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The delivery of therapeutic long non-coding RNAs (lncRNA) to the heart by extracellular vesicles (EVs) is promising for heart repair. H19, a lncRNA acting as a major regulator of gene expression within the cardiovascular system, is alternatively spliced, but the loading of its different splice variants into EVs and their subsequent uptake by recipient cardiac cells remain elusive. Here, we dissected the cellular expression of H19 splice variants and their loading into EVs secreted by Wharton-Jelly mesenchymal stromal/stem cells (WJ-MSCs). We demonstrated that overexpression of the mouse H19 gene in WJ-MSCs induces the expression of H19 splice variants at different levels. Interestingly, EVs isolated from the H19-transfected WJ-MSCs (EV-H19) showed similar expression levels for all tested splice variant sets. In vitro, we further demonstrated that EV-H19 was taken up by cardiomyocytes, fibroblasts, and endothelial cells (ECs). Finally, analysis of EV tropism in living rat myocardial slices indicated that EVs were internalized mostly by cardiomyocytes and ECs. Collectively, our results indicated that EVs can be loaded with different lncRNA splice variants and successfully internalized by cardiac cells.
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Pathologic cardiac hypertrophy is a common consequence of many cardiovascular diseases, including aortic stenosis (AS). AS is known to increase the pressure load of the left ventricle, causing a compensative response of the cardiac muscle, which progressively will lead to dilation and heart failure. At a cellular level, this corresponds to a considerable increase in the size of cardiomyocytes, known as cardiomyocyte hypertrophy, while their proliferation capacity is attenuated upon the first developmental stages. Cardiomyocytes, in order to cope with the increased workload (overload), suffer alterations in their morphology, nuclear content, energy metabolism, intracellular homeostatic mechanisms, contractile activity, and cell death mechanisms. Moreover, modifications in the cardiomyocyte niche, involving inflammation, immune infiltration, fibrosis, and angiogenesis, contribute to the subsequent events of a pathologic hypertrophic response. Considering the emerging need for a better understanding of the condition and treatment improvement, as the only available treatment option of AS consists of surgical interventions at a late stage of the disease, when the cardiac muscle state is irreversible, large animal models have been developed to mimic the human condition, to the greatest extend. Smaller animal models lack physiological, cellular and molecular mechanisms that sufficiently resemblance humans and in vitro techniques yet fail to provide adequate complexity. Animals, such as the ferret (Mustello purtorius furo), lapine (rabbit, Oryctolagus cunigulus), feline (cat, Felis catus), canine (dog, Canis lupus familiaris), ovine (sheep, Ovis aries), and porcine (pig, Sus scrofa), have contributed to research by elucidating implicated cellular and molecular mechanisms of the condition. Essential discoveries of each model are reported and discussed briefly in this review. Results of large animal experimentation could further be interpreted aiming at prevention of the disease progress or, alternatively, at regression of the implicated pathologic mechanisms to a physiologic state. This review summarizes the important aspects of the pathophysiology of LV hypertrophy and the applied surgical large animal models that currently better mimic the condition.
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Estenosis de la Válvula Aórtica , Hipertrofia Ventricular Izquierda , Remodelación Ventricular , Animales , Humanos , Estenosis de la Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/metabolismo , Modelos Animales de Enfermedad , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Especificidad de la Especie , Función Ventricular Izquierda , Presión VentricularRESUMEN
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.
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Insuficiencia Cardíaca , MicroARNs , Animales , Ratones , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/metabolismo , MicroARNs/metabolismo , Miocardio/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/genéticaRESUMEN
Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: The human placenta is a unique organ sustaining fetal growth but is also the source of severe pathologies, such as preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.
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Placenta , Preeclampsia , Embarazo , Femenino , Humanos , Células Endoteliales , Hipoxia , Isquemia , Dispositivos Laboratorio en un Chip , HormonasRESUMEN
Traditionally, to quantify permeability of a biological barrier, the initial slope is used, based on the assumption of sink condition (concentration of the donor is constant, and the receiver increases less than 10%). With on-a-chip barrier models, this assumption fails in cell-free or leaky conditions, which requires the use of the exact solution. To encounter a time delay from performing the assay and acquiring the data, we present a protocol with the exact equation modified to incorporate a time offset.
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Bioensayo , Dispositivos Laboratorio en un Chip , PermeabilidadRESUMEN
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.
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Proteínas de Unión al ADN , Insuficiencia Cardíaca , Ratones , Animales , Humanos , Proteínas de Unión al ADN/metabolismo , Miocitos Cardíacos/metabolismo , Reparación del ADN/genética , Daño del ADN/genética , Insuficiencia Cardíaca/genética , EndonucleasasRESUMEN
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.
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Enfermedades Cardiovasculares , MicroARNs , ARN Largo no Codificante , Humanos , ARN no Traducido/genética , ARN Largo no Codificante/genética , MicroARNs/genética , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/terapiaRESUMEN
Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism.
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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.
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Vesículas Extracelulares , MicroARNs , Animales , Comunicación Celular , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
AIMS: Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV). Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a 'foetal gene programme' contributing to pathological cardiac remodelling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodelling in response to pressure overload induced by pulmonary artery banding (PAB). METHODS AND RESULTS: In this study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived haemodynamic parameters as well as molecular remodelling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control-banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV pressure-overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass, and impaired cardiac function. RNA-sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV. CONCLUSION: Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressu re overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodelling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.
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Insuficiencia Cardíaca , Disfunción Ventricular Derecha , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Humanos , Ratones , ARN/metabolismo , Tamoxifeno/metabolismo , Factores de Transcripción/metabolismo , Disfunción Ventricular Derecha/genética , Disfunción Ventricular Derecha/metabolismo , Función Ventricular Derecha , Presión Ventricular , Remodelación VentricularRESUMEN
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.
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Insuficiencia Cardíaca , MicroARNs , ARN Largo no Codificante , Arritmias Cardíacas , Corazón , Insuficiencia Cardíaca/genética , Humanos , Inflamación/genética , MicroARNs/genéticaRESUMEN
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.
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The heart has been the center of numerous transcriptomic studies in the past decade. Even though our knowledge of the key organ in our cardiovascular system has significantly increased over the last years, it is still not fully understood yet. In recent years, extensive efforts were made to understand the genetic and transcriptomic contribution to cardiac function and failure in more detail. The advent of Next Generation Sequencing (NGS) technologies has brought many discoveries but it is unable to comprehend the finely orchestrated interactions between and within the various cell types of the heart. With the emergence of single-cell sequencing more than 10 years ago, researchers gained a valuable new tool to enable the exploration of new subpopulations of cells, cell-cell interactions, and integration of multi-omic approaches at a single-cell resolution. Despite this innovation, it is essential to make an informed choice regarding the appropriate technique for transcriptomic studies, especially when working with myocardial tissue. Here, we provide a primer for researchers interested in transcriptomics using NGS technologies.
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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 < 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.
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Displasia Ventricular Derecha Arritmogénica/genética , Biomarcadores/metabolismo , Cardiomiopatías/genética , MicroARNs/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Proyectos PilotoRESUMEN
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.
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Envejecimiento Prematuro/genética , Senescencia Celular/genética , Inestabilidad Genómica/genética , Envejecimiento/genética , Animales , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Endonucleasas/genética , Endonucleasas/metabolismo , Femenino , Corazón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Miocardio/metabolismoRESUMEN
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.
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ARN Largo no Codificante , Transcriptoma , Genoma , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ARN , SinteníaRESUMEN
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.