction, ultimately causing heart failure and adult death. Importantly, GGPPS was down‐regulated in the hypertrophic hearts of mice subjected to transverse aortic constriction (TAC) and in failing human hearts. Moreover, HPLC–MS/MS detection revealed that the myocardial farnesyl diphosphate (FPP):geranylgeranyl diphosphate (GGPP) ratio was enhanced after pressure overload. Our observations conclude that the alteration of protein prenylation promotes cardiomyocyte hypertrophic growth, which acts as a potential cause for pathogenesis of heart failure and may provide a new molecular target for hypertrophic heart disease clinical therapy. Copyright

Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice

ABSTRACT The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.

Synergistic regulation of p53 by Mdm2 and Mdm4 is critical in cardiac endocardial cushion morphogenesis during heart development

Congenital heart defects (CHDs) are the most prevalent human birth defects. More than 85% of CHDs are thought to result from a combination of genetic susceptibilities and environmental stress. However, the stress‐related signalling pathways involved remain largely unknown. The p53 transcription factor is a key tumour suppressor and a central regulator of the cellular stress responses. p53 activities are tightly regulated by its inhibitors Mdm2 and Mdm4 at the post‐translational level. Here we used the Cre–loxP system to delete Mdm2 (Tie2Cre;Mdm2FM/FM) or one copy of both Mdm2 and Mdm4 (Tie2Cre;Mdm2FM/+; Mdm4+/−) in endothelial/endocardial cells and their derivatives in mice to examine the regulation of the p53/Mdm2–Mdm4 pathway during vascular and cardiovascular development. The Tie2Cre;Mdm2FM/FM mice died before embryonic day 10.5 (E10.5) and displayed severe vascular defects. On the other hand, the Tie2Cre;Mdm2FM/+; Mdm4+/− mice displayed atrial and ventricular septal defects (ASD, VSD) of the heart, leading to severe heart dysfunction and postnatal death. During cardiac endocardial cushion morphogenesis, p53 activation was associated with defects in both the epithelial‐mesenchymal transition (EMT) of the endocardial cells and the post‐EMT proliferation of the mesenchymal cells, and the valvuloseptal phenotypes of the Tie2Cre;Mdm2FM/+; Mdm4+/− mice were fully rescued by deletion of one copy of p53. Strikingly, maternal exposure to low‐dose X‐rays in C57BL/6 mice mimicked the congenital heart malformations seen in the Tie2Cre;Mdm2FM/+; Mdm4+/− model, which was also dependent on p53 status, establishing a link between maternal exposures and CHD susceptibility through the p53 pathway. These data revealed a new regulatory mechanism in cardiac endocardial cushion morphogenesis and suggested a possible cause of CHDs due to environmental stress. Copyright

Cardiac Ablation of Rheb1 Induces Impaired Heart Growth, Endoplasmic Reticulum-Associated Apoptosis and Heart Failure in Infant Mice

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from cardiomyocytes of post-natal mice resulted in malignant arrhythmias, heart failure, and premature death of these mice. In addition, heart growth impairment, aberrant metabolism relative gene expression, and increased cardiomyocyte apoptosis were observed in Rheb1-knockout mice prior to the development of heart failure and arrhythmias. Also, protein kinase B (PKB/Akt) signaling was enhanced in Rheb1-knockout mice, and removal of phosphatase and tensin homolog (Pten) significantly prolonged the survival of Rheb1-knockouts. Furthermore, signaling via the mammalian target of rapamycin complex 1 (mTORC1) was abolished and C/EBP homologous protein (CHOP) and phosphorylation levels of c-Jun N-terminal kinase (JNK) were increased in Rheb1 mutant mice. In conclusion, this study demonstrates that Rheb1 is important for maintaining cardiac function in post-natal mice via regulation of mTORC1 activity and stress on the endoplasmic reticulum. Moreover, activation of Akt signaling helps to improve the survival of mice with advanced heart failure. Thus, this study provides direct evidence that Rheb1 performs multiple important functions in the heart of the post-natal mouse. Enhancing Akt activity improves the survival of infant mice with advanced heart failure.

WDR1-regulated actin dynamics is required for outflow tract and right ventricle development

Outflow tract (OFT) anomalies account for about 30% of human congenital heart defects detected at birth. The second heart field (SHF) progenitors contribute to OFT and right ventricle (RV) development, but the process largely remains unknown. WDR1 (WD-repeat domain 1) is a major co-factor of actin depolymerizing factor (ADF)/cofilin that actively disassembles ADF/cofilin-bound actin filaments. Its function in embryonic heart development has been unknown. Using Wdr1 floxed mice and Nkx2.5-Cre, we deleted Wdr1 in embryonic heart (Wdr1F/F;Nkx2.5-Cre) and found that these mice exhibited embryonic lethality, and hypoplasia of OFT and RV. To investigate the role of WDR1 in OFT and RV development, we generated SHF progenitors-specific Wdr1 deletion mice (shfKO). shfKO mice began to die at embryonic day 11.5 (E11.5), and displayed decreased size of the proximal OFT and RV at E10.5. In shfKO embryos, neither the number of SHF cells deployment to OFT nor cell proliferation and the cell number were changed, whereas the cellular organization and myofibrillar assembly of cardiomyocytes were severely disrupted. In the proximal OFT and RV of both shfKO and Wdr1F/F;Nkx2.5-Cre embryos, cardiomyocytes were dissociated from the outer compact myocardial layer and loosely and disorderly arranged into multilayered myocardium. Our results demonstrate that WDR1 is indispensable for normal OFT and RV development, and suggest that WDR1-mediated actin dynamics functions in controlling the size of OFT and RV, which might through regulating the spatial arrangement of cardiomyocytes.