Genes, long non-coding RNAs, and nucleic-acid based therapies: how genetics is changing the present and future of cardiovascular diseases

For the podcast associated with this article, please visit https://academic.oup.com/eurheartj/pages/Podcasts. This Focus Issue on arrhythmias and genetics contains a State of the Art Review article entitled ‘Physiological pacing: mechanisms, clinical indications, and perspectives’ by Zachary Whinnett from Imperial College London in the UK, and colleagues in which the authors review the evolving field of physiological pacing.1 Normal cardiac function depends on a highly efficient cardiac conduction system. Conduction system disease impairs cardiac function, which can reduce exercise capacity, cause symptoms such as breathlessness and fatigue, and, if severe, result in syncope and death. Pacemakers were originally developed to treat profound bradycardia caused by complete heart block. However, pacing the right ventricle leads to non-physiological activation of the ventricular myocardium, reducing global cardiac performance. Early developments in physiological pacing focused on promoting intrinsic activation, delivering more physiological heart rate responses during exercise, and coordinating atrial and ventricular activation. More recently, the focus has shifted to delivering pacing which preserves or restores normal ventricular activation patterns, including atrio-biventricular pacing, conduction system pacing by His-bundle pacing, left bundle branch pacing, left septal pacing, and Bachmann’s bundle pacing.2,3 Adoption of these more physiological approaches might reduce the adverse effects of pacing, as well as expanding pacing indications to treatment of other forms of conduction system disease. Advancements in gene editing technology hold the promise of revolutionizing the management of cardiovascular and metabolic disorders by targeting the genetically regulated pathways underlying these diseases. In a second State of the Art Review article entitled ‘Gene therapy and genome editing for lipoprotein disorders’, Chen Gurevitz from Icahn School of Medicine at Mount Sinai in New York, NY, USA, and colleagues revisit major monogenic and polygenic disorders of lipoprotein metabolism, including familial hypercholesterolaemia, elevated lipoprotein(a), and familial chylomicronaemia syndrome, and discuss the genetic-based therapies for management.4 Genetic factors play a critical role in the development of lipoprotein disorders, which significantly contribute to atherosclerotic cardiovascular disease (ASCVD). Traditional management of these conditions has relied on lipid-lowering therapies, which require lifelong adherence.5–9 Recent advancements in gene addition and editing technologies offer novel and potentially transformative approaches for treating lipoprotein disorders by targeting the relevant genetic pathways for each disease. RNA-based, gene addition, and gene editing therapies, including clustered regularly interspaced short palindromic repeats (CRISPR), base editing, and interventions whereby, are highlighted for their potential to provide durable treatments which overcome the adherence challenge. Integration of machine learning for risk prediction10–12 and the use of polygenic risk scores to enhance risk stratification13–16 further demonstrate the promise of personalized approaches, and overall potential for gene-based treatments to revolutionize ASCVD prevention and management. Though it is hypothesized that only a proportion of long non-coding RNAs (lncRNAs) are functional, it is definitively established that several are essential in cardiac homeostasis or as mediators of cardiovascular complications. In a Viewpoint article entitled ‘Missing ‘lncs’: how insufficient scrutiny can lead to misrepresentation of long non-coding RNAs (lncRNAs) and their function in cardiovascular disease’, Matthew Bennett, Igor Ulitsky, and Andrew H. Baker from the University of Edinburgh in the UK, and Weizmann Institute of Science in Rehovot, Israel, provide a critical perspective that will help cardiovascular reviewers and editors navigate the complexities involved in lncRNA research.17 A typical lncRNA is expressed only in particular cell types or states, with a lower expression, a lower conservation level across species, and a more complex set of possible transcript variants (isoforms) when compared with a typical protein-coding gene. Many lncRNAs are also expressed from genome regions with overlapping genes or other functionality. For instance, they may overlap regulatory DNA elements bound by transcription factors (e.g. enhancers that boost nearby gene transcription) or be processed into small RNAs (including microRNAs). Further, advances in protein-coding gene annotation methods have indicated that some lncRNAs produce small functional peptides and thus require re-classification as protein-coding genes. The combination of these features presents a substantial challenge for gene annotation and experimental study. Which isoforms are relevant? Which may produce a small RNA? Do answers to these questions vary across cell types? Which techniques are appropriate to assess this? How will this affect interpretation of experimental data? Some answers to these questions can be sought in easily accessible genomics resources and by scrutinizing the lncRNA gene locus in a genome browser. However, relying solely on broad-brush approaches may downplay their complexity in many cases. Cardiac amyloidosis (CA) is a progressive disease associated with a high incidence of heart failure and arrhythmias, and has a poor prognosis.18–20 Cardiac amyloidosis is often advanced at the time of diagnosis. Therefore, its early diagnosis will have a significant prognostic effect on life expectancy. In a Clinical Research article entitled ‘Atrial amyloidosis identified by biopsy in atrial fibrillation: prevalence and clinical presentation’, Kodai Shinzato from Saga University in Japan, and colleagues aim to assess the prevalence of CA in patients with non-valvular atrial fibrillation (AF) and to test the hypothesis that early-stage CA can be identified through atrial biopsy.21 Atrial biopsy was performed on 578 patients during AF ablation. The amyloid type was assessed using immunohistochemistry. Patients were classified into groups of atrial biopsy-detected CA (abio-CA) and non-CA, with an additional 58 patients clinically diagnosed with CA comprising the ‘clinical CA group’. Amyloid deposits were identified in atrial samples from 40 patients (7%), including 25 amyloid transthyretin types. Prevalence increased to 20%–40% with advancing age, left ventricular (LV) hypertrophy, and the presence of low-voltage areas in the left atrium. The abio-CA group exhibited a thinner LV posterior wall (P < .001) compared with the clinical CA group. The abio-CA group displayed a thicker LV posterior wall (P < .001) and a higher frequency of low-voltage areas defined as <0.5 mV (P < .001) compared with the non-CA group (Figure 1). Structured Graphical Abstract.21 The authors conclude that atrial biopsy reveals amyloid deposits in 7% of patients undergoing AF ablation, identifying early-stage CA. The manuscript is accompanied by an Editorial by Arnela Saljic, Alisha Niskala, and Thomas Jespersen from the University of Copenhagen in Denmark.22 The authors underscore that the findings by Shinzato et al. suggest that atrial biopsies may serve as a promising prognostic marker for identifying early-stage CA. Biopsies can, without increased risk, be harvested during AF catheter ablation and open chest (atrial appendages) procedures. As patients with CA commonly present with AF and increased risk of stroke and HF, there appears to be a causal link between the comorbidities rather than them existing as separate co-occurring conditions. Early intervention and detection are vital to better understand how CA contributes to these conditions. In combination with current imaging modalities (echocardiography/magnetic resonance imaging) and histological confirmation of amyloid deposits from atrial and ventricular biopsies, initiation of early therapeutic treatments can affect patient outcomes and lead to longer survival times. Sudden cardiac death (SCD) during childhood and adolescence occurs with an estimated frequency of 1–3 in 100 000 person-years. Inherited primary arrhythmia syndromes such as long QT syndrome (LQTS) might be the underlying cause of such tragic events. Long QT syndrome is a genetic disorder altering cardiomyocyte action potential and is associated with an increased risk for life-threatening rhythm disturbances. In a Clinical Research article entitled ‘Long QT syndrome in children and adolescents: risk factors and outcomes in a large German cohort’, Lea Lippert from the Technical University of Munich in Germany, and colleagues describe outcomes in a large contemporary national cohort of children and adolescents with LQTS.23 The authors carried out data analysis of 548 paediatric LQTS patients at 12 German tertiary care centres. The primary outcome was the occurrence of a major arrhythmic event (MAE) defined as SCD, aborted cardiac arrest (ACA), and appropriate implantable cardioverter-defibrillator (ICD) therapy before age 18 years. Patients (49% male) presented at a median age of 6.3 years with incidental findings (27%), symptoms (31%), or for cascade screening (42%). The primary outcome occurred in 10.6% of patients: SCD in 1.3%, ACA in 7.3%, and appropriate ICD therapy in 2%, with an overall event rate of 1.0 per 100 patient-years at risk. During follow-up (before 18 years of age), 92% of patients received cardiac medications. An ICD was implanted in 13.3%, of whom 34% received appropriate therapy. In multivariate analysis, the likelihood of experiencing an MAE was greater in patients with LQT3 (hazard ratio [HR] 2.5, P = .03), syncope (HR 3.0, P < .01), absence of cardiac medication (HR 9.5, P < .01), and QTc ≥500 ms (HR 2.9, P < .01) (Figure 2). The study analyses data of children and adolescents diagnosed with different types of long QT syndrome (LQTS) and describes their clinical course, outcome, and genetics. The primary endpoint, a major arrhythmic event (MAE) before the age of 18 years, was reached by about a tenth of patients. Of those patients who received implantable cardioverter-defibrillators (ICDs) either for primary or for secondary prevention, a significant proportion received appropriate therapy. The diagnosis of LQT3, a history of syncope, absence of medication at the time of the MAE, and a QTc duration ≥500 ms were identified as independent risk factors, as depicted by the corresponding Kaplan–Meier curves for MAE-free survival. IQR, interquartile range; LQT, long QT syndrome; MAE, major arrhythmic event; ICD, implantable cardioverter-defibrillator.23 The authors conclude that paediatric LQTS patients are at risk for life-threatening arrhythmias at <18 years of age and require a meticulous personalized clinical approach. The manuscript is accompanied by an Editorial by Peter J. Schwartz and Carla Spazzolini from Istituto Auxologico Italiano IRCCS in Milan, Italy.24 The authors note that Lippert et al. have the merit of having provided relevant information on the very important population below age 18: there are not many reports, especially recent ones, on this subset of LQTS patients of any genotype. Their study is a welcome contribution to the field as on the one hand it provides a reliable picture of what is likely to happen to young Germans affected by LQTS, and on the other offers a rather unique comparison with their management elsewhere. This comparison offers the possibility for an unabashed and frank analysis of what occurs in different countries and in different centres, which might lead some of them to consider changing their management strategies for the benefit of LQTS patients everywhere. Atrial fibrosis represents a critical determinant in AF pathogenesis. Although endothelial dysfunction is a hallmark feature of AF, the precise mechanisms by which endothelial cells (ECs) contribute to atrial fibrosis remain incompletely understood. In a Translational Science article entitled ‘Endothelial plasticity in atrial fibrosis by integrating single-cell sequencing and genetic lineage tracing’, Zhiwei Zeng from Peking Union Medical College in Beijing, China, provide novel insights into the pathophysiology of atrial fibrosis.25 This study employed single-cell RNA sequencing (scRNA-seq) to analyse intercellular communication networks in atrial tissues from both sinus rhythm (SR) and AF patients. Cdh5-CreERT2;RFP mice were generated to track endothelial plasticity following transverse aortic constriction (TAC). Cell–cell interactions were investigated using isolated human primary atrial ECs and fibroblasts (FBs) in vitro. To elucidate the regulatory role of endothelial-derived transforming growth factor-β1 (TGF-β1) on FB function, endothelial-specific Tgf-β1 knockout (Tgf-β1ECKO) mice were generated. scRNA-seq analysis identified ECs as predominant signal-sending cells with extensive FB connectivity in both SR and AF atrial tissues. During progression of fibrosis, ECs displayed significant mesenchymal activation at the transcriptional level. However, immunofluorescence and high-content screening revealed minimal complete endothelial-to-FB transition. Cell–cell communication analysis and in vitro studies identified TGF-β1 as the key mediator through which mesenchymal-activated ECs (EndoMA) promoted FB proliferation and collagen production. Notably, endothelial-specific Tgf-β1 deletion attenuated TAC-induced atrial fibrosis and reduced AF susceptibility. The authors conclude that EndoMA-derived TGF-β1 critically regulates FB function and drives progression of atrial fibrosis. Targeting endothelial-specific pathways represents a promising therapeutic strategy for attenuating atrial fibrosis in AF pathogenesis. The manuscript is accompanied by an Editorial by Guillermo Luxán and Samuel Sossalla from Goethe University Frankfurt in Germany, and Justus Liebig University Giessen in Germany.26 The authors note that it is undisputed that fibrosis is one of the key players in the pathophysiology of AF, and it is, of course, closely linked to and influences electrical remodelling. Despite its clinical significance, no specific therapies currently target atrial fibrosis directly: an urgent gap highlighted by the DECAAF II trial, which demonstrated that MRI-guided fibrosis ablation did not significantly reduce AF recurrence compared with conventional ablation. This, indeed, underscores the crucial need for a deeper understanding of the underlying pathophysiology as the foundation for developing innovative, fibrosis-targeted treatments, a perspective that is also increasingly emphasized from a clinical standpoint regarding therapeutic implementation. The potential implementation of such a therapeutic approach remains unclear at present, as early intervention would probably be required, ideally before significant fibrosis and ‘fibrosis-dependent AF’ occur. Consequently, future discussions and research will probably focus on concepts such as treating patients at risk and preventive strategies in the context of such approaches. In conclusion, a deeper understanding of the pathophysiology of AF, particularly the roles of endothelial plasticity and fibrosis, is essential for advancing treatment strategies. Familial ST-depression (FSTD) syndrome is a recently identified inherited cardiac disease associated with arrhythmias and systolic dysfunction. The underlying genetic aetiology has remained elusive. In a Translational Science article entitled ‘Gain-of-function enhancer variant near KCNB1 causes familial ST-depression syndrome’, Alex Hørby Christensen from Copenhagen University Hospital, Rigshospitalet in Denmark, and colleagues characterize a non-coding variant near KCNB1 as the cause of the dominantly inherited FSTD syndrome.27 A total of 67 FSTD patients (20 families) were studied. Linkage analysis and whole-genome sequencing (WGS) were initially performed. An identified non-coding variant was functionally characterized in AC16 human cardiomyocytes, muscle tissue, and human myocardium. In silico analyses, luciferase and dCas9-activator/repressor assays, protein–DNA experiments, chromosome conformation capture (4C), and RNA sequencing were also performed. The electrocardiographic (ECG) phenotype was inherited in an autosomal dominant manner in all families. Linkage analysis revealed a single peak on chromosome 20, and WGS identified a single, rare, non-coding variant located 18 kb downstream of KCNB1 on chromosome 20 in all affected individuals. Perfect co-segregation with the ECG phenotype was observed together with full penetrance in all families. The variant creates an MEF2-binding site, and the presence of the variant allele or MEF2 co-expression enhanced transcriptional activity. dCas9-activator/repressor assays showed that KCNB1 was the only gene consistently regulated by the locus, and 4C experiments in AC16 cells and human muscle tissue confirmed the locus–KCNB1 promoter interaction. Expression analysis in human endocardial tissue did not document any change in gene expression, probably explained by expressional heterogeneity. The authors conclude that a gain-of-function enhancer variant creates a hyperactive regulatory locus that interacts with the KCNB1 promoter and causes FSTD. This is the first time that KCNB1 has been implicated in human cardiac electrophysiology and arrhythmogenesis. The manuscript is accompanied by an Editorial by Kristina H. Haugaa and Ivana Haby from the University of Oslo in Norway and Karolinska University Hospital in Stockholm, Sweden.28 The authors highlight that the genetic era has shown us multiple times that previously unseen and unknown syndromes suddenly become visible, explainable, and in many cases even treatable with risk-reducing agents. However, you see only what you look for. Due to the eye-opening studies from our colleagues, we will probably find these patients in every centre and region. Should we regard the new entity as a typical ion channel disease? Probably not. A significant number of patients with FSTD syndrome had concomitant and progressive LV dysfunction, indicating the overlap between ion channel and functional dysfunction. Interestingly, the ECG ST-depressions were also slowly progressive over time, which is not seen in other ion channelopathies. These findings add to previous reports showing that altered ion channel function is associated with subtle or overt changes in cardiac function and structure, exemplified by Brugada syndrome and the LQTS. The impressive work from this group has covered the chain from clinical presentation, to definition, to inheritance pattern, and they have now in this translational work demonstrated the genetic causative variant. The issue is also complemented by two Discussion Forum contributions. In a commentary entitled ‘Gender-specific and in potential causal and from Clinical College of Medical Medical and Medical University in China, on the recently entitled and by et from University in and colleagues in a separate The editors that this issue of the will be of to its reports from the to and for help with of this