Endothelin-B Receptors and Left Ventricular Dysfunction after Regional versus Global Ischaemia-Reperfusion in Rat Hearts

Background. Endothelin-1 (ET-1) is implicated in left ventricular dysfunction after ischaemia-reperfusion. ETA and ETB receptors mediate diverse actions, but it is unknown whether these actions depend on ischaemia type and duration. We investigated the role of ETB receptors after four ischaemia-reperfusion protocols in isolated rat hearts. Methods. Left ventricular haemodynamic variables were measured in the Langendorff-perfused model after 40-and 20-minute regional or global ischaemia, followed by 30-minute reperfusion. Wild-type (n = 39) and ETB-deficient (n = 41) rats were compared. Infarct size was measured using fluorescent microspheres after regional ischaemia-reperfusion. Results. Left ventricular dysfunction was more prominent in ETBdeficient rats, particularly after regional ischaemia. Infarct size was smaller (P = 0.006) in wild-type (31.5 ± 4.4%) than ETBdeficient (45.0 ± 7.3%) rats after 40 minutes of regional ischaemia-reperfusion. Although the recovery of left ventricular function was poorer after 40-minute ischaemia-reperfusion, end-diastolic pressure in ETB-deficient rats was higher after 20 than after 40 minutes of regional ischaemia-reperfusion. Conclusion. ETB receptors exert cytoprotective effects in the rat heart, mainly after regional ischaemia-reperfusion. Longer periods of ischaemia suppress the recovery of left ventricular function after reperfusion, but the role of ETB receptors may be more important during the early phases

Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase

Rationale: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. Results: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2–3 months old) 3-MST−/− mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST−/− mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST−/− mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST−/− mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST−/− mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. Conclusions: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis

Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis

Καινοτόμα δεδομένα στο ρόλο του μονοξειδίου του αζώτου και του υδροθείου στη μυοκαρδιακή προστασία

Aim: We sought to evaluate the role and the underlying molecular mechanisms of endogenous produced or exogenous administrated gasotransmitters, NO and H₂S, on i) the pathophysiology of myocardial ischemia (I)- reperfusion (R) injury; ii) PostC mimicking effects in myocardial infarction and iii) their physiological role in cigarette smoking (CS). The aims were assessed by performing four separate experimental studies. Methods and Results: In the first study oxidative stress injury by H₂O₂ in cardiomyocytes or in vivo myocardial I-R injury increased PYK2 activity and eNOS phosphorylation on both the inhibitory site Y656 and on the activator site S1176. Activity assays demonstrated that Y657 exerts a dominant effect over S1176. Pharmacological blockade of PYK2 alleviated eNOS inhibition and reduced myocardial infarct size in wild-type, but not in eNOS KO mice. In the second and third study low dose IV NTG and NaHS reduced infarct size in anesthetized male rabbits without affecting haemodynamics. However differences were detected in the downstream pathways. On one hand NTG’s protection was abolished by PI3K and NOS inhibition and NTG had no effect on infarct size in eNOS KO mice. Moreover, NTG failed to increase cardiac cGMP levels while PKG inhibition did not reverse NTG’s infarct limiting effects. Mechanistically, NTG protected through a CypD-dependent manner as it failed to confer additional protection in hearts from CypD KO mice. Interestingly, NTG minimized nitro-oxidative stress due to an eNOS mediated mechanism. On the other hand NOS inhibition did not alter the effect of NaHS in rabbit hearts; however, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. NaHS increased cardiac cGMP levels and PKG inhibition abrogated its protective effects. In addition, it enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner and did not exert cardioprotection in PLN KO mice. In the fourth study exposure of mice to CS increased blood pressure and up regulated the NOS/sGC/cGMP pathway whilst decreased H₂S generating enzymes expression. Exposure to CS did not increase the infarction compared to the room air (RA)-exposed group. PreC but not PostC application was beneficial for both CS and RA vs non-conditioned animals. PreC preserved its effects in mice exposed to CS, as it does not affect the cardioprotective signaling; in contrast to PostC. Conclusions: i) PYK2 is a pivotal regulator of eNOS function in the pathophysiology of myocardial infarction; ii) Exogenous administration of NO and H₂S donors reduces myocardial infarct size. The two gaseous molecules act different in the upstream pathways, but both preserve mitochondrial integrity and rescue cardiomyocytes from death and iii) CS is a new comorbidity that blunts PostC beneficial effects in the myocardium due to impaired activation of the Akt/eNOS/cGMP axis that occurs in parallel to enhanced oxidative stress.Σκοπός: Να διερευνήσουμε το ρόλο των ενδογενών παραγόμενων και εξωγενώς χορηγούμενων αέριων διαβιβαστών, NO και H₂S, i) στη παθοφυσιολογία του εμφράγματος του μυοκαρδίου, ii) στην ικανότητά τους να μιμούνται το PostC και iii) το φυσιολογικό τους ρόλο στο κάπνισμα. Οι σκοποί προσεγγίστηκαν με την διενέργεια τεσσάρων διακριτών μελετών. Μέθοδοι και Αποτελέσματα: Στην πρώτη μελέτη η επαγωγή οξειδωτικού στρες σε καρδιομυοκύτταρα ή η in vivo επαγωγή εμφράγματος του μυοκαρδίου ενεργοποίησε την PYK2 και φωσφορυλίωσε την eNOS τόσο στην ανασταλτική Y656 όσο και στη θέση ενεργοποίησης S1176. Με δοκιμασίες ενεργότητας η θέση Y657 αποδείχθηκε κυρίαρχη σε σχέση με τη θέση S1176. Η φαρμακολογική αναστολή της PYK2 ανέστρεψε την αναστολή, της eNOS και μείωσε την έκταση του εμφράγματος στους μύες άγριου τύπου, αλλά όχι στους eNOS KO μύες. Στη δεύτερη και τρίτη μελέτη η χορήγηση Νιτρογλυκερίνης και NaHS μείωσαν την έκταση του εμφράγματος σε in vivo κονίκλους. Ωστόσο η μοριακή σηματοδότηση των δύο μορίων διαφοροποιήθηκε. Η καρδιοπροστατευτική δράση της νιτρογλυκερίνης ανατράπηκε κατόπιν αναστολής των PI3K και NOS ενώ η χορήγησή της δε μείωσε την έκταση του εμφράγματος σε μύες eNOS KO. Επιπρόσθετα, η Νιτρογλυκερίνη δεν αύξησε τα επίπεδα του καρδιακού cGMP ενώ η αναστολή της PKG δεν ανέτρεψε την προστατευτική της δράση. Μηχανιστικά, η Νιτρογλυκερίνη προστάτευσε μέσω ενός εξαρτώμενου από την CypD τρόπου καθώς δεν οδήγησε σε επιπρόσθετη προστασία σε CypD KO μύες. Ενδιαφέρον αποτελεί το γεγονός ότι η δράση της σχετίστηκε με μία εξαρτώμενη από την eNOS μείωση του νιτρο-οξειδωτικού στρες. Αντίθετα η αναστολή των NOS δεν επηρέασε την ευεργετική δράση του NaHS στην ομάδα των κονίκλων, ωστόσο, στους μύες η γενετική ή φαρμακολογική αναστολή της eNOS ανέτρεψε τη δράση του NaHS. Το NaHS αύξησε τα καρδιακά επίπεδα του cGMP και η αναστολή της PKG ανέστρεψε την προστατευτική του δράση. Επιπρόσθετα, το NaHS ενεργοποίησε τη φωσφολαμβάνη (PLN), με τρόπο εξαρτώμενο από την PKG και δεν μείωσε την έκταση του εμφράγματος σε μύες PLN KO. Στην τέταρτη μελέτη η έκθεση μυών σε καπνό τσιγάρου αύξησε την αρτηριακή πίεση και ενεργοποίησε το μονοπάτι NOS/sGC/cGMP ενώ μείωσε την έκφραση των ενζύμων που παράγουν H2S. Η έκθεση στο κάπνισμα δεν αύξησε την έκταση του εμφράγματος σε σχέση με την ομάδα έκθεσης σε αέρα δωματίου. Η εφαρμογή του PreC αλλά όχι του PostC ήταν αποτελεσματική στους μύες υπό έκθεση σε καπνό τσιγάρου. Το PreC διατήρησε την καρδιοπροστατευτική του δράση καθώς δεν επηρέασε την σηματοδότηση επιβίωσης σε αντίθεση με το PostC. Συμπεράσματα: i) η PYK2 αποτελεί κυρίαρχο ρυθμιστή της eNOS στο έμφραγμα του μυοκαρδίου; ii) Η εξωγενής χορήγηση δοτών ΝΟ και H₂S μειώνει την έκταση του εμφράγματος. Οι δύο αέριοι διαβιβαστές λειτουργούν με διακριτό μοριακό μηχανισμό, ωστόσο αμφότεροι διατηρούν την καρδιακή μιτοχονδριακή ακεραιότητα και iii) Το κάπνισμα αποτελεί μία νέα συννοσυρότητα η οποία αναστέλλει την ευεργετική δράση του PostC στο μυοκάρδιο επιδρώντας στα σηματοδοτικά μονοπάτια διάσωσης