RESUMEN
BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Terapia Genética/métodos , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/terapia , Complejo Correpresor Histona Desacetilasa y Sin3/biosíntesis , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Metilación , Ratones , Hipertensión Arterial Pulmonar/genética , Ratas , Ratas Sprague-Dawley , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismoRESUMEN
BACKGROUND: The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown. METHODS: We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice. RESULTS: Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and ß-catenin. CONCLUSIONS: We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform.
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Proteínas Portadoras/metabolismo , Ciclo Celular/fisiología , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Regeneración/fisiología , Hormonas Tiroideas/metabolismo , Animales , Humanos , Ratones , Transfección , Proteínas de Unión a Hormona TiroideRESUMEN
Inhibition of pulmonary fibrosis (PF) by restoring sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a) expression using targeted gene therapy may be a potentially powerful new treatment approach for PF. Here, we found that SERCA2a expression was significantly decreased in lung samples from patients with PF and in the bleomycin (BLM) mouse model of PF. In the BLM-induced PF model, intratracheal aerosolized adeno-associated virus serotype 1 (AAV1) encoding for human SERCA2a (AAV1.hSERCA2a) reduces lung fibrosis and associated vascular remodeling. SERCA2a gene therapy also decreases right ventricular pressure and hypertrophy in both prevention and curative protocols. In vitro, we observed that SERCA2a overexpression inhibits fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition induced by transforming growth factor ß (TGF-ß1). Thus, pro-fibrotic gene expression is prevented by blocking nuclear factor κB (NF-κB)/interleukin-6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3) activation. This effect is signaled toward an inhibitory mechanism of small mother against decapentaplegic (SMAD)/TGF-ß signaling through the repression of OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) and Forkhead box M1 (FOXM1). Interestingly, this cross-inhibition leads to an increase of SKI and SnoN expression, an auto-inhibitory feedback loop of TGF-ß signaling. Collectively, our results demonstrate that SERCA2a gene transfer attenuates bleomycin (BLM)-induced PF by blocking the STAT3/FOXM1 pathway and promoting the SNON/SKI Axis. Thus, SERCA2a gene therapy may be a potential therapeutic target for PF.
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Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transducción de Señal , Animales , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Proteína Forkhead Box M1/metabolismo , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Fibrosis Pulmonar/terapia , Factor de Transcripción STAT3/metabolismoRESUMEN
Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by the progressive obstruction of the distal pulmonary arteries (PA). Structural and functional alteration of pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) contributes to PA wall remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and, ultimately, death. Here, we found that decreased expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) in the lung samples of PAH patients was associated with the down-regulation of bone morphogenetic protein receptor type 2 (BMPR2) and the activation of signal transducer and activator of transcription 3 (STAT3). Our results showed that the antiproliferative properties of SERCA2a are mediated through the STAT3/BMPR2 pathway. At the molecular level, transcriptome analysis of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 amongst the most highly regulated transcription factors. Using a specific siRNA and a potent pharmacological STAT3 inhibitor (STAT3i, HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity in PASMCs and PAECs. In vivo, we used a validated and efficient model of severe PAH induced by unilateral left pneumonectomy combined with monocrotaline (PNT/MCT) to further evaluate the therapeutic potential of single and combination therapies using adeno-associated virus (AAV) technology and a STAT3i. We found that intratracheal delivery of AAV1 encoding SERCA2 or BMPR2 alone or STAT3i was sufficient to reduce the mean PA pressure and vascular remodeling while improving RV systolic pressures, RV ejection fraction, and cardiac remodeling. Interestingly, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or STAT3i enhanced the beneficial effects of SERCA2a. Finally, we used cardiac magnetic resonance imaging to measure RV function and found that therapies using AAV1.hSERCA2a alone or combined with STAT3i significantly inhibited RV structural and functional changes in PNT/MCT-induced PAH. In conclusion, our study demonstrated that combination therapies using SERCA2a gene transfer with a STAT3 inhibitor could represent a new promising therapeutic alternative to inhibit PAH and to restore BMPR2 expression by limiting STAT3 activity.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Pulmón/efectos de los fármacos , Hipertensión Arterial Pulmonar/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Terapia Genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Remodelación Vascular/efectos de los fármacosRESUMEN
BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
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Adenosina/análogos & derivados , Insuficiencia Cardíaca/enzimología , Infarto del Miocardio/enzimología , Miocitos Cardíacos/enzimología , Regeneración , Función Ventricular Izquierda , Remodelación Ventricular , Adenosina/metabolismo , Adulto , Anciano , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Señalización del Calcio , Estudios de Casos y Controles , Línea Celular , Proliferación Celular , Desmetilación , Modelos Animales de Enfermedad , Femenino , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Sus scrofaRESUMEN
AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.
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Proteínas de Unión al Calcio/genética , Fibroblastos/metabolismo , Fibrosis/genética , Miocardio/patología , Miofibroblastos/metabolismo , Animales , Diferenciación Celular , Regulación hacia Abajo , Humanos , Masculino , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Proteína smad3/genética , Porcinos , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. OBJECTIVE: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. METHODS: Male rats received subcutaneous injection of MCT (60â¯mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCTâ¯+â¯S2a, Nâ¯=â¯8) or saline (MCT, Nâ¯=â¯9). Age-matched rats served as controls (CTRL, Nâ¯=â¯7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (Pâ¯<â¯.01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (Pâ¯<â¯.05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH. CONCLUSION: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.
Asunto(s)
Aerosoles/administración & dosificación , Arritmias Cardíacas/terapia , Técnicas de Transferencia de Gen , Hipertensión Arterial Pulmonar/terapia , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/uso terapéutico , Tráquea/metabolismo , Potenciales de Acción , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/fisiopatología , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Terapia Genética , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Masculino , Canales de Potasio/genética , Canales de Potasio/metabolismo , Hipertensión Arterial Pulmonar/complicaciones , Hipertensión Arterial Pulmonar/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-DawleyRESUMEN
CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.
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Diferenciación Celular/genética , Interleucina-8/genética , Músculo Esquelético/metabolismo , Ribonucleasas/genética , Adulto , Animales , Western Blotting , Línea Celular , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Interleucina-8/metabolismo , Microscopía Fluorescente , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Mioblastos/citología , Mioblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Systemic inhibition of miR-21 has proven effective against myocardial fibrosis and dysfunction, while studies in cardiac myocytes suggested a protective role in this cell type. Considering potential implications for therapy, we aimed to determine the cell fraction where miR-21 exerts its pathological activity. We developed a viral vector-based strategy for gene targeting of nonmyocyte cardiac cells in vivo and compared global to cardiac myocyte-specific and nonmyocyte-specific deletion of miR-21 in chronic left ventricular pressure overload. Murine moloney virus and serotype 9 of adeno-associated virus were engineered to encode improved Cre recombinase for genetic deletion in miR-21fl/fl mice. Pericardial injection of murine moloney virus-improved Cre recombinase to neonates achieved highly selective genetic ablation of miR-21 in nonmyocyte cardiac cells, identified as cardiac fibroblasts and endothelial cells. Upon left ventricular pressure overload, cardiac function was only preserved in mice with miR-21 deficiency in nonmyocyte cardiac cells, but not in mice with global or cardiac myocyte-specific ablation. Our data demonstrate that miR-21 exerts its pathologic activity directly in cardiac nonmyocytes and encourage further development of antimiR-21 therapy toward cellular tropism.
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Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Cardiopatías/terapia , Ventrículos Cardíacos/fisiopatología , MicroARNs/genética , Remodelación Ventricular , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Técnicas de Inactivación de Genes , Células HEK293 , Cardiopatías/genética , Insuficiencia Cardíaca , Humanos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Miocitos Cardíacos/metabolismoAsunto(s)
Técnicas de Transferencia de Gen , MicroARNs/metabolismo , Infarto del Miocardio/terapia , Miocitos Cardíacos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Ratones , Ratones Transgénicos , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , ARN Mensajero/genética , Ratas , TransfecciónRESUMEN
Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits.
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Transportadoras de Casetes de Unión a ATP/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , HumanosRESUMEN
This review focuses on multidrug resistance protein 4 (MRP4 or ABCC4) that has recently been shown to play a role in cAMP homeostasis, a key-pathway in vascular biology and in platelet functions. In vascular system, recent data provide evidence that inhibition of MRP4 prevents human coronary artery smooth muscle cell proliferation in vitro and in vivo, as well as human pulmonary artery smooth muscle cell proliferation in vitro and pulmonary hypertension in mice in vivo. In the heart, MRP4 silencing in adult rat ventricular myocytes results in an increase in intracellular cAMP levels leading to enhanced cardiomyocyte contractility. However, a prolonged inhibition of MRP4 can promote cardiac hypertrophy. In addition, secreted cAMP, through its metabolite adenosine, prevents adrenergically induced cardiac hypertrophy and fibrosis. Finally, MRP4 inhibition in platelets induces a moderate thrombopathy. The localization of MRP4 underlines the emerging concept of cAMP compartmentalization in platelets, which is a major regulatory mechanism in other cells. cAMP storage in platelet dense granules might limit the cAMP cytosolic concentration upon adenylate cyclase activation, a necessary step to induce platelet activation. In this review, we discuss the therapeutic potential of direct pharmacological inhibition of MRP4 in atherothrombotic disease, via its vasodilating and antiplatelet effects.
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Enfermedades Cardiovasculares/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Animales , Plaquetas/metabolismo , HumanosRESUMEN
BACKGROUND: Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. METHODS AND RESULTS: Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus-mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction-induced cardiac hypertrophy and improved cardiac function. CONCLUSIONS: Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus-miR-378 may prove to be an effective therapeutic strategy in myocardial disease.
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Cardiomegalia/patología , Cardiomegalia/fisiopatología , MicroARNs/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Transducción de Señal/fisiología , Adenoviridae/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Proteína Adaptadora GRB2/antagonistas & inhibidores , Proteína Adaptadora GRB2/fisiología , MicroARNs/genética , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Quinasas/fisiología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores de Somatomedina/antagonistas & inhibidores , Receptores de Somatomedina/fisiologíaRESUMEN
Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.
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Caspasa 8/metabolismo , Ventrículos Cardíacos/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Activación Enzimática , Fluorescencia , Daño por Reperfusión Miocárdica/sangre , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Fenantridinas/metabolismo , Ratas , Ratas Endogámicas WKY , Factor de Necrosis Tumoral alfa/sangre , Remodelación VentricularRESUMEN
Pulmonary hypertension (PH) is a devastating disease, characterized by complex remodeling of the pulmonary vasculature. PH is classified into five groups based on different etiology, pathology, as well as therapy and prognosis. Animal models are essential for the study of underlying mechanisms, pathophysiology, and preclinical testing of new therapies for PH. The complexity of the disease with different clinical entities dictates the necessity for more than one animal model to resemble PH, as a single model cannot imitate the broad spectrum of human PH.Here we describe a detailed protocol for creating a rat model of PH with right ventricular (RV) failure. Furthermore, we present how to characterize it hemodynamically by invasive measurements of RV and pulmonary arterial (PA) pressures. Animals subjected to this model display severe pulmonary vascular remodeling and RV dysfunction. In this model, rats undergo a single subcutaneous injection of Sugen (SU5416, a vascular endothelial growth factor inhibitor) and are immediately exposed to chronic hypoxia in a hypoxia chamber for 3-6 weeks. This Sugen/Hypoxia rat model resembles Group 1 PH.
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Modelos Animales de Enfermedad , Insuficiencia Cardíaca , Hipertensión Pulmonar , Hipoxia , Animales , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/etiología , Ratas , Hipoxia/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Pirroles/farmacología , Indoles/farmacología , Disfunción Ventricular Derecha/fisiopatología , Disfunción Ventricular Derecha/etiología , Hemodinámica , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Masculino , Humanos , Remodelación Vascular , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.
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Movimiento Celular , Proliferación Celular , Células Endoteliales , Miocitos del Músculo Liso , Arteria Pulmonar , Humanos , Arteria Pulmonar/citología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Músculo Liso Vascular/citologíaRESUMEN
Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.
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AMP Cíclico/metabolismo , Homeostasis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Miocitos Cardíacos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Western Blotting , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/genética , Cardiomegalia/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Ecocardiografía , Femenino , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Cyclic nucleotides (e.g., cAMP and cGMP) are ubiquitous second messengers that affect multiple cell functions including vascular tone and vascular cell proliferation. After production, different processes can regulate the concentration of cyclic nucleotides. Cyclic nucleotides' degradation by phosphodiesterase (PDE) enzymes has well-known roles in regulating cyclic nucleotides concentrations. Recently, recognition of ATP-binding cassette (ABC) transporter contribution to both local and global regulation of cAMP has been acknowledged. Recent data support an important role of cyclic nucleotide efflux in the pathobiology of pulmonary hypertension, thus suggesting that inhibition of cyclic nucleotide efflux proteins might be a useful strategy to prevent and treat PH.
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AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hipertensión Pulmonar/etiología , Animales , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Propionatos/uso terapéutico , Quinolinas/uso terapéutico , Transducción de Señal/fisiologíaRESUMEN
Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.
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Ritmo Circadiano , Cardiopatías , Macrófagos , Melatonina , Glándula Pineal , Trastornos del Sueño del Ritmo Circadiano , Ganglio Cervical Superior , Animales , Humanos , Ratones , Cardiopatías/fisiopatología , Melatonina/metabolismo , Glándula Pineal/patología , Glándula Pineal/fisiopatología , Sueño , Trastornos del Sueño del Ritmo Circadiano/fisiopatología , Ganglio Cervical Superior/patología , Ganglio Cervical Superior/fisiopatología , Macrófagos/inmunología , FibrosisRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that control expression of complementary target mRNAs. A growing number of miRNAs has been implicated in the pathogenesis of cardiac diseases, mostly based not on functional data, but on the observation that they are dysregulated in diseased myocardium. Consequently, our knowledge regarding a potential cardiac role of the majority of miRNAs is limited. Here, we report the development of an assay format that allows the simultaneous analysis of several hundred molecules with regard to their phenotypic effect on primary rat cardiomyocytes. Using automated microscopy and an edge detection algorithm, this assay achieved high reproducibility and a robust assessment of cardiomyocyte size as a key parameter. Screening a library of synthetic miRNAs revealed several miRNAs previously not recognized as pro- or anti-hypertrophic. Out of these, we selected nine miRNAs and confirmed the pro-hypertrophic potential of miR-22, miR-30c, miR-30d, miR-212, miR-365 and the anti-hypertrophic potential of miR-27a, miR-27b and miR-133a. Quantitative analysis of the expression level of pro-hypertrophic miRNAs in primary cardiomyocytes indicated a rather low level of correlation of the phenotypic effects of individual miRNAs and their expression level. This assay allows the automated determination of cell size in primary cardiomyocytes and permitted the identification of a set of miRNAs capable of regulating cardiomyocyte hypertrophy. Elucidating their mechanism of action should provide insight into mechanisms underlying the cardiomyocyte hypertrophic response. This article is part of a Special Issue entitled 'Possible Editorial'.