RESUMEN
RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.
Asunto(s)
Cardiomegalia/prevención & control , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Miocitos Cardíacos/enzimología , Sistemas de Mensajero Secundario , Adenoviridae/genética , Animales , Animales Recién Nacidos , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/patología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Masculino , Microdominios de Membrana/enzimología , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Interferencia de ARN , Ratas Sprague-Dawley , Ratas Wistar , Sistemas de Mensajero Secundario/efectos de los fármacos , Factores de Tiempo , Transducción Genética , TransfecciónRESUMEN
Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Miocardio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas de Anclaje a la Quinasa A/análisis , Proteínas de Anclaje a la Quinasa A/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/análisis , Humanos , Miocardio/citología , Miocardio/enzimología , Miocardio/ultraestructura , Fosforilación , Mapas de Interacción de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/análisisRESUMEN
Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Fosfodiesterasa 3/farmacología , Proteínas 14-3-3/genética , Sitios de Unión/genética , Cromatografía en Gel , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Activación Enzimática/fisiología , Células HEK293 , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Isoproterenol/farmacología , Inhibidores de Fosfodiesterasa 3/metabolismo , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation-contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has 'global' effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Insuficiencia Cardíaca/enzimología , Miocardio/enzimología , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Isoenzimas/metabolismo , Ratones , Miocitos Cardíacos/enzimología , Fosforilación , RatasRESUMEN
In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 microM, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised approximately 22 and approximately 43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.
Asunto(s)
AMP Cíclico/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Animales , Vasos Coronarios/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Hidrólisis , Masculino , Ratones , Ratones Endogámicos ICR , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Inhibidores de Fosfodiesterasa 3 , Inhibidores de Fosfodiesterasa 5 , Fosfodiesterasa I/antagonistas & inhibidores , Fosfodiesterasa I/metabolismo , Purinas/farmacología , Citrato de Sildenafil , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from approximately 2 microM to approximately 80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from approximately 1 microM to approximately 40 nM) relieves the inhibition of PDE3A, increasing its activity by approximately 5-fold. This causes a decrease in oocyte cAMP (from approximately 700 nM to approximately 140 nM), leading to the resumption of meiosis.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/fisiología , Meiosis/fisiología , Oocitos/fisiología , Animales , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Femenino , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Humanos , Hormona Luteinizante/farmacología , Hormona Luteinizante/fisiología , Meiosis/efectos de los fármacos , Ratones , Oocitos/efectos de los fármacos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiologíaRESUMEN
Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Miocitos Cardíacos/enzimología , Animales , Línea Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Hidrólisis , Isoenzimas/metabolismo , Cinética , Spodoptera , Fracciones Subcelulares/enzimologíaRESUMEN
Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , AMP Cíclico/metabolismo , Miocardio/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/química , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Señalización del Calcio , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismoRESUMEN
PDE3A cyclic nucleotide phosphodiesterases regulate cAMP- and cGMP-mediated intracellular signaling in cardiac myocytes. We used antibodies to different regions of PDE3A to demonstrate the presence of three PDE3A isoforms in these cells. These isoforms, whose apparent molecular weights are 136,000, 118,000, and 94,000 ("PDE3A-136," "PDE3A-118," and "PDE3A-94"), are identical save for the deletion of different lengths of N-terminal sequence containing two membrane-association domains and sites for phosphorylation/activation by protein kinase B ("PK-B") and protein kinase A ("PK-A"). PDE3A-136 contains both membrane-association domains and the PK-B and PK-A sites. PDE3A-118 contains only the downstream membrane-association domain and the PK-A sites. PDE3A-94 lacks both membrane localization domains and the PK-B and PK-A sites. The three isoforms are translated from two mRNAs derived from the PDE3A1 gene: PDE3A-136 is translated from PDE3A1 mRNA, whereas PDE3A-118 and PDE3A-94 are translated from PDE3A2 mRNA. Experiments involving in vitro transcription/translation indicate that PDE3A-118 and PDE3A-94 may be translated from different AUGs in PDE3A2 mRNA. These findings suggest that alternative transcriptional and post-transcriptional processing of the PDE3A gene results in the generation of two mRNAs and three protein isoforms in cardiac myocytes that differ with respect to intracellular localization and may be regulated through different signaling pathways.