Probes for INS

Single-cell technology has become an indispensable tool in cardiovascular research since its first introduction in 2009. Here, we highlight the recent remarkable progress in using single-cell technology to study transcriptomic and epigenetic heterogeneity in cardiac disease and development. We then introduce the key concepts in single-cell multi-omics modalities that apply to cardiovascular research. Lastly, we discuss some of the trending concepts in single-cell technology that are expected to propel cardiovascular research to the next phase of single-cell research.

Background Hyperglycaemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycaemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. However, diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Purpose (1) Characterise the expression of SGLT1 in the myocardium, (2) investigate if SGLT1 is involved in a glucotoxicity injury during IRI, and (3) whether inhibiting SGLT1 with an SGLT inhibitor may reduce infarct size. Methods RT-PCR and in-situ hybridization (RNAScope) combined with Immunoflurescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22 mmol) on myocardium at reperfusion and Canagliflozin (CANA) a non-selective SGLT inhibitor (1000 nmol/L to block both the SGLT1 receptor and SGLT 2 receptor and 5 nmol/L to block the SGLT2 receptor only) was introduced following ischaemia at two different concentrations, at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results RT-PCR found SGLT1 mRNA is expressed in whole myocardium and in individual cardiac chambers. RNAscope detected SGLT1 mRNA is expressed homogenously within intact myocardium, particularly evident within the vasculature. Importantly, hyperglycaemia (22 mmol) at reperfusion increased infarct size (51.80 ± 3.52% vs 40.80 ± 2.89%; p-value: 0.026) compared to normoglycaemia, low dose CANA (5 nmol/L) did not attenuate infarct size in low glucose or high glucose, whereas high CANA concentration (1μmoL/L) significantly reduced infarct size in high glucose (22 mM) when administered at reperfusion (P value = 0.0047). Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycaemia appears augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs)

Although many studies have addressed the regulatory circuits affecting neuronal activities, local non-synaptic mechanisms that determine neuronal excitability remain unclear. Here, we found that microglia prevented overactivation of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) at steady state. Microglia constitutively released platelet-derived growth factor (PDGF) B, which signaled via PDGFRα on neuronal cells and promoted their expression of Kv4.3, a key subunit that conducts potassium currents. Ablation of microglia, conditional deletion of microglial PDGFB, or suppression of neuronal PDGFRα expression in the PVN elevated the excitability of pre-sympathetic neurons and sympathetic outflow, resulting in a profound autonomic dysfunction. Disruption of the PDGFBMG-Kv4.3Neuron pathway predisposed mice to develop hypertension, whereas central supplementation of exogenous PDGFB suppressed pressor response when mice were under hypertensive insult. Our results point to a non-immune action of resident microglia in maintaining the balance of sympathetic outflow, which is important in preventing cardiovascular diseases.

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β-treated human AEECs, cardiac-specific TGF-β-transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β-treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β-transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

Background Diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Hyperglycemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. Purpose Investigate if SGLT1 is involved in a glucotoxicity injury during IRI and whether inhibiting SGLT1 with an SGLT1 inhibitor may reduce infarct size. Method 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 isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22mmol) on myocardium at reperfusion. Canagliflozin (CANA) a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 receptor and SGLT2 and 5nmol/L to block only the SGLT2 receptor) 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 We showed that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. we demonstrate that hyperglycemia at reperfusion is injurious to myocardium with an increase of myocardial infarction. Our data reveal that glucose exacerbation of injury appears to be mediated via SGLT1. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model is abrogated through the administration of a clinically available mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, but by the SGLT2-selective concentration. Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this incre Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): The government of saudi Arabia

Introduction: Myocardial infarction causes several types of injury to the myocardium including lethal cell injury and ‘no-reflow’ (NRF) /microvascular obstruction (MVO). Nonselective Rho Kinase (ROCK1/2) inhibitors such as Fasudil, ameliorate myocardial ischemia/reperfusion (I/R) injury but cause unwanted hypotension. Selective ROCK2 inhibitors (e.g.: KD025) are safe in clinical trials without causing haemodynamic compromise, however they have not been investigated in myocardial I/R. ROCK inhibitors prevent vascular smooth muscle cell (VSMC) contraction; such VSMC contraction/coronary spasm being features of ‘no-reflow’ (NRF) and microvascular obstruction (MVO). At present, there are limited therapies to improve ischemic MVO outcomes, and prognosis is poor. Hypothesis: Using a rat model, we hypothesised that, i) ROCK2 mRNA is expressed in myocardium and coronary vasculature and ii) The selective ROCK2 inhibitor KD025, would reduce infarct size (IS%) and NRF% (MVO) following I/R. Methods: RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in myocardium and coronary vasculature. Male SD rats underwent in-vivo myocardial infarction with 30min ischemia, 180min reperfusion. 15min prior to reperfusion, the ROCK inhibitors Fasudil and KD025 or vehicle (DMSO) were administered i.p. For IS%, myocardium was stained with TTC, and regions not perfused with 1.5% Thioflavin S (NRF%), were visualised under UV light. Results: RNAscope confirmed the presence of ROCK2 mRNA within myocardium and VSMC of coronary arteries. Fasudil (10mg/kg) vs control significantly reduced regional IS% (30.3±4.4 vs52.9±3.8,p=0.02, n=15) and area of NRF% (12.4±2.8 vs28.6±2.2, p=0.001, n=15). However, there was significant hypotension;- Mean BP (mmHg) (72±3.9) vs control (84±2.3, p=0.007). KD025 (100mg/kg) did not reduce IS%, but significantly reduced the area of NRF% vs control (18.4±2.8 vs28.6±2.2,p=0.02, n=14) without hypotensive effect. Conclusions: Our results suggest that ROCK2 may be a prospective target in the management of coronary circulation reperfusion injury and ischemic MVO.

Mural cells in ascending aortic aneurysms undergo phenotypic changes that promote extracellular matrix destruction and structural weakening. To explore this biology, we analyzed the transcriptional features of thoracic aortic tissue.Single-nuclear RNA sequencing was performed on 13 samples from human donors, 6 with thoracic aortic aneurysm, and 7 without aneurysm. Individual transcriptomes were then clustered based on transcriptional profiles. Clusters were used for between-disease differential gene expression analyses, subcluster analysis, and analyzed for intersection with genetic aortic trait data.We sequenced 71 689 nuclei from human thoracic aortas and identified 14 clusters, aligning with 11 cell types, predominantly vascular smooth muscle cells (VSMCs) consistent with aortic histology. With unbiased methodology, we found 7 vascular smooth muscle cell and 6 fibroblast subclusters. Differentially expressed genes analysis revealed a vascular smooth muscle cell group accounting for the majority of differential gene expression. Fibroblast populations in aneurysm exhibit distinct behavior with almost complete disappearance of quiescent fibroblasts. Differentially expressed genes were used to prioritize genes at aortic diameter and distensibility genome-wide association study loci highlighting the genes JUN, LTBP4 (latent transforming growth factor beta-binding protein 1), and IL34 (interleukin 34) in fibroblasts, ENTPD1, PDLIM5 (PDZ and LIM domain 5), ACTN4 (alpha-actinin-4), and GLRX in vascular smooth muscle cells, as well as LRP1 in macrophage populations.Using nuclear RNA sequencing, we describe the cellular diversity of healthy and aneurysmal human ascending aorta. Sporadic aortic aneurysm is characterized by differential gene expression within known cellular classes rather than by the appearance of novel cellular forms. Single-nuclear RNA sequencing of aortic tissue can be used to prioritize genes at aortic trait loci.