Phosphorylation of Shox2 Is Required for Its Function to Control Sinoatrial Node Formation

Background: Inactivation of Shox2, a member of the short‐stature 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 B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2‐hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p‐Shox2a) in cell culture as well as in the developing mouse and human SAN. Site‐directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal‐regulated 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 Shox2a‐S92AS110A, mutant in the developing heart resulted in down‐regulation of Nkx2.5 in wild‐type 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

Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5

The pacemaker is composed of specialized cardiomyocytes located within the sinoatrial node (SAN), and is responsible for originating and regulating the heart beat. Recent advances towards understanding the SAN development have been made on the genetic control and gene interaction within this structure. Here we report that the Shox2 homeodomain transcription factor is restrictedly expressed in the sinus venosus region including the SAN and the sinus valves during embryonic heart development. Shox2 null mutation results in embryonic lethality due to cardiovascular defects, including an abnormal low heart beat rate (bradycardia) and severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation. Genetically, the lack of Tbx3 and Hcn4 expression, along with ectopic activation of Nppa, Cx40, and Nkx2-5 in the Shox2−/− SAN region, indicates a failure in SAN differentiation. Furthermore, Shox2 overexpression in Xenopus embryos results in extensive repression of Nkx2-5 in the developing heart, leading to a reduced cardiac field and aberrant heart formation. Reporter gene expression assays provide additional evidence for the repression of Nkx2-5 promoter activity by Shox2. Taken together our results demonstrate that Shox2 plays an essential role in the SAN and pacemaker development by controlling a genetic cascade through the repression of Nkx2-5

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline