Functional Characterisation of the Rare SCN5A p.E1225K Variant, Segregating in a Brugada Syndrome Familial Case, in Human Cardiomyocytes from Pluripotent Stem Cells.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Pathogenic rare mutations in the SCN5A gene, encoding the alpha-subunit of the voltage-dependent cardiac Na+ channel protein (Nav1.5), are identified in 20% of BrS patients, affecting the correct function of the channel. To date, even though hundreds of SCN5A variants have been associated with BrS, the underlying pathogenic mechanisms are still unclear in most cases. Therefore, the functional characterization of the SCN5A BrS rare variants still represents a major hurdle and is fundamental to confirming their pathogenic effect. Human cardiomyocytes (CMs) differentiated from pluripotent stem cells (PSCs) have been extensively demonstrated to be reliable platforms for investigating cardiac diseases, being able to recapitulate specific traits of disease, including arrhythmic events and conduction abnormalities. Based on this, in this study, we performed a functional analysis of the BrS familial rare variant NM_198056.2:c.3673G>A (NP_932173.1:p.Glu1225Lys), which has been never functionally characterized before in a cardiac-relevant context, as the human cardiomyocyte. Using a specific lentiviral vector encoding a GFP-tagged SCN5A gene carrying the specific c.3673G>A variant and CMs differentiated from control PSCs (PSC-CMs), we demonstrated an impairment of the mutated Nav1.5, thus suggesting the pathogenicity of the rare BrS detected variant. More broadly, our work supports the application of PSC-CMs for the assessment of the pathogenicity of gene variants, the identification of which is increasing exponentially due to the advances in next-generation sequencing methods and their massive use in genetic testing.

Regulation of STAT3 and its role in cardioprotection by conditioning: focus on non-genomic roles targeting mitochondrial function.

Ischemia-reperfusion injury (IRI) is one of the biggest challenges for cardiovascular researchers given the huge death toll caused by myocardial ischemic disease. Cardioprotective conditioning strategies, namely pre- and post-conditioning maneuvers, represent the most important strategies for stimulating pro-survival pathways essential to preserve cardiac health. Conditioning maneuvers have proved to be fundamental for the knowledge of the molecular basis of both IRI and cardioprotection. Among this evidence, the importance of signal transducer and activator of transcription 3 (STAT3) emerged. STAT3 is not only a transcription factor but also exhibits non-genomic pro-survival functions preserving mitochondrial function from IRI. Indeed, STAT3 is emerging as an influencer of mitochondrial function to explain the cardioprotection phenomena. Studying cardioprotection, STAT3 proved to be crucial as an element of the survivor activating factor enhancement (SAFE) pathway, which converges on mitochondria and influences their function by cross-talking with other cardioprotective pathways. Clearly there are still some functional properties of STAT3 to be discovered. Therefore, in this review, we highlight the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with processes aimed at maintaining mitochondrial functions, including the regulation of the electron transport chain, the production of reactive oxygen species, the homeostasis of Ca2+ and the inhibition of opening of mitochondrial permeability transition pore. Then we consider the role of STAT3 and the parallels between STA3/STAT5 in cardioprotection by conditioning, giving emphasis to the human heart and confounders.

Percutaneous Coronary Intervention (PCI) Reprograms Circulating Extracellular Vesicles from ACS Patients Impairing Their Cardio-Protective Properties.

Extracellular vesicles (EVs) are promising therapeutic tools in the treatment of cardiovascular disorders. We have recently shown that EVs from patients with Acute Coronary Syndrome (ACS) undergoing sham pre-conditioning, before percutaneous coronary intervention (PCI) were cardio-protective, while EVs from patients experiencing remote ischemic pre-conditioning (RIPC) failed to induce protection against ischemia/reperfusion Injury (IRI). No data on EVs from ACS patients recovered after PCI are currently available. Therefore, we herein investigated the cardio-protective properties of EVs, collected after PCI from the same patients. EVs recovered from 30 patients randomly assigned (1:1) to RIPC (EV-RIPC) or sham procedures (EV-naive) (NCT02195726) were characterized by TEM, FACS and Western blot analysis and evaluated for their mRNA content. The impact of EVs on hypoxia/reoxygenation damage and IRI, as well as the cardio-protective signaling pathways, were investigated in vitro (HMEC-1 + H9c2 co-culture) and ex vivo (isolated rat heart). Both EV-naive and EV-RIPC failed to drive cardio-protection both in vitro and ex vivo. Consistently, EV treatment failed to activate the canonical cardio-protective pathways. Specifically, PCI reduced the EV-naive Dusp6 mRNA content, found to be crucial for their cardio-protective action, and upregulated some stress- and cell-cycle-related genes in EV-RIPC. We provide the first evidence that in ACS patients, PCI reprograms the EV cargo, impairing EV-naive cardio-protective properties without improving EV-RIPC functional capability.

Angiotensin-converting enzyme 2: a key enzyme in key organs.

2020 marked the 20th anniversary of the discovery of the angiotensin-converting enzyme 2 (ACE2). This major event that changed the way we see the renin-angiotensin system today could have passed quietly. Instead, the discovery that ACE2 is a major player in the severe acute respiratory syndrome coronavirus 2 pandemic has blown up the literature regarding this enzyme. ACE2 connects the classical arm renin-angiotensin system, consisting mainly of angiotensin II peptide and its AT1 receptor, with a protective arm, consisting mainly of the angiotensin 1-7 peptide and its Mas receptor. In this brief article, we have reviewed the literature to describe how ACE2 is a key protective arm enzyme in the function of many organs, particularly in the context of brain and cardiovascular function, as well as in renal, pulmonary and digestive homeostasis. We also very briefly review and refer to recent literature to present an insight into the role of ACE2 in determining the course of coronavirus diseases 2019.

Extracellular vesicles (EVs) in ischemic conditioning and angiogenesis: Focus on endothelial derived EVs.

During myocardial ischemia, timely reperfusion is critical to limit infarct area and the overall loss of cardiac contractile function. However, reperfusion further exacerbates the damage of the ischemic heart. This type of injury is known as ischemia-reperfusion injury (IRI). Ischemic conditioning is a procedure which consists of brief cycles of ischemia and reperfusion in order to protect the myocardium against IRI. Remote ischemic conditioning (RIC), namely transient brief episodes of ischemia at a remote site before a subsequent damaging ischemia/reperfusion procedure of the target organ (e.g., the heart), protects against IRI. However, how the stimulus of RIC is transduced from the remote organ to the ischemic heart is still unknown. Recently, extracellular vesicles (EVs) have been proposed to have a role in the RIC procedure. The endothelium releases EVs and is also one of the tissues mostly exposed to EVs during their journey to the target organ. Moreover, EVs may have important roles in angiogenesis and, therefore, in the remodeling of post-ischemic organs. Here we analyze how EVs may contribute to the overall cardioprotective effect and the implication of the endothelium and its EVs in RIC mediated acute cardioprotection as well as in angiogenesis.