RESUMEN
Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by ß-adrenergic receptors (ß-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known. Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro 20-1724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P < 0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of ß-AR responses is shifted toward PDE3 during CH.
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Canales de Calcio Tipo L/metabolismo , Cardiomegalia/enzimología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Citosol/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Ventrículos Cardíacos/patología , Cinética , Masculino , Modelos Biológicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Fenotipo , Inhibidores de Fosfodiesterasa 4/farmacología , Ratas WistarRESUMEN
AIM: To obtain a quantitative expression profile of the main genes involved in the cAMP-signaling cascade in human control atria and in different cardiac pathologies. METHODS AND RESULTS: Expression of 48 target genes playing a relevant role in the cAMP-signaling cascade was assessed by RT-qPCR. 113 samples were obtained from right atrial appendages (RAA) of patients in sinus rhythm (SR) with or without atrium dilation, paroxysmal atrial fibrillation (AF), persistent AF or heart failure (HF); and left atrial appendages (LAA) from patients in SR or with AF. Our results show that right and left atrial appendages in donor hearts or from SR patients have similar expression values except for AC7 and PDE2A. Despite the enormous chamber-dependent variability in the gene-expression changes between pathologies, several distinguishable patterns could be identified. PDE8A, PI3Kγ and EPAC2 were upregulated in AF. Different phosphodiesterase (PDE) families showed specific pathology-dependent changes. CONCLUSION: By comparing mRNA-expression patterns of the cAMP-signaling cascade related genes in right and left atrial appendages of human hearts and across different pathologies, we show that 1) gene expression is not significantly affected by cardioplegic solution content, 2) it is appropriate to use SR atrial samples as controls, and 3) many genes in the cAMP-signaling cascade are affected in AF and HF but only few of them appear to be chamber (right or left) specific. TOPIC: Genetic changes in human diseased atria. TRANSLATIONAL PERSPECTIVE: The cyclic AMP signaling pathway is important for atrial function. However, expression patterns of the genes involved in the atria of healthy and diseased hearts are still unclear. We give here a general overview of how different pathologies affect the expression of key genes in the cAMP signaling pathway in human right and left atria appendages. Our study may help identifying new genes of interest as potential therapeutic targets or clinical biomarkers for these pathologies and could serve as a guide in future gene therapy studies.
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AMP Cíclico/metabolismo , Variación Genética , Atrios Cardíacos/metabolismo , Sistemas de Mensajero Secundario/genética , Anciano , Alelos , Apéndice Atrial/metabolismo , Fibrilación Atrial/complicaciones , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Biomarcadores , Susceptibilidad a Enfermedades , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Humanos , Masculino , Persona de Mediana Edad , Proteoma , Proteómica/métodosRESUMEN
BACKGROUND: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac ß-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. METHODS: We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. RESULTS: PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but ß-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. CONCLUSIONS: Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Expresión Génica , Insuficiencia Cardíaca/etiología , Miocardio/metabolismo , Remodelación Ventricular/genética , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Terapia Genética , Vectores Genéticos/genética , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Pruebas de Función Cardíaca , Humanos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fenotipo , Receptores Adrenérgicos beta/metabolismo , Transducción Genética , Remodelación Ventricular/efectos de los fármacosRESUMEN
The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.
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Arritmias Cardíacas/etiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Cardiomegalia/etiología , AMP Cíclico/metabolismo , Acoplamiento Excitación-Contracción , Factor-23 de Crecimiento de Fibroblastos , Proteínas Klotho , Masculino , Ratones , Nefrectomía , Cultivo Primario de Células , Ratas WistarRESUMEN
Acute heart failure (HF) and in particular, cardiogenic shock are associated with high morbidity and mortality. A therapeutic dilemma is that the use of positive inotropic agents, such as catecholamines or phosphodiesterase-inhibitors, is associated with increased mortality. Newer drugs, such as levosimendan or omecamtiv mecarbil, target sarcomeres to improve systolic function putatively without elevating intracellular Ca2+. Although meta-analyses of smaller trials suggested that levosimendan is associated with a better outcome than dobutamine, larger comparative trials failed to confirm this observation. For omecamtiv mecarbil, Phase II clinical trials suggest a favourable haemodynamic profile in patients with acute and chronic HF, and a Phase III morbidity/mortality trial in patients with chronic HF has recently begun. Here, we review the pathophysiological basis of systolic dysfunction in patients with HF and the mechanisms through which different inotropic agents improve cardiac function. Since adenosine triphosphate and reactive oxygen species production in mitochondria are intimately linked to the processes of excitation-contraction coupling, we also discuss the impact of inotropic agents on mitochondrial bioenergetics and redox regulation. Therefore, this position paper should help identify novel targets for treatments that could not only safely improve systolic and diastolic function acutely, but potentially also myocardial structure and function over a longer-term.
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Cardiotónicos/uso terapéutico , Acoplamiento Excitación-Contracción/efectos de los fármacos , Insuficiencia Cardíaca/tratamiento farmacológico , Choque Cardiogénico/tratamiento farmacológico , Enfermedad Aguda , Animales , Antioxidantes/efectos adversos , Antioxidantes/uso terapéutico , Calcio/metabolismo , Cardiotónicos/efectos adversos , Estudios de Casos y Controles , Catecolaminas/efectos adversos , Catecolaminas/uso terapéutico , Ensayos Clínicos como Asunto , Diástole/efectos de los fármacos , Dobutamina/efectos adversos , Dobutamina/uso terapéutico , Perros , Metabolismo Energético/efectos de los fármacos , Insuficiencia Cardíaca/mortalidad , Humanos , Mitocondrias/metabolismo , Modelos Animales , Contracción Miocárdica/efectos de los fármacos , Óxidos de Nitrógeno/efectos adversos , Óxidos de Nitrógeno/uso terapéutico , Oxidación-Reducción/efectos de los fármacos , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/uso terapéutico , Placebos/administración & dosificación , Receptores Adrenérgicos/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Choque Cardiogénico/mortalidad , Simendán/efectos adversos , Simendán/uso terapéutico , Porcinos , Sístole/efectos de los fármacos , Urea/efectos adversos , Urea/análogos & derivados , Urea/uso terapéuticoRESUMEN
Cardiac failure is a common complication in cancer survivors treated with anthracyclines. Here we followed up cardiac function and excitation-contraction (EC) coupling in an in vivo doxorubicin (Dox) treated mice model (iv, total dose of 10â¯mg/Kg divided once every three days). Cardiac function was evaluated by echocardiography at 2, 6 and 15â¯weeks after the last injection. While normal at 2 and 6â¯weeks, ejection fraction was significantly reduced at 15â¯weeks. In order to evaluate the underlying mechanisms, we measured [Ca2+]i transients by confocal microscopy and action potentials (AP) by patch-clamp technique in cardiomyocytes isolated at these times. Three phases were observed: 1/depression and slowing of the [Ca2+]i transients at 2â¯weeks after treatment, with occurrence of proarrhythmogenic Ca2+ waves, 2/compensatory state at 6â¯weeks, and 3/depression on [Ca2+]i transients and cell contraction at 15â¯weeks, concomitant with in-vivo defects. These [Ca2+]i transient alterations were observed without cellular hypertrophy or AP prolongation and mirrored the sarcoplasmic reticulum (SR) Ca2+ load variations. At the molecular level, this was associated with a decrease in the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression and enhanced RyR2 phosphorylation at the protein kinase A (PKA, pS2808) site (2 and 15â¯weeks). RyR2 phosphorylation at the Ca2+/calmodulin dependent protein kinase II (CaMKII, pS2814) site was enhanced only at 2â¯weeks, coinciding with the higher incidence of proarrhythmogenic Ca2+ waves. Our study highlighted, for the first time, the progression of Dox treatment-induced alterations in Ca2+ handling and identified key components of the underlying Dox cardiotoxicity. These findings should be helpful to understand the early-, intermediate-, and late- cardiotoxicity already recorded in clinic in order to prevent or treat at the subclinical level.
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Cardiotoxicidad/fisiopatología , Doxorrubicina/efectos adversos , Acoplamiento Excitación-Contracción , Potenciales de Acción , Animales , Calcio/metabolismo , Señalización del Calcio , Pruebas de Función Cardíaca , Masculino , Ratones Endogámicos C57BL , Retículo Sarcoplasmático/metabolismo , Factores de TiempoRESUMEN
AIMS: Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to ß-adrenergic receptor (ß-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model. METHODS AND RESULTS: Real-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Förster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1â¯Hz allowing simultaneous measurement of intracellular Ca2+ and sarcomere shortening. The expression of the different PDE4 subfamilies was assessed by Western blot in pig right ventricles and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective ß-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D subfamilies are expressed in pig ventricles. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects. CONCLUSIONS: Our results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or ß-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress.
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AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Acoplamiento Excitación-Contracción/genética , Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Familia de Multigenes , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Fosfodiesterasa 3/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Receptores Adrenérgicos beta/metabolismo , PorcinosRESUMEN
NEW FINDINGS: What is the central question of this study? Can imipramine, an antidepressant agent that is a cationic amphiphilic drug that interferes with the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system, be validated as a new detubulating tool? What is the main finding and its importance? Imipramine was validated as a more efficient and less toxic detubulating agent of cardiomyocytes than formamide. New insights are provided on how PI(4,5)P2 is crucial to maintaining T-tubule attachment to the cell surface and on the cardiotoxic effects of imipramine overdoses. ABSTRACT: Cardiac T-tubules are membrane invaginations essential for excitation-contraction coupling (ECC). Imipramine, like other cationic amphiphilic drugs, interferes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system connected to the cell surface. Our main purpose was to validate imipramine as a new detubulating agent in cardiomyocytes. Staining adult rat ventricular myocytes (ARVMs) with di-4-ANEPPS, we showed that unlike formamide, imipramine induces a complete detubulation with no impact on cell viability. Using the patch-clamp technique, we observed a â¼40% decrease in cell capacitance after imipramine pretreatment and a reduction of ICa,L amplitude by â¼72%. These parameters were not affected in atrial cells, excluding direct side effects of imipramine. ß-Adrenergic receptor (ß-AR) stimulation of the remaining ICa,L with isoproterenol (Iso) was still effective. ECC was investigated in ARVMs loaded with Fura-2 and paced at 1 Hz, allowing simultaneous measurement of the Ca2+ transient (CaT) and sarcomere shortening (SS). Amplitude of both CaT and SS was decreased by imipramine and partially restored by Iso. Furthermore, detubulated cells exhibited Ca2+ homeostasis perturbations. Real-time cAMP variations induced by Iso using a Förster resonance energy transfer biosensor revealed â¼27% decreased cAMP elevation upon ß-AR stimulation. To conclude, we validated a new cardiomyocyte detubulation method using imipramine, which is more efficient and less toxic than formamide. This antidepressant agent induces the hallmark effects of detubulation on ECC and its ß-AR stimulation. Besides, we provide new insights on how an imipramine overdose may affect cardiac function and suggest that PI(4,5)P2 is crucial for maintaining T-tubule structure.
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Formamidas/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Imipramina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Antidepresivos/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Acoplamiento Excitación-Contracción/efectos de los fármacos , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Isoproterenol/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Wistar , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismoRESUMEN
RATIONALE: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown. OBJECTIVE: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction. METHODS AND RESULTS: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal ß-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated ß-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction. CONCLUSIONS: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction.
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Arritmias Cardíacas/metabolismo , Cardiotónicos/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/biosíntesis , Isoproterenol/toxicidad , Contracción Miocárdica/fisiología , Infarto del Miocardio/metabolismo , Animales , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/prevención & control , Catecolaminas/toxicidad , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/antagonistas & inhibidores , Perros , Femenino , Imidazoles/farmacología , Masculino , Ratones , Ratones Transgénicos , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/fisiopatología , Triazinas/farmacologíaRESUMEN
Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during ß-adrenergic stimulation by isoprenaline, a non-selective agonist of ß-adrenergic receptors. The whole-cell patchâ»clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.
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Canales de Calcio Tipo L/metabolismo , Miocitos Cardíacos/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Desacopladores/farmacología , Potenciales de Acción , Agonistas Adrenérgicos beta/farmacología , Animales , Señalización del Calcio , Células Cultivadas , Humanos , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ionóforos de Protónes/farmacología , Ratas , Ratas WistarRESUMEN
AIMS/HYPOTHESIS: CUG-binding protein 1 (CUGBP1) is a multifunctional RNA-binding protein that regulates RNA processing at several stages including translation, deadenylation and alternative splicing, as well as RNA stability. Recent studies indicate that CUGBP1 may play a role in metabolic disorders. Our objective was to examine its role in endocrine pancreas function through gain- and loss-of-function experiments and to further decipher the underlying molecular mechanisms. METHODS: A mouse model in which type 2 diabetes was induced by a high-fat diet (HFD; 60% energy from fat) and mice on a standard chow diet (10% energy from fat) were compared. Pancreas-specific CUGBP1 overexpression and knockdown mice were generated. Different lengths of the phosphodiesterase subtype 3B (PDE3B) 3' untranslated region (UTR) were cloned for luciferase reporter analysis. Purified CUGBP1 protein was used for gel shift experiments. RESULTS: CUGBP1 is present in rodent islets and in beta cell lines; it is overexpressed in the islets of diabetic mice. Compared with control mice, the plasma insulin level after a glucose load was significantly lower and glucose clearance was greatly delayed in mice with pancreas-specific CUGBP1 overexpression; the opposite results were obtained upon pancreas-specific CUGBP1 knockdown. Glucose- and glucagon-like peptide1 (GLP-1)-stimulated insulin secretion was significantly attenuated in mouse islets upon CUGBP1 overexpression. This was associated with a strong decrease in intracellular cAMP levels, pointing to a potential role for cAMP PDEs. CUGBP1 overexpression had no effect on the mRNA levels of PDE1A, 1C, 2A, 3A, 4A, 4B, 4D, 7A and 8B subtypes, but resulted in increased PDE3B expression. CUGBP1 was found to directly bind to a specific ATTTGTT sequence residing in the 3' UTR of PDE3B and stabilised PDE3B mRNA. In the presence of the PDE3 inhibitor cilostamide, glucose- and GLP-1-stimulated insulin secretion was no longer reduced by CUGBP1 overexpression. Similar to CUGBP1, PDE3B was overexpressed in the islets of diabetic mice. CONCLUSIONS/INTERPRETATION: We conclude that CUGBP1 is a critical regulator of insulin secretion via activating PDE3B. Repressing this protein might provide a potential strategy for treating type 2 diabetes.
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Proteínas CELF1/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Insulina/metabolismo , Animales , Western Blotting , Proteínas CELF1/genética , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Inmunoprecipitación , Secreción de Insulina , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Estabilidad del ARN/genética , Estabilidad del ARN/fisiología , ARN Mensajero/genéticaRESUMEN
The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.
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Cardiomegalia/enzimología , Mutación Missense , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Remodelación Ventricular , Sustitución de Aminoácidos , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Miocardio/patología , Miocitos Cardíacos/patología , Ratas , Ratas Sprague-Dawley , Sarcómeros/enzimología , Sarcómeros/genéticaRESUMEN
Cyclic AMP regulates a multitude of cellular responses and orchestrates a network of intracellular events. In the heart, cAMP is the main second messenger of the ß-adrenergic receptor (ß-AR) pathway producing positive chronotropic, inotropic, and lusitropic effects during sympathetic stimulation. Whereas short-term stimulation of ß-AR/cAMP is beneficial for the heart, chronic activation of this pathway triggers pathological cardiac remodeling, which may ultimately lead to heart failure (HF). Cyclic AMP is controlled by two families of enzymes with opposite actions: adenylyl cyclases, which control cAMP production and phosphodiesterases, which control its degradation. The large number of families and isoforms of these enzymes, their different localization within the cell, and their organization in macromolecular complexes leads to a high level of compartmentation, both in space and time, of cAMP signaling in cardiac myocytes. Here, we review the expression level, molecular characteristics, functional properties, and roles of the different adenylyl cyclase and phosphodiesterase families expressed in heart muscle and the changes that occur in cardiac hypertrophy and failure.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Insuficiencia Cardíaca/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Animales , AMP Cíclico/biosíntesis , Humanos , Hidrólisis , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.
Title: Les phosphodiestérases des nucléotides cycliques - Cibles thérapeutiques dans l'hypertrophie et l'insuffisance cardiaques. Abstract: Les phosphodiestérases des nucléotides cycliques (PDE) modulent la régulation neuro-hormonale de la fonction cardiaque en dégradant l'AMPc et le GMPc. Dans les cardiomyocytes, de multiples isoformes de PDE, aux propriétés enzymatiques et aux localisations subcellulaires différentes, régulent localement les niveaux de nucléotides cycliques et les fonctions cellulaires associées. Cette organisation est fortement perturbée au cours de l'hypertrophie et de l'insuffisance cardiaque à fraction d'éjection réduite (IC), ce qui peut contribuer à la progression de la maladie. Sur le plan clinique, l'inhibition des PDE a été considérée comme une approche prometteuse pour compenser la désensibilisation aux catécholamines qui accompagne l'IC. Bien que des inhibiteurs de la PDE3, tels que la milrinone ou l'énoximone, puissent être utilisés cliniquement pour améliorer la fonction systolique et soulager les symptômes de l'IC aiguë, leur utilisation chronique s'est avérée préjudiciable. D'autres PDE, telles que les PDE1, PDE2, PDE4, PDE5, PDE9 et PDE10, sont apparues comme de nouvelles cibles potentielles pour le traitement de l'IC, chacune ayant un rôle unique dans les voies de signalisation locales des nucléotides cycliques. Dans cette revue, nous décrivons la signalisation de l'AMPc et du GMPc dans les cardiomyocytes et présentons les différentes familles de PDE exprimées dans le cÅur ainsi que leurs modifications dans l'hypertrophie cardiaque pathologique et dans l'IC. Nous évaluons également les résultats issus de modèles précliniques ainsi que les données cliniques indiquant l'utilisation d'inhibiteurs ou d'activateurs de PDE spécifiques qui pourraient avoir un potentiel thérapeutique dans l'IC.
Asunto(s)
Cardiomegalia , Insuficiencia Cardíaca , Inhibidores de Fosfodiesterasa , Humanos , Cardiomegalia/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Animales , Inhibidores de Fosfodiesterasa/uso terapéutico , Inhibidores de Fosfodiesterasa/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Terapia Molecular Dirigida/métodos , GMP Cíclico/metabolismo , GMP Cíclico/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/fisiologíaRESUMEN
The cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective ß-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Quinolinas/farmacología , Proteínas Portadoras , AMP Cíclico/química , AMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Cinética , Unión Proteica/efectos de los fármacos , Quinolinas/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
BACKGROUND: Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by ß-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown. METHODS AND RESULTS: Mice lacking PI3Kγ (PI3Kγ(-/-)) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective ß(2)-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after ß(2)-adrenergic receptor activation in PI3Kγ(-/-) cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from ß-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A-mediated phosphorylation of L-type calcium channel (Ca(v)1.2) and phospholamban. In PI3Kγ(-/-) cardiomyocytes, Ca(v)1.2 and phospholamban were hyperphosphorylated, leading to increased Ca(2+) spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ(-/-) cardiomyocytes showed spontaneous Ca(2+) release events and developed arrhythmic calcium transients. CONCLUSIONS: PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.
Asunto(s)
Catecolaminas/toxicidad , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Taquicardia Ventricular/enzimología , Taquicardia Ventricular/prevención & control , Animales , Animales Recién Nacidos , Biorretroalimentación Psicológica/fisiología , Señalización del Calcio/genética , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Técnicas de Sustitución del Gen , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/enzimologíaRESUMEN
Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.