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1.
Molecules ; 29(16)2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-39202903

RESUMO

Cilostazol is a phosphodiesterase III inhibitor characterized by poor solubility. This limitation can be overcome by using a drug carrier capable of delivering the drug to the target site. Cyclodextrins are essential as drug carriers because of their outstanding complexation abilities and their capacity to improve drug bioavailability. This study comprises two stages: The first involves verifying different cyclodextrins and their complexation abilities towards cilostazol. This was accomplished using molecular docking simulations (MDS) and density functional theory (DFT). Both techniques indicate that the largest Sulfobutyl Ether-ß-Cyclodextrin forms the most stable complex with cilostazol. Additionally, other important parameters of the complex are described, including binding sites, dominant interactions, and thermodynamic parameters such as complexation enthalpy, Gibbs free energy, and Gibbs free energy of solvation. The second stage involves a binding study between cilostazol and Phosphodiesterse3 (PDE3). This study was conducted using molecular docking simulations, and the most important energetic parameters are detailed. This is the first such report, and we believe that the results of our predictions will pave the way for future drug development efforts using cyclodextrin-cilostazol complexes as potential therapeutics.


Assuntos
Cilostazol , Ciclodextrinas , Simulação de Acoplamento Molecular , Inibidores da Fosfodiesterase 3 , Termodinâmica , Cilostazol/química , Inibidores da Fosfodiesterase 3/química , Inibidores da Fosfodiesterase 3/farmacologia , Ciclodextrinas/química , Sítios de Ligação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Ligação Proteica , Humanos
2.
Biomolecules ; 14(8)2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-39199373

RESUMO

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic ß-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.


Assuntos
AMP Cíclico , Fatores de Troca do Nucleotídeo Guanina , Compostos de Sulfonilureia , Humanos , Compostos de Sulfonilureia/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/química , Hipoglicemiantes/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Sítios de Ligação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo
3.
Cells ; 13(13)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38994957

RESUMO

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.


Assuntos
Proteínas de Ancoragem à Quinase A , Plaquetas , Proteínas Quinases Dependentes de AMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Epoprostenol , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Humanos , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epoprostenol/metabolismo , Epoprostenol/farmacologia , Fosforilação , AMP Cíclico/metabolismo , Transdução de Sinais
4.
Clin Cancer Res ; 30(16): 3603-3621, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38864850

RESUMO

PURPOSE: Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor with KIT or PDGFRA driver mutations, is typically treated with tyrosine kinase inhibitors (TKI). However, resistance to TKIs due to secondary mutations is a common challenge in advanced GISTs. In addition, there are currently no effective therapies for several other molecular subtypes, such as succinate dehydrogenase-deficient GISTs. Therefore, novel therapeutic strategies are needed. EXPERIMENTAL DESIGN: To address this need, we tested the efficacy of a novel non-TKI compound, OPB-171775, using patient-derived xenograft models of GISTs. In parallel, we sought to elucidate the mechanism of action of the compound. RESULTS: Our study revealed that OPB-171775 exhibited significant efficacy against GISTs regardless of their KIT mutation status by inducing complex formation between phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12), which are highly expressed in GISTs, leading to SLFN12 RNase-mediated cell death. Furthermore, we identified the activation of general control non-derepressible 2 and its downstream response as an effector pathway of SLFN12 in mediating anticancer activity and revealed potential pharmacodynamic markers. CONCLUSIONS: These findings suggest that OPB-171775, with its significant efficacy, could potentially serve as a novel and effective treatment option for advanced GISTs, particularly those resistant to TKIs.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Resistencia a Medicamentos Antineoplásicos , Tumores do Estroma Gastrointestinal , Inibidores de Proteínas Quinases , Ensaios Antitumorais Modelo de Xenoenxerto , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Humanos , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Camundongos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Mutação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos
5.
Int J Nanomedicine ; 19: 4411-4427, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38774028

RESUMO

Background: Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation and joint destruction. Despite progress in RA therapy, it remains difficult to achieve long-term remission in RA patients. Phosphodiesterase 3B (Pde3b) is a member of the phosphohydrolyase family that are involved in many signal transduction pathways. However, its role in RA is yet to be fully addressed. Methods: Studies were conducted in arthritic DBA/1 mice, a suitable mouse strain for collagen-induced rheumatoid arthritis (CIA), to dissect the role of Pde3b in RA pathogenesis. Next, RNAi-based therapy with Pde3b siRNA-loaded liposomes was assessed in a CIA model. To study the mechanism involved, we investigated the effect of Pde3b knockdown on macrophage polarization and related signaling pathway. Results: We demonstrated that mice with CIA exhibited upregulated Pde3b expression in macrophages. Notably, intravenous administration of liposomes loaded with Pde3b siRNA promoted the macrophage anti-inflammatory program and alleviated CIA in mice, as indicated by the reduced inflammatory response, synoviocyte infiltration, and bone and cartilage erosion. Mechanistic study revealed that depletion of Pde3b increased cAMP levels, by which it enhanced PKA-CREB-C/EBPß pathway to transcribe the expression of anti-inflammatory program-related genes. Conclusion: Our results support that Pde3b is involved in the pathogenesis of RA, and Pde3b siRNA-loaded liposomes might serve as a promising therapeutic approach against RA.


Assuntos
Artrite Experimental , Artrite Reumatoide , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Terapia Genética , Lipossomos , Macrófagos , Animais , Masculino , Camundongos , Artrite Experimental/genética , Artrite Experimental/prevenção & controle , Artrite Experimental/terapia , Artrite Reumatoide/genética , Artrite Reumatoide/terapia , Artrite Reumatoide/induzido quimicamente , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Lipossomos/química , Lipossomos/administração & dosagem , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos DBA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/administração & dosagem , Transdução de Sinais/efeitos dos fármacos
6.
J Med Chem ; 67(3): 2049-2065, 2024 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-38284310

RESUMO

Human genetic evidence shows that PDE3B is associated with metabolic and dyslipidemia phenotypes. A number of PDE3 family selective inhibitors have been approved by the FDA for various indications; however, given the undesirable proarrhythmic effects in the heart, selectivity for PDE3B inhibition over closely related family members (such as PDE3A; 48% identity) is a critical consideration for development of PDE3B therapeutics. Selectivity for PDE3B over PDE3A may be achieved in a variety of ways, including properties intrinsic to the compound or tissue-selective targeting. The high (>95%) active site homology between PDE3A and B represents a massive obstacle for obtaining selectivity at the active site; however, utilization of libraries with high molecular diversity in high throughput screens may uncover selective chemical matter. Herein, we employed a DNA-encoded library screen to identify PDE3B-selective inhibitors and identified potent and selective boronic acid compounds bound at the active site.


Assuntos
DNA , Coração , Humanos , Domínio Catalítico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3
7.
Biochem Biophys Res Commun ; 696: 149489, 2024 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-38244313

RESUMO

Lung cancer has a high incidence rate and requires more effective treatment strategies and drug options for clinical patients. EGFR is a common genetic alteration event in lung cancer that affects patient survival and drug strategy. Our study discovered aberrant aldolase A (ALDOA) expression and dysfunction in lung cancer patients with EGFR mutations. In addition to investigating relevant metabolic processes like glucose uptake, lactate production, and ATPase activity, we examined multi-omics profiles (transcriptomics, proteomics, and pull-down assays). It was observed that phosphodiesterase 3A (PDE3A) enzyme and ALDOA exhibit correlation, and furthermore, they impact M2 macrophage polarization through ß-catenin and downstream ID3. In addition to demonstrating the aforementioned mechanism of action, our experiments discovered that the PDE3 inhibitor trequinsin has a substantial impact on lung cancer cell lines with EGFR mutants. The trequinsin medication was found to decrease the M2 macrophage polarization status and several cancer phenotypes, in addition to transduction. These findings have potential prognostic and therapeutic applications for clinical patients with EGFR mutation and lung cancer.


Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Frutose-Bifosfato Aldolase/genética , beta Catenina/genética , beta Catenina/metabolismo , Transdução de Sinais/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Linhagem Celular Tumoral , Mutação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Inibidoras de Diferenciação/genética
8.
Naunyn Schmiedebergs Arch Pharmacol ; 397(7): 4911-4925, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38165426

RESUMO

Cuproptosis is a new Cu-dependent programmed cell death manner that has shown regulatory functions in many tumor types, however, its mechanism in bladder cancer remains unclear. Here, we reveal that Phosphodiesterase 3B (PDE3B), a cuproptosis-associated gene, could reduce the invasion and migration of bladder cancer. PDE3B is downregulated in bladder cancer tissues, which is correlated with better prognosis. Conversely, overexpression of PDE3B in bladder cancer cell could significantly resist invasion and migration, which is consistent with the TCGA database results. Future study demonstrate the anti-cancer effect of PDE3B is mediated by Keratin 6B (KRT6B) which leads to the keratinization. Therefore, PDE3B can reduce KRT6B expression and inhibit the invasion and migration of bladder cancer. Meanwhile, increased expression of PDE3B was able to enhance the sensitivity of Cuproptosis drug thiram. This study show that PDE3B/KRT6B is a potential cancer therapeutic target and PDE3B activation is able to increase the sensitivity of bladder cancer cells to copper ionophores.


Assuntos
Cobre , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Queratina-6 , Neoplasias da Bexiga Urinária , Humanos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cobre/metabolismo , Cobre/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Regulação Neoplásica da Expressão Gênica , Queratina-6/metabolismo , Queratina-6/genética , Invasividade Neoplásica , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
11.
Cells ; 12(11)2023 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-37296663

RESUMO

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.


Assuntos
Sistemas CRISPR-Cas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Miócitos Cardíacos , Animais , Camundongos , Ratos , Sistemas CRISPR-Cas/genética , AMP Cíclico/metabolismo , Dietilestilbestrol , Miócitos Cardíacos/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Isoformas de Proteínas/metabolismo
13.
Commun Biol ; 6(1): 504, 2023 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-37165086

RESUMO

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.


Assuntos
Coração , Células Cultivadas , Animais , Camundongos , Fibroblastos/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Células Musculares/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Transdução de Sinais , Sobrevivência Celular
14.
Int Immunopharmacol ; 119: 110157, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37086679

RESUMO

Synovial samples collected from 30 rheumatoid arthritis (RA) patients and 30 normal controls were used to isolate fibroblast-like synoviocytes (FLSs) and named FLS-RA and FLS-Normal, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to detect circ_0007707 expression. Effects of circ_0007707 silencing on cell proliferation and apoptosis were evaluated using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (Edu), and flow cytometry assays. Levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Increased circ_0007707 expression was observed in synovial samples from RA patients and FLS-RA cells. Functional analysis showed circ_0007707 silencing restrained cell proliferation, induced cell apoptosis, and decreased cell inflammatory response in FLS-RA cells. Mechanistic analysis revealed the sponge function of circ_0007707 on miR-27b-3p, and miR-27b-3p inhibition weakened circ_0007707 knockdown-mediated effects on FLS-RA cell proliferation, apoptosis, and inflammatory response. Circ_0007707 could mediate PDE3B expression via sponging miR-27b-3p, and PDE3B overturned miR-27b-3p mimic-mediated effects on FLS-RA cell proliferation, apoptosis, and inflammatory response. Circ_0007707 mediated cell apoptosis and inflammatory response in FLS-RA cells through the miR-27b-3p/PDE3B axis, indicating the potential function of circ_0007707 as a target for RA treatment.


Assuntos
Artrite Reumatoide , MicroRNAs , Sinoviócitos , Humanos , Sinoviócitos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Inflamação/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proliferação de Células/fisiologia , Apoptose/genética , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo
15.
Hypertension ; 80(6): 1171-1179, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37035914

RESUMO

Hypertension with brachydactyly (HTNB) represents an autosomal dominant form of hypertension. It is a rare syndrome, in which the blood pressure can rise by more than 50 mmHg. If untreated, the patients die of stroke by the age of 50 years. In HTNB, vascular smooth muscle cell proliferation is increased, vasodilation compromised, and the kidney not affected. Surprisingly, after decades of hypertension, HTNB is not associated with hypertension-induced cardiac damage. HTNB is caused by gain-of-function mutations in the PDE3A (phosphodiesterase 3A) gene. The mutant enzymes are hyperactive. PDE3A (phosphodiesterase 3A) hydrolyzes and thereby terminates cyclic adenosine monophosphate signaling in defined cellular compartments. The cardioprotective effect involves local changes of cyclic adenosine monophosphate signaling and inhibition of Ca2+ reuptake into the sarcoplasmic reticulum of cardiac myocytes. This review introduces HTNB and discusses how insight into the molecular mechanisms underlying HTNB could contribute to a better understanding of blood pressure control and lead to PDE3A-directed strategies for the treatment of essential hypertension and the prevention of hypertension-induced cardiac damage. A focus will be on cAMP (cyclic adenosine monophosphate) signaling compartments.


Assuntos
Hipertensão , Humanos , Pessoa de Meia-Idade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Hipertensão/genética , Mutação , Miócitos Cardíacos , Monofosfato de Adenosina
16.
Circulation ; 147(16): 1221-1236, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-36876489

RESUMO

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Insuficiência Cardíaca , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Humanos , Camundongos , Cálcio/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Insuficiência Cardíaca/metabolismo , Células HEK293 , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
17.
Eur J Pharmacol ; 944: 175562, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-36736940

RESUMO

Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF. Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to ß-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling. We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Insuficiência Cardíaca , Ratos , Animais , Ratos Wistar , Células Endoteliais , NG-Nitroarginina Metil Éster , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Artérias Mesentéricas , 3',5'-AMP Cíclico Fosfodiesterases
18.
Naunyn Schmiedebergs Arch Pharmacol ; 396(4): 669-682, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36445386

RESUMO

Levosimendan (up to 10 µM) given alone failed to increase force of contraction in isolated electrically stimulated (1 Hz) left atrial (LA) preparations from wild-type mice. Only in the additional presence of 0.1 µM rolipram, an inhibitor of the activity of phosphodiesterase IV, levosimendan increased force of contraction in LA and increased the phosphorylation state of phospholamban at amino acid serine 16. Levosimendan alone increased the beating rate in isolated spontaneously beating right atrial preparations from mice and this effect was potentiated by rolipram. The positive inotropic and the positive chronotropic effects of levosimendan in mouse atrial preparations were attenuated by 10 µM propranolol. Finally, we studied the contractile effects of levosimendan in isolated electrically stimulated (1 Hz) right atrial preparations from the human atrium (HAP), obtained during cardiac surgery. We detected concentration-dependent positive inotropic effects of levosimendan alone that reached plateau at 1 µM levosimendan in HAP (n = 11). Levosimendan shortened time of tension relaxation in HAP. Cilostamide (1 µM), an inhibitor of phosphodiesterase III, or propranolol (10 µM) blocked the positive inotropic effect of levosimendan in HAP. Levosimendan (1 µM) alone increased in HAP the phosphorylation state of phospholamban. In conclusion, we present evidence that levosimendan acts via phosphodiesterase III inhibition in the human atrium leading to phospholamban phosphorylation and thus explaining the positive inotropic effects of levosimendan in HAP.


Assuntos
Fibrilação Atrial , Propranolol , Humanos , Camundongos , Animais , Simendana/farmacologia , Rolipram/farmacologia , Fosforilação , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Propranolol/farmacologia , Contração Miocárdica , Cardiotônicos/farmacologia
20.
Circulation ; 146(23): 1758-1778, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36259389

RESUMO

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.


Assuntos
Insuficiência Cardíaca , Hipertensão , Células-Tronco Pluripotentes Induzidas , Humanos , Ratos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Microtomografia por Raio-X , Células-Tronco Pluripotentes Induzidas/metabolismo , Hipertensão/complicações , Hipertensão/genética , Miócitos Cardíacos/metabolismo , Cardiomegalia , RNA
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