Empagliflozin prevents heart failure through inhibition of the NHE1-NO pathway, independent of SGLT2.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) constitute the only medication class that consistently prevents or attenuates human heart failure (HF) independent of ejection fraction. We have suggested earlier that the protective mechanisms of the SGLT2i Empagliflozin (EMPA) are mediated through reductions in the sodium hydrogen exchanger 1 (NHE1)-nitric oxide (NO) pathway, independent of SGLT2. Here, we examined the role of SGLT2, NHE1 and NO in a murine TAC/DOCA model of HF. SGLT2 knockout mice only showed attenuated systolic dysfunction without having an effect on other signs of HF. EMPA protected against systolic and diastolic dysfunction, hypertrophy, fibrosis, increased Nppa/Nppb mRNA expression and lung/liver edema. In addition, EMPA prevented increases in oxidative stress, sodium calcium exchanger expression and calcium/calmodulin-dependent protein kinase II activation to an equal degree in WT and SGLT2 KO animals. In particular, while NHE1 activity was increased in isolated cardiomyocytes from untreated HF, EMPA treatment prevented this. Since SGLT2 is not required for the protective effects of EMPA, the pathway between NHE1 and NO was further explored in SGLT2 KO animals. In vivo treatment with the specific NHE1-inhibitor Cariporide mimicked the protection by EMPA, without additional protection by EMPA. On the other hand, in vivo inhibition of NOS with L-NAME deteriorated HF and prevented protection by EMPA. In conclusion, the data support that the beneficial effects of EMPA are mediated through the NHE1-NO pathway in TAC/DOCA-induced heart failure and not through SGLT2 inhibition.

Dissecting the Molecular Mechanisms Driving Electropathology in Atrial Fibrillation: Deployment of RNA Sequencing and Transcriptomic Analyses.

Despite many efforts to treat atrial fibrillation (AF), the most common progressive and age-related cardiac tachyarrhythmia in the Western world, the efficacy is still suboptimal. A plausible reason for this is that current treatments are not directed at underlying molecular root causes that drive electrical conduction disorders and AF (i.e., electropathology). Insights into AF-induced transcriptomic alterations may aid in a deeper understanding of electropathology. Specifically, RNA sequencing (RNA-seq) facilitates transcriptomic analyses and discovery of differences in gene expression profiles between patient groups. In the last decade, various RNA-seq studies have been conducted in atrial tissue samples of patients with AF versus controls in sinus rhythm. Identified differentially expressed molecular pathways so far include pathways related to mechanotransduction, ECM remodeling, ion channel signaling, and structural tissue organization through developmental and inflammatory signaling pathways. In this review, we provide an overview of the available human AF RNA-seq studies and highlight the molecular pathways identified. Additionally, a comparison is made between human RNA-seq findings with findings from experimental AF model systems and we discuss contrasting findings. Finally, we elaborate on new exciting RNA-seq approaches, including single-nucleotide variants, spatial transcriptomics and profiling of different populations of total RNA, small RNA and long non-coding RNA.

Quaking regulates circular RNA production in cardiomyocytes.

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that are gaining increasing attention for their roles in various pathophysiological processes. The RNA-binding protein quaking (QKI) has been identified as a regulator of circRNA formation. In this study, we investigate the role of QKI in the formation of circRNAs in the heart by performing RNA-sequencing on Qki-knockout mice. Loss of QKI resulted in the differential expression of 17% of the circRNAs in adult mouse hearts. Interestingly, the majority of the QKI-regulated circRNAs (58%) were derived from genes undergoing QKI-dependent splicing, indicating a relationship between back-splicing and linear splicing. We compared these QKI-dependent circRNAs with those regulated by RBM20, another cardiac splicing factor essential for circRNA formation. We found that QKI and RBM20 regulate the formation of a distinct, but partially overlapping set of circRNAs in the heart. Strikingly, many shared circRNAs were derived from the Ttn gene, and they were regulated in an opposite manner. Our findings indicate that QKI not only regulates alternative splicing in the heart but also the formation of circRNAs.

Splicing factors in the heart: Uncovering shared and unique targets.

Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the splicing factor RNA-binding protein 20 (RBM20) cause a severe form of familial dilated cardiomyopathy has sparked a great interest in alternative splicing in the field of cardiology. Since then, identification of splicing factors controlling alternative splicing in the heart has grown at a rapid pace. Despite the intriguing observation that a certain overlap exists between the targets of some splicing factors, an integrated and systematic analysis of their splicing networks is missing. Here, we compared the splicing networks of individual splicing factors by re-analyzing original RNA-sequencing data from eight previously published mouse models, in which a single splicing factor has been genetically deleted (i.e. HNRNPU, MBNL1/2, QKI, RBM20, RBM24, RBPMS, SRSF3, SRSF4). We show that key splicing events in Camk2d, Ryr2, Tpm1, Tpm2 and Pdlim5 require the combined action of the majority of these splicing factors. Additionally, we identified common targets and pathways among splicing factors, with the largest overlap between the splicing networks of MBNL, QKI and RBM24. We also re-analyzed a large-scale RNA-sequencing study on hearts of 128 heart failure patients. Here, we observed that MBNL1, QKI and RBM24 expression varied greatly. This variation in expression correlated with differential splicing of their downstream targets as found in mice, suggesting that aberrant splicing by MBNL1, QKI and RBM24 might contribute to the disease mechanism in heart failure.

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes.

AIMS: In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. METHODS AND RESULTS: We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. CONCLUSION: Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

Inhibition of minor intron splicing reduces Na+ and Ca2+ channel expression and function in cardiomyocytes.

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure.

Extracellular matrix remodeling in animal models of anthracycline-induced cardiomyopathy: a meta-analysis.

As in other cardiomyopathies, extracellular matrix (ECM) remodeling plays an important role in anthracycline-induced cardiomyopathy. To understand the pattern and timing of ECM remodeling pathways, we conducted a systematic review in which we describe protein and mRNA markers for ECM remodeling that are differentially expressed in the hearts of animals with anthracycline-induced cardiomyopathy. We included 68 studies in mice, rats, rabbits, and pigs with follow-up of 0.1-8.2 human equivalent years after anthracycline administration. Using meta-analysis, we found 29 proteins and 11 mRNAs that were differentially expressed in anthracycline-induced cardiomyopathy compared to controls. Collagens, matrix metalloproteinases (MMPs), inflammation markers, transforming growth factor ß signaling markers, and markers for cardiac hypertrophy were upregulated, whereas the protein kinase B (AKT) pro-survival pathway was downregulated. Their expression patterns over time from single time point studies were studied with meta-regression using human equivalent years as the time scale. Connective tissue growth factor showed an early peak in expression but remained upregulated at all studied time points. Brain natriuretic peptide (BNP) and MMP9 protein levels increased in studies with longer follow-up. Significant associations were found for higher atrial natriuretic peptide with interstitial fibrosis and for higher BNP and MMP2 protein levels with left ventricular systolic function.

Dietary restriction in the long-chain acyl-CoA dehydrogenase knockout mouse.

Patients with a disorder of mitochondrial long-chain fatty acid ß-oxidation (FAO) have reduced fasting tolerance and may present with hypoketotic hypoglycemia, hepatomegaly, (cardio)myopathy and rhabdomyolysis. Patients should avoid a catabolic state because it increases reliance on FAO as energy source. It is currently unclear whether weight loss through a reduction of caloric intake is safe in patients with a FAO disorder. We used the long-chain acyl-CoA dehydrogenase knockout (LCAD KO) mouse model to study the impact of dietary restriction (DR) on the plasma metabolite profile and cardiac function. For this, LCAD KO and wild type (WT) mice were subjected to DR (70% of ad libitum chow intake) for 4 weeks and compared to ad libitum chow fed mice. We found that DR had a relatively small impact on the plasma metabolite profile of WT and LCAD KO mice. Echocardiography revealed a small decrease in left ventricular systolic function of LCAD KO mice, which was most noticeable after DR, but there was no evidence of DR-induced cardiac remodeling. Our results suggest that weight loss through DR does not have acute and detrimental consequences in a mouse model for FAO disorders.

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes.

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor-ß (TGF-ß). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-ß receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-ß-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.

Low miR-19b-1-5p Expression Is Related to Aspirin Resistance and Major Adverse Cardio- Cerebrovascular Events in Patients With Acute Coronary Syndrome.

Background Because of a nonresponse to aspirin (aspirin resistance), patients with acute coronary syndrome (ACS) are at increased risk of developing recurrent event. The in vitro platelet function tests have potential limitations, making them unsuitable for the detection of aspirin resistance. We investigated whether miR-19b-1-5p could be utilized as a biomarker for aspirin resistance and future major adverse cardio-cerebrovascular (MACCE) events in patients with ACS. Methods and Results In this cohort study, patients with ACS were enrolled from multiple tertiary hospitals in Christchurch, Hong Kong, Sarawak, and Singapore between 2011 and 2015. MiR-19b-1-5p expression was measured from buffy coat of patients with ACS (n=945) by reverse transcription quantitative polymerase chain reaction. Platelet function was determined by Multiplate aggregometry testing. MACCE was collected over a mean follow-up time of 1.01±0.43 years. Low miR-19b-1-5p expression was found to be related to aspirin resistance as could be observed from sustained platelet aggregation in the presence of aspirin (-Log-miR-19b-1-5p, [unstandardized beta, 44.50; 95% CI, 2.20-86.80; P<0.05]), even after adjusting for age, sex, ethnicity, and prior history of stroke. Lower miR-19b-1-5p expression was independently associated with a higher risk of MACCE (-Log-miR-19b-1-5p, [hazard ratio, 1.85; 95% CI, 1.23-2.80; P<0.05]). Furthermore, a significant interaction was noted between the inverse miR-19b-1-5p expression and family history of premature coronary artery disease (P=0.01) on the risk of MACCE. Conclusions Lower miR-19b-1-5p expression was found to be associated with sustained platelet aggregation on aspirin, and a higher risk of MACCE in patients with ACS. Therefore, miR-19b-1-5p could be a suitable marker for aspirin resistance and might predict recurrence of MACCE in patients with ACS.

Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart.

RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster. OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo. METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa. CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.

Functional modulation of atrio-ventricular conduction by enhanced late sodium current and calcium-dependent mechanisms in Scn5a1798insD/+ mice.

AIMS: SCN5A mutations are associated with arrhythmia syndromes, including Brugada syndrome, long QT syndrome type 3 (LQT3), and cardiac conduction disease. Long QT syndrome type 3 patients display atrio-ventricular (AV) conduction slowing which may contribute to arrhythmogenesis. We here investigated the as yet unknown underlying mechanisms. METHODS AND RESULTS: We assessed electrophysiological and molecular alterations underlying AV-conduction abnormalities in mice carrying the Scn5a1798insD/+ mutation. Langendorff-perfused Scn5a1798insD/+ hearts showed prolonged AV-conduction compared to wild type (WT) without changes in atrial and His-ventricular (HV) conduction. The late sodium current (INa,L) inhibitor ranolazine (RAN) normalized AV-conduction in Scn5a1798insD/+ mice, likely by preventing the mutation-induced increase in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) concentrations. Indeed, further enhancement of [Na+]i and [Ca2+]i by the Na+/K+-ATPase inhibitor ouabain caused excessive increase in AV-conduction time in Scn5a1798insD/+ hearts. Scn5a1798insD/+ mice from the 129P2 strain displayed more severe AV-conduction abnormalities than FVB/N-Scn5a1798insD/+ mice, in line with their larger mutation-induced INa,L. Transverse aortic constriction (TAC) caused excessive prolongation of AV-conduction in FVB/N-Scn5a1798insD/+ mice (while HV-intervals remained unchanged), which was prevented by chronic RAN treatment. Scn5a1798insD/+-TAC hearts showed decreased mRNA levels of conduction genes in the AV-nodal region, but no structural changes in the AV-node or His bundle. In Scn5a1798insD/+-TAC mice deficient for the transcription factor Nfatc2 (effector of the calcium-calcineurin pathway), AV-conduction and conduction gene expression were restored to WT levels. CONCLUSIONS: Our findings indicate a detrimental role for enhanced INa,L and consequent calcium dysregulation on AV-conduction in Scn5a1798insD/+ mice, providing evidence for a functional mechanism underlying AV-conduction disturbances secondary to gain-of-function SCN5A mutations.

MiR-223-3p and miR-122-5p as circulating biomarkers for plaque instability.

BACKGROUND: In this study, we discovered and validated candidate microRNA (miRNA) biomarkers for coronary artery disease (CAD). METHOD: Candidate tissue-derived miRNAs from atherosclerotic plaque material in patients with stable coronary artery disease (SCAD) (n=14) and unstable coronary artery disease (UCAD) (n=25) were discovered by qPCR-based arrays. We validated differentially expressed miRNAs, along with seven promising CAD-associated miRNAs from the literature, in the serum of two large cohorts (n=395 and n=1000) of patients with SCAD and UCAD and subclinical atherosclerosis (SubA) and controls, respectively. RESULT: From plaque materials (discovery phase), miR-125b-5p and miR-193b-3p were most upregulated in SCAD, whereas miR-223-3p and miR-142-3p were most upregulated in patients with UCAD. Subsequent validation in serum from patients with UCAD, SCAD, SubA and controls demonstrated significant upregulation of miR-223-3p, miR-133a-3p, miR-146-3p and miR-155-5p. The ischaemia-related miR-499-5p was also highly upregulated in patients with UCAD compared with the other groups (SCAD OR 20.63 (95% CI 11.16 to 38.15), SubA OR 96.10 (95% CI 40.13 to 230.14) and controls OR 15.73 (95% CI 7.80 to 31.72)). However, no significant difference in miR-499-5p expression was observed across SCAD, SubA and controls. MiR-122-5p was the only miRNA to be significantly upregulated in the serum of both patients with UCAD and SCAD. CONCLUSION: In conclusion, miR-122-5p and miR-223-3p might be markers of plaque instability.

circRNAprofiler: an R-based computational framework for the downstream analysis of circular RNAs.

BACKGROUND: Circular RNAs (circRNAs) are a newly appreciated class of non-coding RNA molecules. Numerous tools have been developed for the detection of circRNAs, however computational tools to perform downstream functional analysis of circRNAs are scarce. RESULTS: We present circRNAprofiler, an R-based computational framework that runs after circRNAs have been identified. It allows to combine circRNAs detected by multiple publicly available annotation-based circRNA detection tools and to analyze their expression, genomic context, evolutionary conservation, biogenesis and putative functions. CONCLUSIONS: Overall, the circRNA analysis workflow implemented by circRNAprofiler is highly automated and customizable, and the results of the analyses can be used as starting point for further investigation in the role of specific circRNAs in any physiological or pathological condition.

Loss-of-function variants in myocardin cause congenital megabladder in humans and mice.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.

Conserved NPPB+ Border Zone Switches From MEF2- to AP-1-Driven Gene Program.

BACKGROUND: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb, encoding B-type natriuretic peptide, after infarction. METHODS: Transgenic reporter mice were used to identify the Nppb-positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. RESULTS: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB, ANKRD1, DES, UCHL1, JUN, and FOXP1. Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. CONCLUSIONS: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1-driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

Circular RNAs open a new chapter in cardiovascular biology.

Circular RNAs (circRNAs) are emerging as a new class of non-coding RNA molecules. This unusual class of RNA species is generated by a back-splicing event of one or two exons, resulting in a covalently closed circRNA molecule. Owing to their circular form, circRNAs are protected from degradation by exonucleases and have greater stability than linear RNA. Advances in computational analysis of RNA sequencing have revealed that thousands of different circRNAs are expressed in a wide range of mammalian tissues, including the cardiovascular system. Moreover, numerous circRNAs are expressed in a disease-specific manner. A great deal of progress has been made in understanding the biogenesis and function of these circRNAs. In this Review, we discuss the current understanding of circRNA biogenesis and function, with a particular emphasis on the cardiovascular system.

AAV9-mediated Rbm24 overexpression induces fibrosis in the mouse heart.

The RNA-binding protein Rbm24 has recently been identified as a pivotal splicing factor in the developing heart. Loss of Rbm24 in mice disrupts cardiac development by governing a large number of muscle-specific splicing events. Since Rbm24 knockout mice are embryonically lethal, the role of Rbm24 in the adult heart remained unexplored. Here, we used adeno-associated viruses (AAV9) to investigate the effect of increased Rbm24 levels in adult mouse heart. Using high-resolution microarrays, we found 893 differentially expressed genes and 1102 differential splicing events in 714 genes in hearts overexpressing Rbm24. We found splicing differences in cardiac genes, such as PDZ and Lim domain 5, Phospholamban, and Titin, but did not find splicing differences in previously identified embryonic splicing targets of Rbm24, such as skNAC, αNAC, and Coro6. Gene ontology enrichment analysis demonstrated increased expression of extracellular matrix (ECM)-related and immune response genes. Moreover, we found increased expression of Tgfß-signaling genes, suggesting enhanced Tgfß-signaling in these hearts. Ultimately, this increased activation of cardiac fibroblasts, as evidenced by robust expression of Periostin in the heart, and induced extensive cardiac fibrosis. These results indicate that Rbm24 may function as a regulator of cardiac fibrosis, potentially through the regulation of TgfßR1 and TgfßR2 expression.

Circular RNAs in the cardiovascular system.

Until recently considered as rare, circular RNAs (circRNAs) are emerging as important regulators of gene expression. They are ubiquitously expressed and represent a novel branch of the family of non-coding RNAs. Recent investigations showed that circRNAs are regulated in the cardiovascular system and participate in its physiological and pathological development. In this review article, we will provide an overview of the role of circRNAs in cardiovascular health and disease. After a description of the biogenesis of circRNAs, we will summarize what is known of the expression, regulation and function of circRNAs in the cardiovascular system. We will then address some technical aspects of circRNAs research, discussing how artificial intelligence may aid in circRNAs research. Finally, the potential of circRNAs as biomarkers of cardiovascular disease will be addressed and directions for future research will be proposed.

Nur77 protects against adverse cardiac remodelling by limiting neuropeptide Y signalling in the sympathoadrenal-cardiac axis.

Aims: Cardiac remodelling and heart failure are promoted by persistent sympathetic activity. We recently reported that nuclear receptor Nur77 may protect against sympathetic agonist-induced cardiac remodelling in mice. The sympathetic co-transmitter neuropeptide Y (NPY) is co-released with catecholamines and is a known cardiac modulator and predictor of heart failure mortality. Recently, transcriptome analyses revealed NPY as a putative target of Nur77. In this study, we assess whether Nur77 modulates adverse cardiac remodelling via NPY signalling. Methods and results: Nur77 represses NPY expression in the PC12 adrenal chromaffin cell line. Accordingly, NPY levels are higher in adrenal gland, plasma, and heart from Nur77-KO compared to wild-type mice. Conditioned medium from Nur77-silenced chromaffin cells and serum from Nur77-KO mice induce marked hypertrophy in cultured neonatal rat cardiomyocytes, which is inhibited by the NPY type 1 receptor (NPY1R) antagonist BIBO3304. In cardiomyocytes from Nur77-KO mice, intracellular Ca2+ is increased partially via the NPY1R. This is independent from elevated circulating NPY since cardiomyocyte-specific Nur77-deficient mice (CM-KO) do not have elevated circulating NPY, but do exhibit BIBO3304-sensitive, increased cardiomyocyte intracellular Ca2+. In vivo, this translates to NPY1R antagonism attenuating cardiac calcineurin activity and isoproterenol-induced cardiomyocyte hypertrophy and fibrosis in full-body Nur77-KO mice, but not in CM-KO mice. Conclusions: The cardioprotective action of Nur77 can be ascribed to both inhibition of circulating NPY levels and to cardiomyocyte-specific modulation of NPY-NPY1R signalling. These results reveal the underlying mechanism of Nur77 as a promising modifier gene in heart failure.