Probes for INS

BACKGROUND The long saphenous vein (LSV) is commonly utilised in CABG surgery to facilitate revascularisation. However, over time these grafts develop intimal hyperplasia (IH) and accelerated atherosclerosis, leading to stenosis and occlusion. A common feature of IH is vascular calcification (VC) within the affected vessel. Recently, the matricellular protein osteopontin (OPN) has been implicated in this process at endothelial injury sites in porcine models, but this has not been expanded to humans. Consecutively, studies have implicated the arterial haemodynamic environment as a major driver of the pro-inflammatory conditions facilitating VC and IH. As such, treatment with a synthetic glucocorticoid, dexamethasone, which has proven beneficial in inhibiting IH in murine models, may beneficially modulate this process in humans. This work aims to assess the role of OPN on VC and IH in an ex vivo model, whether dexamethasone can modulate this process, and whether detection of VC in situ can act as a novel clinical monitoring approach to graft patency.

Introduction: Hyperglycemia is a common finding in ACS patients in both diabetic and non-diabetic, it is considered a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury is not fully understood, whether the Sodium Glucose Co-Transporter 1(SGLT1) plays a role in increase injury, before and/or after reperfusion, remains to be elucidated. SGLT2 inhibitors clinical trials have shown significant improvements in cardiovascular outcomes in diabetic and non-diabetic, yet the mechanism is not fully understood and whether SGLT1 plays a role in infarct augmentation remains to be elucidated. Hypothesis: High glucose at reperfusion leads to excess myocardial injury and the increased injury is mediated through the activity of SGLT1. Methods: RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and Zucker diabetic rats. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose on myocardium at reperfusion. Canagliflozin a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 and SGLT2 transporter and 5nmol/L to block only the SGLT2 transposer) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results: Our data reveal that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model and it is abrogated through the administration of both mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, and through the administration of novel specific SGLT1 inhibitor, Mizagliflozin. Conclusions: We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

Heart development is controlled by a complex transcriptional network composed of transcription factors and epigenetic regulators. Mutations in key developmental transcription factor MESP1 and chromatin factors, such as PRC1 and cohesin components, have been found in human congenital heart diseases (CHDs), although their functional mechanism during heart development remains elusive. Here, we find that MESP1 interacts with RING1A/RING1, the core component of PRC1. RING1A depletion impairs human cardiomyocyte differentiation, and cardiac abnormalities similar to those in patients with MESP1 mutations were observed in Ring1A knockout mice. Mechanistically, MESP1 associates with RING1A to activate cardiogenic genes through promoter-enhancer interactions regulated by cohesin and CTCF and histone acetylation mediated by p300. Importantly, CHD mutations of MESP1 significantly affect such mechanisms and impair target gene activation. Together, our results demonstrate the importance of MESP1-RING1A complex in heart development and provide insights into the pathogenic mechanisms of CHDs caused by mutations in MESP1, PRC1, and cohesin components.

The regenerative capacity of the mammalian heart is poor with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate is lost. Therefore, understanding regulatory programs that can induce post-mitotic cardiomyocytes into a proliferative state is essential to enhance cardiac regeneration. Here we report the forkhead transcription factor, foxm1, is required for cardiomyocyte proliferation after injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured zebrafish hearts revealed that foxm1 expression is increased in border zone cardiomyocytes. Decreased cardiomyocyte proliferation and expression of cell cycle genes in foxm1 mutant hearts was observed, suggesting it is required for cell cycle checkpoints. Subsequent analysis of a candidate Foxm1 target gene, cenpf, revealed this microtubule and kinetochore binding protein is also required for cardiac regeneration. Moreover, cenpf mutants show increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.

Organ morphogenesis involves dynamic changes of tissue properties while cells adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis remains unclear. Here, we studied the formation of the zebrafish atrioventricular canal (AVC) where cardiac valves develop. We show that the AVC forms within a zone of tissue convergence associated with the increased activation of the actomyosin meshwork and cell-orientation changes. We demonstrate that tissue convergence occurs with a reduction of cell volume triggered by mechanical forces and the mechanosensitive channel TRPP2/TRPV4. Finally, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that multiple force-sensitive signaling pathways converge to modulate cell volume. We conclude that cell volume reduction is a key cellular feature activated by mechanotransduction during cardiovascular morphogenesis. This work further identifies how mechanical forces and extracellular matrix influence tissue remodeling in developing organs.