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Non-coding RNAs (ncRNAs), such as miRNAs and long non-coding RNAs (lncRNAs) have been reported as regulators of cardiovascular pathophysiology. Their transient effect and diversified mechanisms of action offer a plethora of therapeutic opportunities for cardiovascular diseases (CVDs). However, physicochemical RNA features such as charge, stability, and structural organization hinder efficient on-target cellular delivery. Here, we highlight recent preclinical advances in ncRNA delivery for the cardiovascular system using non-viral approaches. We identify the unmet needs and advance possible solutions towards clinical translation. Finding the optimal delivery vehicle and administration route is vital to improve therapeutic efficacy and safety; however, given the different types of ncRNAs, this may ultimately not be frameable within a one-size-fits-all approach.

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Heart failure has reached epidemic proportions in a progressively ageing population. The molecular mechanisms underlying heart failure remain elusive, but evidence indicates that DNA damage is enhanced in failing hearts. Here, we tested the hypothesis that endogenous DNA repair in cardiomyocytes is critical for maintaining normal cardiac function, so that perturbed repair of spontaneous DNA damage drives early onset of heart failure. To increase the burden of spontaneous DNA damage, we knocked out the DNA repair endonucleases xeroderma pigmentosum complementation group G (XPG) and excision repair cross-complementation group 1 (ERCC1), either systemically or cardiomyocyte-restricted, and studied the effects on cardiac function and structure. Loss of DNA repair permitted normal heart development but subsequently caused progressive deterioration of cardiac function, resulting in overt congestive heart failure and premature death within 6 months. Cardiac biopsies revealed increased oxidative stress associated with increased fibrosis and apoptosis. Moreover, gene set enrichment analysis showed enrichment of pathways associated with impaired DNA repair and apoptosis, and identified TP53 as one of the top active upstream transcription regulators. In support of the observed cardiac phenotype in mutant mice, several genetic variants in the ERCC1 and XPG gene in human GWAS data were found to be associated with cardiac remodelling and dysfunction. In conclusion, unrepaired spontaneous DNA damage in differentiated cardiomyocytes drives early onset of cardiac failure. These observations implicate DNA damage as a potential novel therapeutic target and highlight systemic and cardiomyocyte-restricted DNA repair-deficient mouse mutants as bona fide models of heart failure.

Cardiovascular disease (CVD) remains the leading cause of death worldwide. A deeper characterization of regional transcription patterns within different heart chambers may aid to improve our understanding of the molecular mechanisms involved in myocardial function and further, our ability to develop novel therapeutic strategies. Here, we used RNA sequencing to determine differentially expressed protein coding (PC) and long non-coding (lncRNA) transcripts within the heart chambers across seven vertebrate species and identified evolutionarily conserved chamber specific genes, lncRNAs and pathways. We investigated lncRNA homologs based on sequence, secondary structure, synteny and expressional conservation and found most lncRNAs to be conserved by synteny. Regional co-expression patterns of transcripts are modulated by multiple factors, including genomic overlap, strandedness and transcript biotype. Finally, we provide a community resource designated EvoACTG, which informs researchers on the conserved yet intertwined nature of the coding and non-coding cardiac transcriptome across popular model organisms in CVD research.

As mediators of intercellular communication, extracellular vesicles containing molecular cargo, such as microRNAs, are secreted by cells and taken up by recipient cells to influence their cellular phenotype and function. Here we report that cardiac stress-induced differential microRNA content, with miR-200c-3p being one of the most enriched, in cardiomyocyte-derived extracellular vesicles mediates functional cross-talk with endothelial cells. Silencing of miR-200c-3p in mice subjected to chronic increased cardiac pressure overload resulted in attenuated hypertrophy, smaller fibrotic areas, higher capillary density, and preserved cardiac ejection fraction. We were able to maximally rescue microvascular and cardiac function with very low doses of antagomir, which specifically silences miR-200c-3p expression in non-myocyte cells. Our results reveal vesicle transfer of miR-200c-3p from cardiomyocytes to cardiac endothelial cells, underlining the importance of cardiac intercellular communication in the pathophysiology of heart failure.

Heart disease represents one of the main challenges in modern medicine with insufficient treatment options. Whole genome sequencing allowed for the discovery of several classes of non-coding RNA (ncRNA) and widened our understanding of disease regulatory circuits. The intrinsic ability of long ncRNAs (lncRNAs) and circular RNAs (circRNAs) to regulate gene expression by a plethora of mechanisms make them candidates for conceptually new treatment options. However, important questions remain to be addressed before we can fully exploit the therapeutic potential of these molecules. Increasing our knowledge of their mechanisms of action and refining the approaches for modulating lncRNAs expression are just a few of the challenges we face. The accurate identification of novel lncRNAs is hampered by their relatively poor cross-species sequence conservation and their low and context-dependent expression pattern. Nevertheless, progress has been made in their annotation in recent years, while a few experimental studies have confirmed the value of lncRNAs as new mechanisms in the development of cardiac hypertrophy and other cardiovascular diseases. Here, we explore cardiac lncRNA biology and the evidence that this class of molecules has therapeutic benefit to treat cardiac hypertrophy.

We investigated the transcriptomic landscape of the murine myocardium along the course of natural aging and in three distinct mouse models of premature aging with established aging-related cardiac dysfunction. Genome-wide total RNA-seq was performed and the expression patterns of protein-coding genes and non-coding RNAs were compared between hearts from naturally aging mice, mice with cardiac-specific deficiency of a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity and mice with reduced telomere length. Our results demonstrate that no dramatic changes are evident in the transcriptomes of naturally senescent murine hearts until two years of age, in contrast to the transcriptome of accelerated aged mice. Additionally, these mice displayed model-specific alterations of the expression levels of protein-coding and non-coding genes with hardly any overlap with age-related signatures. Our data demonstrate very limited similarities between the transcriptomes of all our murine aging models and question their reliability to study human cardiovascular senescence.

Contains fulltext : 229383.pdf (Publisher’s version ) (Open Access) Obesity leads to an amplified risk of disease and contributes to the occurrence of type 2 diabetes, fatty liver disease, coronary heart disease, stroke, chronic kidney disease and various types of cancer. MicroRNAs (miRNAs), small non-coding RNA molecules of 20-25 nucleotides, can remain stable in plasma and have been studied as potential (predictive) biomarkers for obesity and related metabolic disorders. The aim of this study was to identify circulating miRNAs as biomarkers for obesity status and metabolic alterations in women. Circulating miR-216a and miR-155-5p were selected by miRNA expression profiling and validated by real time quantitative PCR in a validation cohort of 60 obese women and 60 normal weight-age-matched control women. This was supplemented by correlation analysis of the candidate miRNA and anthropometric variables, blood biochemistry and lipid profile markers. Circulating miR-216a was validated as a biomarker of obesity status with significantly reduced levels in obese women. Interestingly, this was associated with a negative correlation between the plasma miR-216a content and body mass index (BMI), waist circumference, mean arterial pressure (MAP), triglycerides, ratio of total cholesterol/high density lipoprotein (HDL)-cholesterol and high sensitivity-C reactive protein (hs-CRP).Taken together, we provide evidence for an abnormally expressed circulating miRNA, miR-216a, with additive value as a predictive marker for obesity that correlates with metabolic alterations presented by lipid profile and inflammatory markers.

Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life. F.D.M. is supported by HS-BAFTA and Kootstra fellowships of Maastricht University and a CVON-ARENA-PRIME fellowship. L.M. is supported by the fund Innovative Medical Research of the University of Münster Medical School (RÜ121510). M.R.H. is supported by a Juan de la Cierva contract from the Spanish Ministerio de Ciencia, Innovación y Universidades (IJC2019-040798-I). R.M.N. is the beneficiary of a predoctoral contract from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/ 05027). V.A. is supported by the Spanish Ministerio de Ciencia e Innovación (PID2019-108489RB-I00) and the Instituto de Salud Carlos III (ISCIII) (AC16/ 00091) as member of the ERA-CVD JCT2016 EXPERT Network (European Union’s Horizon 2020 Framework Programme), with cofunding from the European Regional Development Fund (“Una manera de hacer Europa”). The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the Ministry for Research, Science and Innovation (MICIN), the ISCIII, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence. C.B. was supported by the Deutsche Forschungsgemeinschaft (DFG) (BA5631/ 2-1). T.T. was supported by the European Research Council (ERC) Consolidator Grant LONGHEART, by ERA-CVD JCT2016 EXPERT, and the DFG (TH903/ 22-1). D.J.D., M.S., and L.J.D.W. acknowledge support from the Dutch CardioVascular Initiative: the Netherlands Heart Foundation, Dutch Federation of University Medical Centers, ZonMW, and the Royal Netherlands Academy of Sciences (CVON-ARENA-PRIME, CVON-RACE-V, CVON-PREDICT-2). B.S. acknowledges funding by the Netherlands Heart Foundation (Dr. Dekker 2014T105 and CVON-SHE-PREDICTS-HF) and a VIDI Award 917.14.363 from the Dutch Research Council (NWO). A.S.A. was funded by Association Française contres les Myopathies (AFM 18802). F.D.M., T.T., and L.J.D.W. are supported by ERA-CVD JCT2016 EXPERT. M.S. is funded by the DFG (RTG2220, Project 281125614) and Marie Skłodowska-Curie Grant Agreement 81371. L.J.D.W. was further supported by ERC Consolidator Grant 311549 CALMIRS, a VICI Award 918-156-47 from NWO and Marie Skłodowska-Curie Grant Agreements 813716 and 765274. Sí

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b~25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b~25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b~25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b~25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b~25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart., Myocardial regeneration and proliferation of heart muscle cells is limited to a short period after birth early postnatal life, after which heart muscle cells can only grow in size and not in number. Here, the authors identified that the expression level of an endogenous microRNA cluster in heart muscle promotes the passage of the proliferative state to adult heart growth, and modulating the expression of this cluster can stimulate heart regeneration after myocardial infarction.

Aims Arrhythmogenic cardiomyopathy (AC) is one of the most common inherited cardiomyopathies, characterized by progressive fibro-fatty replacement in the myocardium. Clinically, AC manifests itself with ventricular arrhythmias, syncope, and sudden death and shows wide inter- and intra-familial variability. Among the causative genes identified so far, those encoding for the desmosomal proteins plakophilin-2 (PKP2), desmoplakin (DSP), and desmoglein-2 (DSG2) are the most commonly mutated. So far, little is known about the molecular mechanism(s) behind such a varied spectrum of phenotypes, although it has been shown that the causative mutations not only lead to structural abnormalities but also affect the miRNA profiling of cardiac tissue. Here, we aimed at studying the pathogenic effects of a nonsense mutation of the desmoglein-2 gene, both at the structural level and in terms of miRNA expression pattern.Methods and results We generated transgenic mice with cardiomyocyte-specific overexpression of a FLAG-tagged human desmoglein-2 harbouring the Q558* nonsense mutation found in an AC patient. The hearts of these mice showed signs of fibrosis, decrease in desmosomal size and number, and reduction of the Wnt/beta-catenin signalling. Genome-wide RNA-Seq performed in Tg-hQ hearts and non-transgenic hearts revealed that 24 miRNAs were dysregulated in transgenic animals. Further bioinformatic analyses for selected miRNAs suggested that miR-217-5p, miR-499-5p, and miR-708-5p might be involved in the pathogenesis of the disease.Conclusion Down-regulation of the canonical Wnt/beta-catenin signalling might be considered a common key event in the AC pathogenesis. We identified the miRNA signature in AC hearts, with miR-708-5p and miR-217-5p being the most up-regulated and miR-499-5p the most down-regulated miRNAs. All of them were predicted to be involved in the regulation of the Wnt/beta-catenin pathway and might reveal the potential pathophysiology mechanisms of AC, as well as be useful as therapeutic targets for the disease.

Randomized clinical trials initially used heart failure (HF) patients with low left ventricular ejection fraction (LVEF) to select study populations with high risk to enhance statistical power. However, this use of LVEF in clinical trials has led to oversimplification of the scientific view of a complex syndrome. Descriptive terms such as ‘HFrEF’ (HF with reduced LVEF), ‘HFpEF’ (HF with preserved LVEF), and more recently ‘HFmrEF’ (HF with mid-range LVEF), assigned on arbitrary LVEF cut-off points, have gradually arisen as separate diseases, implying distinct pathophysiologies. In this article, based on pathophysiological reasoning, we challenge the paradigm of classifying HF according to LVEF. Instead, we propose that HF is a heterogeneous syndrome in which disease progression is associated with a dynamic evolution of functional and structural changes leading to unique disease trajectories creating a spectrum of phenotypes with overlapping and distinct characteristics. Moreover, we argue that by recognizing the spectral nature of the disease a novel stratification will arise from new technologies and scientific insights that will shape the design of future trials based on deeper understanding beyond the LVEF construct alone.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease characterized by progressive myocardial fibro-fatty replacement, arrhythmias and risk of sudden death. Its diagnosis is challenging and often it is achieved after disease onset or postmortem. In this study, we sought to identify circulating microRNAs (miRNAs) differentially expressed in ARVC patients compared to healthy controls. In the pilot study, we screened the expression of 754 miRNAs from 21 ARVC patients and 20 healthy controls. After filtering the miRNAs considering a log fold-change cut-off of ±1, p-value &lt, 0.05, we selected five candidate miRNAs for a subsequent validation study in which we used TaqMan-based real-time PCR to analyse samples from 37 ARVC patients and 30 healthy controls. We found miR-185-5p significantly upregulated in ARVC patients. Receiver operating characteristic analysis indicated an area under the curve of 0.854, corroborating the link of this miRNA and ARVC pathophysiology.

Heart failure is among the most progressive diseases and a leading cause of morbidity. Despite several advances in cardiovascular therapies, pharmacological treatments are limited to relieve symptoms without curing cardiac injury. Multiple observations point to the involvement of immune cells as key drivers in the pathophysiology of heart failure. In particular, there is a growing recognition that heart failure is related to a prolonged and insufficiently repressed inflammatory response leading to molecular, cellular, and functional cardiac alterations. Over the last decades, non-coding RNAs are recognized as prominent mediators of cardiac inflammation, affecting the function of several immune cells. In the current review, we explore the contribution of the diverse immune cells in the progression of heart failure, revealing mechanistic functions for non-coding RNAs in cardiac immune cells as a new and exciting field of investigation.

In the last decade, the generation of cardiac disease models based on human-induced pluripotent stem cells (hiPSCs) has become of common use, providing new opportunities to overcome the lack of appropriate cardiac models. Although much progress has been made toward the generation of hiPSC-derived cardiomyocytes (hiPS-CMs), several lines of evidence indicate that two-dimensional (2D) cell culturing presents significant limitations, including hiPS-CMs immaturity and the absence of interaction between different cell types and the extracellular matrix. More recently, new advances in bioengineering and co-culture systems have allowed the generation of three-dimensional (3D) constructs based on hiPSC-derived cells. Within these systems, biochemical and physical stimuli influence the maturation of hiPS-CMs, which can show structural and functional properties more similar to those present in adult cardiomyocytes. In this review, we describe the latest advances in 2D- and 3D-hiPSC technology for cardiac disease mechanisms investigation, drug development, and therapeutic studies.

The prevalence of metabolic syndrome (MetS) and obesity is an alarming health issue worldwide. Obesity is characterized by an excessive accumulation of white adipose tissue (WAT), and it is associated with diminished brown adipose tissue (BAT) activity. Twist1 acts as a negative feedback regulator of BAT metabolism. Therefore, targeting Twist1 could become a strategy for obesity and metabolic disease. Here, we have identified miR-337-3p as an upstream regulator of Twist1. Increased miR-337-3p expression paralleled decreased expression of TWIST1 in BAT compared to WAT. Overexpression of miR-337-3p in brown pre-adipocytes provoked a reduction in Twist1 expression that was accompanied by increased expression of brown/mitochondrial markers. Luciferase assays confirmed an interaction between the miR-337 seed sequence and Twist1 3&prime, UTR. The inverse relationship between the expression of TWIST1 and miR-337 was finally validated in adipose tissue samples from non-MetS and MetS subjects that demonstrated a dysregulation of the miR-337-Twist1 molecular axis in MetS. The present study demonstrates that adipocyte miR-337-3p suppresses Twist1 repression and enhances the browning of adipocytes.

AimsThe pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling.Methods and resultsNeonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity.ConclusionOur data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure.

Pressure overload causes cardiac fibroblast activation and transdifferentiation, leading to increased interstitial fibrosis formation and subsequently myocardial stiffness, diastolic and systolic dysfunction, and eventually heart failure. A better understanding of the molecular mechanisms underlying pressure overload-induced cardiac remodeling and fibrosis will have implications for heart failure treatment strategies. The microRNA (miRNA)-221/222 family, consisting of miR-221-3p and miR-222-3p, is differentially regulated in mouse and human cardiac pathology and inversely associated with kidney and liver fibrosis. We investigated the role of this miRNA family during pressure overload-induced cardiac remodeling. In myocardial biopsies of patients with severe fibrosis and dilated cardiomyopathy or aortic stenosis, we found significantly lower miRNA-221/222 levels as compared to matched patients with nonsevere fibrosis. In addition, miRNA-221/222 levels in aortic stenosis patients correlated negatively with the extent of myocardial fibrosis and with left ventricular stiffness. Inhibition of both miRNAs during AngII (angiotensin II)-mediated pressure overload in mice led to increased fibrosis and aggravated left ventricular dilation and dysfunction. In rat cardiac fibroblasts, inhibition of miRNA-221/222 derepressed TGF-beta (transforming growth factor-beta)-mediated profibrotic SMAD2 (mothers against decapentaplegic homolog 2) signaling and downstream gene expression, whereas overexpression of both miRNAs blunted TGF-beta-induced profibrotic signaling. We found that the miRNA-221/222 family may target several genes involved in TGF-beta signaling, including JNK1 (c-Jun N-terminal kinase 1), TGF-beta receptor 1 and TGF-beta receptor 2, and ETS-1 (ETS proto-oncogene 1). Our findings show that heart failure-associated downregulation of the miRNA-221/222 family enables profibrotic signaling in the pressure-overloaded heart.

Activation of the immune system in heart failure (HF) has been recognized for over 20 years. Initially, experimental studies demonstrated a maladaptive role of the immune system. However, several phase III trials failed to show beneficial effects in HF with therapies directed against an immune activation. Preclinical studies today describe positive and negative effects of immune activation in HF. These different effects depend on timing and aetiology of HF. Therefore, herein we give a detailed review on immune mechanisms and their importance for the development of HF with a special focus on commonalities and differences between different forms of cardiomyopathies. The role of the immune system in ischaemic, hypertensive, diabetic, toxic, viral, genetic, peripartum, and autoimmune cardiomyopathy is discussed in depth. Overall, initial damage to the heart leads to disease specific activation of the immune system whereas in the chronic phase of HF overlapping mechanisms occur in different aetiologies.

As heart failure with preserved ejection fraction (HFpEF) rises to epidemic proportions, major steps in patient management and therapeutic development are badly needed. With the current position paper we seek to update our view on HFpEF as a highly complex systemic syndrome, from risk factors and mechanisms to long-term clinical manifestations. We will revise recent advances in animal model development, experimental set-ups and basic and translational science approaches to HFpEF research, highlighting their drawbacks and advantages. Directions are provided for proper model selection as well as for integrative functional evaluation from the in vivo setting to in vitro cell function testing. Additionally, we address new research challenges that require integration of higher-order inter-organ and inter-cell communication to achieve a full systems biology perspective of HFpEF.

Objective Strategies improving skeletal muscle mitochondrial capacity are commonly paralleled by improvements in (metabolic) health. We and others previously identified microRNAs regulating mitochondrial oxidative capacity, but data in skeletal muscle are limited. Therefore, the present study aimed to identify novel microRNAs regulating skeletal muscle mitochondrial metabolism. Methods and results We conducted an unbiased, hypothesis-free microRNA silencing screen in C2C12 myoblasts, using >700 specific microRNA inhibitors, and investigated a broad panel of mitochondrial markers. After subsequent validation in differentiated C2C12 myotubes, and exclusion of microRNAs without a human homologue or with an adverse effect on mitochondrial metabolism, 19 candidate microRNAs remained. Human clinical relevance of these microRNAs was investigated by measuring their expression in human skeletal muscle of subject groups displaying large variation in skeletal muscle mitochondrial capacity. Conclusion The results show that that microRNA-320a, microRNA-196b-3p, microRNA-150-5p, and microRNA-34c-3p are tightly related to in vivo skeletal muscle mitochondrial function in humans and identify these microRNAs as targets for improving mitochondrial metabolism., Graphical abstract Image 1, Highlights • A microRNA screen identifies microRNAs modulating muscle mitochondrial metabolism. • Identified microRNAs correlate with in vivo muscle mitochondrial function in humans. • MicroRNAs are potential targets to improve skeletal muscle mitochondrial metabolism.

// Alessandra Lorenzon 1 , Martina Calore 2 , Giulia Poloni 1 , Leon J. De Windt 2 , Paola Braghetta 3 and Alessandra Rampazzo 1 1 University of Padua, Department of Biology, Padua, Italy 2 Maastricht University, Department of Cardiology, Maastricht, The Netherlands 3 University of Padua, Department of Molecular Medicine, Padua, Italy Correspondence to: Alessandra Rampazzo, email: alessandra.rampazzo@unipd.it Keywords: Wnt, β-catenin, microRNAs, arrhythmogenic cardiomyopathy, molecular pathogenesis Received: January 30, 2017 Accepted: April 14, 2017 Published: April 27, 2017 ABSTRACT Wnt/β-catenin signaling pathway plays essential roles in heart development as well as cardiac tissue homoeostasis in adults. Abnormal regulation of this signaling pathway is linked to a variety of cardiac disease conditions, including hypertrophy, fibrosis, arrhythmias, and infarction. Recent studies on genetically modified cellular and animal models document a crucial role of Wnt/β-catenin signaling in the molecular pathogenesis of arrhythmogenic cardiomyopathy (AC), an inherited disease of intercalated discs, typically characterized by ventricular arrhythmias and progressive substitution of the myocardium with fibrofatty tissue. In this review, we summarize the conflicting published data regarding the Wnt/β-catenin signaling contribution to AC pathogenesis and we report the identification of a new potential therapeutic molecule that prevents myocyte injury and cardiac dysfunction due to desmosome mutations in vitro and in vivo by interfering in this signaling pathway. Finally, we underline the potential function of microRNAs, epigenetic regulatory RNA factors reported to participate in several pathological responses in heart tissue and in the Wnt signaling network, as important modulators of Wnt/β-catenin signaling transduction in AC. Elucidation of the precise regulatory mechanism of Wnt/β-catenin signaling in AC molecular pathogenesis could provide fundamental insights for new mechanism-based therapeutic strategy to delay the onset or progression of this cardiac disease.

While cardiomyocytes (CMs) have been the main subject of extensive research, the role of other cardiac cell types, such as fibroblasts and endothelial cells (ECs), received considerable less attention in the pathogenesis of heart failure (HF). MiRNAs have recently emerged as mediators of paracrine signaling by being selectively incorporated in exosomes and exchanged between different cell types. The aim of our study is to investigate a potential paracrine miRNA crosstalk between CMs and cardiac ECs and assess the consequences of such miRNA transfer for cardiac vascular remodeling under pathological conditions. We isolated and characterized exosomes from CMs at baseline or after pathological stimulation with phenylephrine and isoproterenol. Although baseline and stressed CMs secrete miRNA-enriched exosomes at similar rates, comparative analysis of extracellular vesicles from both conditions revealed differential miRNA levels, with miR-200c-3p being highly enriched under stress conditions. Direct transfection of ECs with miR-200c-3p precursor molecules or indirect overexpression through transwell co-culture with stimulated CMs leads to diminished angiogenesis reflected by reduced capacity of ECs to proliferate, migrate, and form tubes. This effect was abrogated when we treated CMs with GW4869, an inhibitor of exosomal biogenesis and release. Next, we tested in vivo two doses of specific miR-200c-3p antagomir, Cy3 labelled, to assess specific target of ECs. FACS analysis on cardiac cells derived from the injected mice, confirmed that a low antagomir dose targets ECs whereas, the high dose of antagomir targets all different cardiac cell types. Moreover, when we treated mice subjected transverse aortic constriction (TAC)-induced cardiac pressure overload with miR-200c-3p antagomir, the animals developed a milder hypertrophic phenotype, smaller fibrotic areas, higher amount of capillaries and preserved cardiac ejection fraction, when compared to untreated pressure overloaded mice. Altogether, our results showing exosomal transfer of miR-200c-3p from CMs to ECs indicate the importance of cardiac intercellular communication in the pathophysiology of HF and identify a potential new therapeutic target for intervention strategies.

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3′ to the 5′ end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5′ end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling., Raso et al. show that miR-148a is elevated in concentric hypertrophy and decreased in dilated cardiomyopathy. Adeno-associated viral delivery of miR-148a protects the mouse heart from cardiac dilation. Mechanistically, miR-148a regulates the sensitivity of the heart to extracellular cytokines.

Heart failure (HF) is the end result of a diverse set of causes such as genetic cardiomyopathies, coronary artery disease, and hypertension and represents the primary cause of hospitalization in Europe. This serious clinical disorder is mostly associated with pathological remodeling of the myocardium, pump failure, and sudden death. While the survival of HF patients can be prolonged with conventional pharmacological therapies, the prognosis remains poor. New therapeutic modalities are thus needed that will target the underlying causes and not only the symptoms of the disease. Under chronic cardiac stress, small noncoding RNAs, in particular microRNAs, act as critical regulators of cardiac tissue remodeling and represent a new class of therapeutic targets in patients suffering from HF. Here, we focus on the potential use of microRNA inhibitors as a new treatment paradigm for HF.

Organogenesis of the vertebrate heart is a highly specialized process involving progressive specification and differentiation of distinct embryonic cardiac progenitor cell populations driven by specialized gene programming events. Likewise, the onset of pathologies in the adult heart, including cardiac hypertrophy, involves the reactivation of embryonic gene programs. In both cases, these intricate genomic events are temporally and spatially regulated by complex signaling networks and gene regulatory networks. Apart from well-established transcriptional mechanisms, increasing evidence indicates that gene programming in both the developing and the diseased myocardium are under epigenetic control by non-coding RNAs (ncRNAs). MicroRNAs regulate gene expression at the post-transcriptional level, and numerous studies have now established critical roles for this species of tiny RNAs in a broad range of aspects from cardiogenesis towards adult heart failure. Recent reports now also implicate the larger family of long non-coding RNAs (lncRNAs) in these processes as well. Here we discuss the involvement of these two ncRNA classes in proper cardiac development and hypertrophic disease processes of the adult myocardium. This article is part of a Special Issue entitled: Non-coding RNAs.

for senior cardiovascular scientists, it is well known and accepted that the American Journal of Physiology ( AJP ) -Heart and Circulatory Physiology publishes important and long-lasting cardiovascular science ([2][1]). Early career investigators and trainees may be less familiar with the history of

The majority of the human genome encodes non-coding RNAs (ncRNAs), species of RNA without protein-coding potential but with powerful regulatory functions in disease onset and progression. Functional studies demonstrate that both coding and ncRNAs underlie various mechanisms in heart disease and that molecules targeting RNA species show promising efficacy in preclinical development. Accompanying the exciting developments in basic RNA biology, an equally provocative field has flourished for the design of RNA-based strategies to generate innovative types of therapeutics against these new "druggable" targets, going beyond our current repertoire of small chemistry or biologics. Here, we review the (bio)chemical basis of RNA-based drug design, provide examples that show promise as translatable drug products in preclinical studies, give an insight in the current barriers that hamper straight-forward clinical translation and discuss future directions that may overcome these hurdles to expand the current pharmacotherapy for myocardial disorders.