RESUMO
AIM: Inhibition of microRNA (miR)-132 effectively prevents and reverses adverse cardiac remodelling, making it an attractive heart failure (HF) target. CDR132L, a synthetic antisense oligonucleotide selectively blocking pathologically elevated miR-132, demonstrated beneficial effects on left ventricular (LV) structure and function in relevant preclinical models, and was safe and well tolerated in a Phase 1b study in stable chronic HF patients. Patients with acute myocardial infarction (MI) and subsequent LV dysfunction and remodelling have limited therapeutic options, and may profit from early CDR132L treatment. METHODS: The HF-REVERT (Phase 2, multicenter, randomized, parallel, 3-arm, placebo-controlled Study to Assess Efficacy and Safety of CDR132L in Patients with Reduced Left Ventricular Ejection Fraction after Myocardial Infarction) evaluates the efficacy and safety of CDR132L in HF patients post-acute MI (n = 280), comparing the effect of 5 and 10 mg/kg CDR132L, administered as three single intravenous doses 28 days apart, in addition to standard of care. Key inclusion criteria are the diagnosis of acute MI, the development of systolic dysfunction (LV ejection fraction ≤45%) and elevated N-terminal pro-B-type natriuretic peptide. The study consists of a 6-month double-blinded treatment period with the primary endpoint LV end-systolic volume index and relevant secondary endpoints, followed by a 6-month open-label observation period. CONCLUSION: The HF-REVERT trial may underpin the concept of miR-132 inhibition to prevent or reverse cardiac remodelling in post-MI HF. The results will inform the design of subsequent outcome trials to test CDR132L in HF.
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Infarto do Miocárdio , Volume Sistólico , Humanos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/complicações , Volume Sistólico/fisiologia , Masculino , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Esquerda/etiologia , Resultado do Tratamento , MicroRNAs , Remodelação Ventricular/efeitos dos fármacos , Pessoa de Meia-Idade , Idoso , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/administração & dosagem , Método Duplo-Cego , Função Ventricular Esquerda/fisiologia , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.
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Oligonucleotídeos , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos/farmacocinéticaRESUMO
We have designed translational animal models to investigate cardiac profibrotic gene signatures. Domestic pigs were treated with cardiotoxic drugs (doxorubicin, DOX, n = 5 or Myocet®, MYO, n = 5) to induce replacement fibrosis via cardiotoxicity. Reactive interstitial fibrosis was triggered by LV pressure overload by artificial isthmus stenosis with stepwise developing myocardial hypertrophy and final fibrosis (Hyper, n = 3) or by LV volume overload in the adverse remodeled LV after myocardial infarction (RemoLV, n = 3). Sham interventions served as controls and healthy animals (Control, n = 3) served as a reference in sequencing study. Myocardial samples from the LV of each group were subjected to RNA sequencing. RNA-seq analysis revealed a clear distinction between the transcriptomes of myocardial fibrosis (MF) models. Cardiotoxic drugs activated the TNF-alpha and adrenergic signaling pathways. Pressure or volume overload led to the activation of FoxO pathway. Significant upregulation of pathway components enabled the identification of potential drug candidates used for the treatment of heart failure, such as ACE inhibitors, ARB, ß-blockers, statins and diuretics specific to the distinct MF models. We identified candidate drugs in the groups of channel blockers, thiostrepton that targets the FOXM1-regulated ACE conversion to ACE2, tyrosine kinases or peroxisome proliferator-activated receptor inhibitors. Our study identified different gene targets involved in the development of distinct preclinical MF protocols enabling tailoring expression signature-based approach for the treatment of MF.
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Cardiomiopatias , Insuficiência Cardíaca , Animais , Transcriptoma , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Cardiomiopatias/metabolismo , Insuficiência Cardíaca/patologia , Cardiotoxicidade/patologia , Doxorrubicina/farmacologia , Fenótipo , Fibrose , Sistemas de Liberação de Medicamentos , Miocárdio/metabolismo , Modelos Animais de DoençasRESUMO
Diabetes mellitus promotes accelerated cardiovascular aging and inflammation, which in turn facilitate the development of cardiomyopathy/heart failure. High glucose-induced oxidative/nitrative stress, activation of various pro-inflammatory, and cell death pathways are critical in the initiation and progression of the changes culminating in diabetic cardiomyopathy. Cannabinoid 2 receptor (CB2R) activation in inflammatory cells and activated endothelium attenuates the pathological changes associated with atherosclerosis, myocardial infarction, stroke, and hepatic cardiomyopathy. In this study, we explored the role of CB2R signaling in myocardial dysfunction, oxidative/nitrative stress, inflammation, cell death, remodeling, and fibrosis associated with diabetic cardiomyopathy in type 1 diabetic mice. Control human heart left ventricles and atrial appendages, similarly to mouse hearts, had negligible CB2R expression determine by RNA sequencing or real-time RT-PCR. Diabetic cardiomyopathy was characterized by impaired diastolic and systolic cardiac function, enhanced myocardial CB2R expression, oxidative/nitrative stress, and pro-inflammatory response (tumor necrosis factor-α, interleukin-1ß, intracellular adhesion molecule 1, macrophage inflammatory protein-1, monocyte chemoattractant protein-1), macrophage infiltration, fibrosis, and cell death. Pharmacological activation of CB2R with a selective agonist attenuated diabetes-induced inflammation, oxidative/nitrative stress, fibrosis and cell demise, and consequent cardiac dysfunction without affecting hyperglycemia. In contrast, genetic deletion of CB2R aggravated myocardial pathology. Thus, selective activation of CB2R ameliorates diabetes-induced myocardial tissue injury and preserves the functional contractile capacity of the myocardium in the diabetic milieu. This is particularly encouraging, since unlike CB1R agonists, CB2R agonists do not elicit psychoactive activity and cardiovascular side effects and are potential clinical candidates in the treatment of diabetic cardiovascular and other complications.
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Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Fibrose , Inflamação/patologia , Camundongos , Estresse Oxidativo , Receptores de Canabinoides/metabolismo , Receptores de Canabinoides/uso terapêuticoRESUMO
Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies, all of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task, in particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and comorbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models do not represent a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on an organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and state-of-the-art animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction, and refinement (3R) as a guiding concept.
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Doenças Cardiovasculares , Humanos , Animais , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/terapia , Projetos de Pesquisa , Modelos AnimaisRESUMO
Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy.
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Caquexia/genética , Fibrose Endomiocárdica/genética , Insuficiência Cardíaca/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Fatores de Transcrição/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Caquexia/metabolismo , Caquexia/fisiopatologia , Caquexia/prevenção & controle , Estudos de Casos e Controles , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/fisiopatologia , Fibrose Endomiocárdica/prevenção & controle , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Testes de Função Cardíaca , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/agonistas , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/deficiência , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Atrofia Muscular/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/deficiênciaRESUMO
BACKGROUND: Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. OBJECTIVES: This study aimed to establish a novel porcine model of pressure-overload-induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. METHODS: This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. RESULTS: At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 µm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132-treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). CONCLUSIONS: The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.
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Antagomirs/administração & dosagem , Doenças da Aorta/complicações , Cardiomegalia/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , Remodelação Ventricular/efeitos dos fármacos , Animais , Aorta Torácica/cirurgia , Cardiomegalia/complicações , Cardiomegalia/diagnóstico , Constrição , Constrição Patológica/complicações , Vasos Coronários , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Injeções Intra-Arteriais , MicroRNAs/genética , MicroRNAs/metabolismo , Stents/efeitos adversos , Suínos , Resultado do TratamentoRESUMO
Doxorubicin is one of the most potent chemotherapeutic agents. However, its clinical use is restricted due to the severe risk of cardiotoxicity, partially attributed to elevated production of reactive oxygen species (ROS). Telomerase canonically maintains telomeres during cell division but is silenced in adult hearts. In non-dividing cells such as cardiomyocytes, telomerase confers pro-survival traits, likely owing to the detoxification of ROS. Therefore, we hypothesized that pharmacological overexpression of telomerase may be used as a therapeutic strategy for the prevention of doxorubicin-induced cardiotoxicity. We used adeno-associated virus (AAV)-mediated gene therapy for long-term expression of telomerase in in vitro and in vivo models of doxorubicin-induced cardiotoxicity. Overexpression of telomerase protected the heart from doxorubicin-mediated apoptosis and rescued cardiac function, which was accompanied by preserved cardiomyocyte size. At the mechanistic level, we observed altered mitochondrial morphology and dynamics in response to telomerase expression. Complementary in vitro experiments confirmed the anti-apoptotic effects of telomerase overexpression in human induced pluripotent stem cell-derived cardiomyocytes after doxorubicin treatment. Strikingly, elevated levels of telomerase translocated to the mitochondria upon doxorubicin treatment, which helped to maintain mitochondrial function. Thus, telomerase gene therapy could be a novel preventive strategy for cardiotoxicity by chemotherapy agents such as the anthracyclines.
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Cardiotoxicidade/genética , Doxorrubicina/efeitos adversos , Neoplasias/tratamento farmacológico , Telomerase/genética , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade/prevenção & controle , Cardiotoxicidade/terapia , Dependovirus/genética , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias/complicações , Neoplasias/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Telomerase/farmacologiaRESUMO
AIMS: Cardiac microRNA-132-3p (miR-132) levels are increased in patients with heart failure (HF) and mechanistically drive cardiac remodelling processes. CDR132L, a specific antisense oligonucleotide, is a first-in-class miR-132 inhibitor that attenuates and even reverses HF in preclinical models. The aim of the current clinical Phase 1b study was to assess safety, pharmacokinetics, target engagement, and exploratory pharmacodynamic effects of CDR132L in patients on standard-of-care therapy for chronic ischaemic HF in a randomized, placebo-controlled, double-blind, dose-escalation study (NCT04045405). METHODS AND RESULTS: Patients had left ventricular ejection fraction between ≥30% and <50% or amino terminal fragment of pro-brain natriuretic peptide (NT-proBNP) >125 ng/L at screening. Twenty-eight patients were randomized to receive CDR132L (0.32, 1, 3, and 10 mg/kg body weight) or placebo (0.9% saline) in two intravenous infusions, 4 weeks apart in four cohorts of seven (five verum and two placebo) patients each. CDR132L was safe and well tolerated, without apparent dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level at ≥1 mg/kg CDR132L. CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients given CDR132L ≥1 mg/kg displayed a median 23.3% NT-proBNP reduction, vs. a 0.9% median increase in the control group. CDR132L treatment induced significant QRS narrowing and encouraging positive trends for relevant cardiac fibrosis biomarkers. CONCLUSION: This study is the first clinical trial of an antisense drug in HF patients. CDR132L was safe and well tolerated, confirmed linear plasma pharmacokinetics with no signs of accumulation, and suggests cardiac functional improvements. Although this study is limited by the small patient numbers, the indicative efficacy of this drug is very encouraging justifying additional clinical studies to confirm the beneficial CDR132L pharmacodynamic effects for the treatment of HF.
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Insuficiência Cardíaca , MicroRNAs , Método Duplo-Cego , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Volume Sistólico , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
AIMS: Cardiac miR-132 activation leads to adverse remodelling and pathological hypertrophy. CDR132L is a synthetic lead-optimized oligonucleotide inhibitor with proven preclinical efficacy and safety in heart failure (HF) early after myocardial infarction (MI), and recently completed clinical evaluation in a Phase 1b study (NCT04045405). The aim of the current study was to assess safety and efficacy of CDR132L in a clinically relevant large animal (pig) model of chronic heart failure following MI. METHODS AND RESULTS: In a chronic model of post-MI HF, slow-growing pigs underwent 90 min left anterior descending artery occlusion followed by reperfusion. Animals were randomized and treatment started 1-month post-MI. Monthly intravenous (IV) treatments of CDR132L over 3 or 5 months (3× or 5×) were applied in a blinded randomized placebo-controlled fashion. Efficacy was evaluated based on serial magnetic resonance imaging, haemodynamic, and biomarker analyses. The treatment regime provided sufficient tissue exposure and CDR132L was well tolerated. Overall, CDR132L treatment significantly improved cardiac function and reversed cardiac remodelling. In addition to the systolic recovery, diastolic function was also ameliorated in this chronic model of HF. CONCLUSION: Monthly repeated dosing of CDR132L is safe and adequate to provide clinically relevant exposure and therapeutic efficacy in a model of chronic post-MI HF. CDR132L thus should be explored as treatment for the broad area of chronic heart failure.
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Insuficiência Cardíaca , Infarto do Miocárdio , Animais , Diástole , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Suínos , Remodelação VentricularRESUMO
Anti-fibrotic therapies are of increasing interest to combat cardiac remodeling and heart failure progression. Recently, anti-fibrotic circular RNAs (circRNAs) have been identified in human and rodent cardiac tissue. In vivo (rodent) experiments proved cardiac anti-fibrotic effects of the natural compounds bufalin and lycorine by downregulating miRNA-671-5p, associated with a theoretic increase in the tissue level of circRNA CDR1as. Accordingly, we hypothesized that both anti-fibrotic drugs may inhibit focal myocardial fibrosis of the remodeled left ventricle (LV) also in a translational large animal model of heart failure (HF). Domestic pigs were repeatedly treated with subcutaneous injections of either bufalin, lycorine, or saline, (n = 5/group) between days 7-21 post acute myocardial infarction (AMI). At the 2-month follow-up, both bufalin and lycorine led to significantly reduced cardiac fibrosis. Bufalin treatment additionally led to smaller end-diastolic volumes, higher LV ejection fraction (EF), and increased expression of CDR1as of the AMI region. Elevated tissue levels of the circRNA CDR1as in the AMI region of the pig heart correlated significantly with LV and right ventricular EF, LV stroke volume, and negatively with infarct size. In conclusion, we successfully identified the circRNA CDR1as in pig hearts and show a significant association with improved LV and RV function by anti-fibrotic therapies in a translational animal model of HF.
Assuntos
Alcaloides de Amaryllidaceae/administração & dosagem , Bufanolídeos/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Fenantridinas/administração & dosagem , RNA Circular/genética , RNA Longo não Codificante/economia , Remodelação Ventricular/efeitos dos fármacos , Alcaloides de Amaryllidaceae/farmacologia , Animais , Bufanolídeos/farmacologia , Modelos Animais de Doenças , Humanos , Injeções Subcutâneas , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Fenantridinas/farmacologia , Volume Sistólico/efeitos dos fármacos , Suínos , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacosRESUMO
AIMS: Pathological cardiac remodelling and subsequent heart failure represents an unmet clinical need. Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes, including that of heart diseases. Here, we report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. METHOD AND RESULTS: Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase but strong sustained repression upon reaching the decompensated phase of heart failure. The translational potential of H19 is highlighted by its repression in a large animal (pig) model of left ventricular hypertrophy, in diseased human heart samples, in human stem cell-derived cardiomyocytes and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses and Chromatin ImmunoPrecipitation DNA-Sequencing, we identified a link between H19 and pro-hypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the anti-hypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity. CONCLUSION: H19 is highly conserved and down-regulated in failing hearts from mice, pigs and humans. H19 gene therapy prevents and reverses experimental pressure-overload-induced heart failure. H19 acts as an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling.
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Cardiopatias , Insuficiência Cardíaca , RNA Longo não Codificante , Animais , Cardiomegalia/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Hipertrofia Ventricular Esquerda , Camundongos , Camundongos Knockout , Miócitos Cardíacos , RNA Longo não Codificante/genética , SuínosRESUMO
Cardiac diseases are the most frequent causes of death in industrialized countries. Pathological remodeling of the heart muscle is caused by several etiologies such as prolonged hypertension or injuries that can lead to myocardial infarction and in serious cases also the death of the patient. The micro-RNA miR-132 has been identified as a master-switch in the development of cardiac hypertrophy and adverse remodeling. In this study, MALDI-TOF mass spectrometry (MS) was utilized to establish a robust and fast method to sensitively detect and accurately quantify anti-microRNA (antimiR) oligonucleotides in blood plasma. An antimiR oligonucleotide isolation protocol containing an ethanol precipitation step with glycogen as oligonucleotide carrier as well as a robust and reproducible MS-analysis procedure has been established. Proteinase K treatment was crucial for releasing antimiR oligonucleotides from plasma- as well as cellular proteins and reducing background derived from biological matrices. AntimiR oligonucleotide detection was achieved from samples of studies in different animal models such as mouse and pig where locked nucleic acids-(LNA)-modified antimiR oligonucleotides have been used to generate pharmacokinetic data.
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Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.
Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , MicroRNAs/genética , Oligonucleotídeos Antissenso/genética , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Humanos , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , SuínosRESUMO
BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Bufanolídeos/farmacologia , Cardiomiopatias/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Fibroblastos/efeitos dos fármacos , Fenantridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diástole , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos Dahl , Selenoproteína P/genética , Selenoproteína P/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Besides the acquisition of pharmacokinetic parameters of antisense oligonucleotide microRNA (miRNA) inhibitors, such as measuring in vivo concentration, their pharmacodynamic characteristics are also of interest. An emerging and straightforward method for studying molecular interactions is microscale thermophoresis (MST). This technique makes it possible to study interactions between miRNAs and various oligonucleotide inhibitors, independent of the chemical modifications of the inhibitors or their respective target structure, with very little sample volume required compared to competitive techniques, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Interaction studies between these inhibitors and their respective target structures were performed, and they allowed the assessment of binding characteristics and parameters, such as EC50 for a number of these inhibitors, with little effort. Furthermore, MST could be utilized for obtaining kinetic binding data of the Argonaute-2 protein with a miRNA, which showed a possible RNA-induced silencing complex (RISC)-mediated turnover of inhibited miRNAs.
RESUMO
Background: A deleterious, late-onset side effect of thoracic radiotherapy is the development of radiation-induced heart disease (RIHD). It covers a spectrum of cardiac pathology including also heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MicroRNA-212 (miR-212) is a crucial regulator of pathologic LVH via FOXO3-mediated pathways in pressure-overload-induced heart failure. We aimed to investigate whether miR-212 and its selected hypertrophy-associated targets play a role in the development of RIHD. Methods: RIHD was induced by selective heart irradiation (50 Gy) in a clinically relevant rat model. One, three, and nineteen weeks after selective heart irradiation, transthoracic echocardiography was performed to monitor cardiac morphology and function. Cardiomyocyte hypertrophy and fibrosis were assessed by histology at week 19. qRT-PCR was performed to measure the gene expression changes of miR-212 and forkhead box O3 (FOXO3) in all follow-up time points. The cardiac transcript level of other selected hypertrophy-associated targets of miR-212 including extracellular signal-regulated kinase 2 (ERK2), myocyte enhancer factor 2a (MEF2a), AMP-activated protein kinase, (AMPK), heat shock protein 40 (HSP40), sirtuin 1, (SIRT1), calcineurin A-alpha and phosphatase and tensin homolog (PTEN) were also measured at week 19. Cardiac expression of FOXO3 and phospho-FOXO3 were investigated at the protein level by Western blot at week 19. Results: In RIHD, diastolic dysfunction was present at every time point. Septal hypertrophy developed at week 3 and a marked LVH with interstitial fibrosis developed at week 19 in the irradiated hearts. In RIHD, cardiac miR-212 was overexpressed at week 3 and 19, and FOXO3 was repressed at the mRNA level only at week 19. In contrast, the total FOXO3 protein level failed to decrease in response to heart irradiation at week 19. Other selected hypertrophy-associated target genes failed to change at the mRNA level in RIHD at week 19. Conclusions: LVH in RIHD was associated with cardiac overexpression of miR-212. However, miR-212 seems to play a role in the development of LVH via FOXO3-independent mechanisms in RIHD. As a central regulator of pathologic remodeling, miR-212 might become a novel target for RIHD-induced LVH and heart failure.
RESUMO
Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end-stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti-hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co-factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy.
Assuntos
Acetiltransferases/metabolismo , Cardiomegalia/fisiopatologia , Elongação Traducional da Cadeia Peptídica , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Miócitos Cardíacos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Chronic kidney disease (CKD) is a public health problem that increases the risk of cardiovascular morbidity and mortality. Heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction is a common cardiovascular complication of CKD. MicroRNA-212 (miR-212) has been demonstrated previously to be a crucial regulator of pathologic LVH in pressure-overload-induced heart failure via regulating the forkhead box O3 (FOXO3)/calcineurin/nuclear factor of activated T-cells (NFAT) pathway. Here we aimed to investigate whether miR-212 and its hypertrophy-associated targets including FOXO3, extracellular signal-regulated kinase 2 (ERK2), and AMP-activated protein kinase (AMPK) play a role in the development of HFpEF in CKD. CKD was induced by 5/6 nephrectomy in male Wistar rats. Echocardiography and histology revealed LVH, fibrosis, preserved systolic function, and diastolic dysfunction in the CKD group as compared to sham-operated animals eight and/or nine weeks later. Left ventricular miR-212 was significantly overexpressed in CKD. However, expressions of FOXO3, AMPK, and ERK2 failed to change significantly at the mRNA or protein level. The protein kinase B (AKT)/FOXO3 and AKT/mammalian target of rapamycin (mTOR) pathways are also proposed regulators of LVH induced by pressure-overload. Interestingly, phospho-AKT/total-AKT ratio was increased in CKD without significantly affecting phosphorylation of FOXO3 or mTOR. In summary, cardiac overexpression of miR-212 in CKD failed to affect its previously implicated hypertrophy-associated downstream targets. Thus, the molecular mechanism of the development of LVH in CKD seems to be independent of the FOXO3, ERK1/2, AMPK, and AKT/mTOR-mediated pathways indicating unique features in this form of LVH.