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.

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.

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.

Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. We aimed to investigate the correlation between the levels of circulating miR-214 and the expression of vascular endothelial growth factor (VEGF) in the pathogenesis of coronary heart disease patients to further explore the mechanism involved in the vasculogenesis. Three different cohorts, including 13 acute myocardial infarction patients, 176 angina pectoris patients, and 127 control subjects, were enrolled to investigate the expression levels of circulating miR-214 in patients with myocardial ischemia and also the relationship between plasma miR-214 and severity of coronary stenosis. Plasma miR-214 levels of participants were examined by real-time quantitative PCR. Simultaneously, plasma cardiac troponin I concentrations were measured by ELISA assays. We further detected the correlation of miR-214 and VEGF by molecular and animal assays. MiR-214 was enriched in not only diseased endothelial progenitor cells (EPCs) but also the plasma of coronary artery disease (CAD) patients. Besides, we found out miR-214 was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-214 to the 39-UTR of VEGF mRNA. Knockdown of miR-214 not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but also further promoted blood flow recovery in ischemic limbs of mice. Circulating miR-214 may be a new biomarker for CAD and as a potential diagnostic tool. And increased miR-214 level may be used to predict the presence and severity of coronary lesions in CAD patients.

eThe 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.

BackgroundThe Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.Principal FindingsHere, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability.ConclusionsThis study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.

Background Myocardial infarction (MI) is the leading cause of death from cardiovascular disease (CVD). Currently, the efficacy for MI treatment remains unsatisfactory. Therefore, it is urgent to develop a novel therapeutic strategy. Methods Left anterior descending arteries (LAD) of mice were ligated to induce MI. Another set of mice were intravenously injected with PTEN inhibitor BPV (1 mg/kg) 1 h after LAD ligation and continued to receive BPV injection daily for the following 6 days. Mice were performed echocardiography 14 days after surgery. Results Mice in MI group displayed an increased expression of PTEN with impaired cardiac function, enhanced cardiomyocyte apoptosis and decreased angiogenesis. BPV treatment significantly improved cardiac function, with reduced cardiomyocyte apoptosis, promoted angiogenesis, and activated PI3K/Akt/vascular endothelial growth factor (VEGF) signaling pathway. Conclusion PTEN inhibitor BPV could effectively prevent myocardial infarction in mice, highlighting its potential as a candidate therapeutic drug.

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