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Phosphorylation of Shox2 Is Required for Its Function to Control Sinoatrial Node Formation
Background: Inactivation of Shox2, a member of the shortâstature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. Methods and Results: A specific interaction between the B56ÎŽ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2âhybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (pâShox2a) in cell culture as well as in the developing mouse and human SAN. Siteâdirected mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signalâregulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2aâS92AS110A, mutant in the developing heart resulted in downâregulation of Nkx2.5 in wildâtype mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. Conclusions: Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation
Altered FGF Signaling Pathways Impair Cell Proliferation and Elevation of Palate Shelves
In palatogenesis, palatal shelves are patterned along the mediolateral axis as well as the anteroposterior axis before the onset of palatal fusion. Fgf10 specifically expressed in lateral mesenchyme of palate maintains Shh transcription in lateral epithelium, while Fgf7 activated in medial mesenchyme by Dlx5, suppressed the expansion of Shh expression to medial epithelium. How FGF signaling pathways regulate the cell behaviors of developing palate remains elusive. In our study, we found that when Fgf8 is ectopically expressed in the embryonic palatal mesenchyme, the elevation of palatal shelves is impaired and the posterior palatal shelves are enlarged, especially in the medial side. The palatal deformity results from the drastic increase of cell proliferation in posterior mesenchyme and decrease of cell proliferation in epithelium. The expression of mesenchymal Fgf10 and epithelial Shh in the lateral palate, as well as the Dlx5 and Fgf7 transcription in the medial mesenchyme are all interrupted, indicating that the epithelial-mesenchymal interactions during palatogenesis are disrupted by the ectopic activation of mesenchymal Fgf8. Besides the altered Fgf7, Fgf10, Dlx5 and Shh expression pattern, the reduced Osr2 expression domain in the lateral mesenchyme also suggests an impaired mediolateral patterning of posterior palate. Moreover, the ectopic Fgf8 expression up-regulates pJak1 throughout the palatal mesenchyme and pErk in the medial mesenchyme, but down-regulates pJak2 in the epithelium, suggesting that during normal palatogenesis, the medial mesenchymal cell proliferation is stimulated by FGF/Erk pathway, while the epithelial cell proliferation is maintained through FGF/Jak2 pathway
Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation
The molecular mechanisms underlying atrial fibrillation, the most common sustained cardiac arrhythmia, remain poorly understood. Genome-wide association studies uncovered a major atrial fibrillation susceptibility locus on human chromosome 4q25 in close proximity to the paired-like homeodomain transcription factor 2 (Pitx2) homeobox gene. Pitx2, a target of the left-sided Nodal signaling pathway that initiates early in development, represses the sinoatrial node program and pacemaker activity on the left side. To address the mechanisms underlying this repressive activity, we hypothesized that Pitx2 regulates microRNAs (miRs) to repress the sinoatrial node genetic program. MiRs are small noncoding RNAs that regulate gene expression posttranscriptionally. Using an integrated genomic approach, we discovered that Pitx2 positively regulates miR-17-92 and miR-106b-25. Intracardiac electrical stimulation revealed that both miR-17-92 and miR-106b-25 deficient mice exhibit pacing-induced atrial fibrillation. Furthermore electrocardiogram telemetry revealed that mice with miR-17-92 cardiac-specific inactivation develop prolonged PR intervals whereas mice with miR-17-92 cardiac-specific inactivation and miR-106b-25 heterozygosity develop sinoatrial node dysfunction. Both arrhythmias are risk factors for atrial fibrillation in humans. Importantly, miR-17-92 and miR-106b-25 directly repress genes, such as Shox2 and Tbx3, that are required for sinoatrial node development. Together, to our knowledge, these findings provide the first genetic evidence for an miR loss-of-function that increases atrial fibrillation susceptibility
A common Shox2âNkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node
In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5(+) domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5(+) domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2(+) cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2(â) cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development
Illustrations and the Eco-Reality of The Velveteen Rabbit
This presentation will offer a look into the Japanese edition of The Velveteen Rabbit, adapted and illustrated by Komako Sakai, as a miniature ecosystem of words and pictures that interact with each other and create meaning through counterpoints. I will analyze primarily the visual narrative, namely Sakaiâs use of color and light, relative sizes, and distance and frames in her paintings. I will also focus on the depiction of and implication about nature and how illustrating techniques contribute to our understanding of the story. By acknowledging the interests of animal others (even toy animals) and identifying whether the natural environment is implicated in the story, such reading engages us in the ethics of human relationships with built and natural environments, as well as nonhuman beings
GW25-e0828 The effects and safety between high-dose Atorvastatin and Rosuvastatin in Acute myocardial infarction patients
Characteristics and dynamics of sandy natural forests in sandy forest-steppe ecotone in the northern area of China
Long-term treatment effect and adverse events of a modified jailed-balloon technique for side branch protection in patients with coronary bifurcation lesions
Abstract Background Percutaneous coronary interventions (PCI) of bifurcation lesions is technically challenging and associated with lower success rates and higher frequency of adverse outcomes. In the present study, we aimed to evaluate the immediate and long-term treatment effect and adverse events of a new modified jailed-balloon technique on side branch (SB) during PCI on coronary bifurcation lesions. Methods This was a prospective study of 60 patients (49 males, 11 females, mean age 66â±â10âyears) with coronary bifurcation lesions treated at the Beijing Hospital between September 2014 and October 2015. They underwent main vessel (MV) stenting and modified jailed-balloon technique on the SB. All patients were followed with hospital visits at 9âmonths. Angiographic success, major adverse cardiac events (MACE), SB occlusion, and angina were evaluated. Results The majority of the patients had acute coronary syndrome (91.7%) and Medina 1.1.1. bifurcation lesions (71.7%). After MV stenting, thrombolysis in myocardial infarction (TIMI) 3 flow was established 100% of MV and 93.3% of SB. No SB occlusion occurred. The jailed SB balloon and wire could be successfully removed in all patients without damage or entrapment. The majority (91.7%) of patients achieved Canadian Cardiovascular Society I stage. There was no MACE during in-hospital stay and 9-month follow-up. Conclusion The modified JBT provided high rate of procedural success, excellent SB protection during MV stenting, and excellent immediate and long-term clinical outcomes
Genetic Regulation of Sinoatrial Node Development and Pacemaker Program in the Venous Pole
The definitive sinoatrial node (SAN), the primary pacemaker of the mammalian heart, develops from part of pro-pacemaking embryonic venous pole that expresses both Hcn4 and the transcriptional factor Shox2. It is noted that ectopic pacemaking activities originated from the myocardial sleeves of the pulmonary vein and systemic venous return, both derived from the Shox2+ pro-pacemaking cells in the venous pole, cause atrial fibrillation. However, the developmental link between the pacemaker properties in the embryonic venous pole cells and the SAN remains largely uncharacterized. Furthermore, the genetic program for the development of heterogeneous populations of the SAN is also under-appreciated. Here, we review the literature for a better understanding of the heterogeneous development of the SAN in relation to that of the sinus venosus myocardium and pulmonary vein myocardium. We also attempt to revisit genetic models pertinent to the development of pacemaker activities in the perspective of a Shox2-Nkx2-5 epistatic antagonism. Finally, we describe recent efforts in deciphering the regulatory networks for pacemaker development by genome-wide approaches
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