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1.
Res Sq ; 2024 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-38463967

RESUMEN

Metal ions are vital components in many proteins for the inference and engineering of protein function, with coordination complexity linked to structural (4-residue predominate), catalytic (3-residue predominate), or regulatory (2-residue predominate) roles. Computational tools for modeling metal ions in protein structures, especially for transient, reversible, and concentration-dependent regulatory sites, remain immature. We present PinMyMetal (PMM), a sophisticated hybrid machine learning system for predicting zinc ion localization and environment in macromolecular structures. Compared to other predictors, PMM excels in predicting regulatory sites (median deviation of 0.34 Å), demonstrating superior accuracy in locating catalytic sites (median deviation of 0.27 Å) and structural sites (median deviation of 0.14 Å). PMM assigns a certainty score to each predicted site based on local structural and physicochemical features independent of homolog presence. Interactive validation through our server, CheckMyMetal, expands PMM's scope, enabling it to pinpoint and validates diverse functional zinc sites from different structure sources (predicted structures, cryo-EM and crystallography). This facilitates residue-wise assessment and robust metal binding site design. The lightweight PMM system demands minimal computing resources and is available at https://PMM.biocloud.top. While currently trained on zinc, the PMM workflow can easily adapt to other metals through expanded training data.

2.
J Cancer ; 15(1): 79-89, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38164275

RESUMEN

Hsp70 (heat shock protein 70) plays critical roles in cancer cell proliferation and apoptosis. Recently, accumulating evidences have demonstrated the cancer promoting effects of Hsp70 in bladder cancer. The development of novel therapeutic approaches targeting Hsp70 thus received great attention from researchers. In this study, we demonstrated that silibinin, a natural polyphenolic flavonoid isolated from the milk thistle, targeted Hsp70 by inhibiting its transcription in bladder cancer cells. We also demonstrated that knockdown of endogenous Hsp70 enhanced silibinin-induced apoptosis, while overexpression of exogenous Hsp70 could partially reverse the effects of silibinin-induced cell apoptosis. Furthermore, we found that silibinin could activate HSF1/Hsp70-regulated mitochondrial apoptotic pathway. Mechanically, silibinin inhibited the interaction between Apaf-1 and Hsp70, thus increasing the recruitment of pro caspase-9. Results from in vivo study demonstrated that silibinin suppressed the growth of bladder cancer xenografts, which was accompanied with the activation of caspase-3 and downregulation of HSF1 and Hsp70. Taken together, our data indicates that silibinin induces mitochondrial apoptosis via inhibiting HSF1/Hsp70 pathway and also suggests the therapeutic potential of silibinin in the treatment of bladder cancer.

3.
Circ Res ; 133(11): 902-923, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37850368

RESUMEN

BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].


Asunto(s)
AMP Cíclico , Miocitos Cardíacos , Ratones , Animales , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Adenosina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Péptidos/metabolismo
5.
Cardiovasc Res ; 118(12): 2703-2717, 2022 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-34550322

RESUMEN

AIMS: Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. METHODS AND RESULTS: RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodelling of cultured human saphenous vein segments. CONCLUSIONS: Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signalling and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.


Asunto(s)
Músculo Liso Vascular , Lesiones del Sistema Vascular , Animales , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacología , Proliferación Celular , Células Cultivadas , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Ciclina D1/metabolismo , Células Endoteliales/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , ARN Mensajero/metabolismo , Remodelación Vascular , Lesiones del Sistema Vascular/tratamiento farmacológico , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/metabolismo
6.
Eur J Pharmacol ; 908: 174368, 2021 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-34302816

RESUMEN

Allopurinol, a xanthine oxidase (XO) inhibitor, is reported to alleviate myocardial ischemia/reperfusion (I/R) injury by reducing the production of reactive oxygen species (ROS). As an XO-derived product, H2O2 can act as a substrate of vascular peroxidase 1 (VPO1) to induce the generation of hypochlorous acid (HOCl), a potent oxidant. This study aims to explore whether the XO/VPO1 pathway is involved in the anti-oxidative effects of allopurinol on the myocardial I/R injury. In a rat heart model of I/R, allopurinol alleviated I/R oxidative injury accompanied by decreased XO activity, XO-derived products (H2O2 and uric acid), and VPO1 expression (mRNA and protein). In a cardiac cell model of hypoxia/reoxygenation (H/R), allopurinol or XO siRNA reduced H/R injury concomitant with decreased XO activity, VPO1 expression as well as the XO and VPO1-derived products (H2O2, uric acid, and HOCl). Although knockdown of VPO1 could also exert a beneficial effect on H/R injury, it did not affect XO activity, XO expression, and XO-derived products. Based on these observations, we conclude that the novel pathway of XO/VPO1 is responsible for, at least partly, myocardial I/R-induced oxidative injury, and allopurinol exerted the cardioprotective effects on myocardial I/R injury via inhibiting the XO/VPO1 pathway.


Asunto(s)
Alopurinol , Xantina Oxidasa , Animales , Peróxido de Hidrógeno , Ratas
7.
Proc Natl Acad Sci U S A ; 118(31)2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34312235

RESUMEN

Abdominal aortic aneurysm (AAA) is characterized by aorta dilation due to wall degeneration, which mostly occurs in elderly males. Vascular aging is implicated in degenerative vascular pathologies, including AAA. Cyclic nucleotide phosphodiesterases, by hydrolyzing cyclic nucleotides, play critical roles in regulating vascular structure remodeling and function. Cyclic nucleotide phosphodiesterase 1C (PDE1C) expression is induced in dedifferentiated and aging vascular smooth muscle cells (SMCs), while little is known about the role of PDE1C in aneurysm. We observed that PDE1C was not expressed in normal aorta but highly induced in SMC-like cells in human and murine AAA. In mouse AAA models induced by Angiotensin II or periaortic elastase, PDE1C deficiency significantly decreased AAA incidence, aortic dilation, and elastin degradation, which supported a causative role of PDE1C in AAA development in vivo. Pharmacological inhibition of PDE1C also significantly suppressed preestablished AAA. We showed that PDE1C depletion antagonized SMC senescence in vitro and/or in vivo, as assessed by multiple senescence biomarkers, including senescence-associated ß-galactosidase activity, γ-H2AX foci number, and p21 protein level. Interestingly, the role of PDE1C in SMC senescence in vitro and in vivo was dependent on Sirtuin 1 (SIRT1). Mechanistic studies further showed that cAMP derived from PDE1C inhibition stimulated SIRT1 activation, likely through a direct interaction between cAMP and SIRT1, which leads to subsequent up-regulation of SIRT1 expression. Our findings provide evidence that PDE1C elevation links SMC senescence to AAA development in both experimental animal models and human AAA, suggesting therapeutical significance of PDE1C as a potential target against aortic aneurysms.


Asunto(s)
Aneurisma de la Aorta Abdominal/enzimología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Angiotensina II/toxicidad , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Biomarcadores , Senescencia Celular , AMP Cíclico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Histonas , Masculino , Ratones , Ratones Noqueados para ApoE , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Regulación hacia Arriba , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
8.
Eur J Pharmacol ; 900: 174013, 2021 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-33766620

RESUMEN

RATIONALE: Higenamine (HG), is one of the main active components in many widely used Chinese herbs, and a common ingredient of health products in Europe and North America. Several groups, including our own, have previously shown the beneficial effects of HG against cardiomyocyte death during acute ischemic damage. However, the effect of HG on chronic cardiac remodeling, such as cardiac fibrosis, remains unknown. OBJECTIVE: Herein, we aim to investigate the role of HG in cardiac fibrosis in vivo as well as its cellular and molecular mechanisms. METHODS AND RESULTS: Chronic pressure overload with transverse aortic constriction (TAC) significantly increased cardiac hypertrophy, fibrosis, and cardiac dysfunction in mice, which were significantly attenuated by HG. Consistently, cardiac fibrosis induced by the chronic infusion of isoproterenol (ISO), was also significantly reduced by HG. Interestingly, our results showed that HG had no effect on adult mouse CM hypertrophy in vitro. However, HG suppressed the activation of cardiac fibroblasts (CFs) in vitro. Furthermore, TGF-ß1-induced expression of ACTA2, a marker of fibroblast activation, was significantly suppressed by HG. Concomitantly, HG inhibited TGF-ß1-induced phosphorylation of Smad2/3 in CFs. HG also reduced the expression of extracellular matrix molecules such as collagen I and collagen III. To our surprise, the inhibitory effect of HG on CFs activation was independent of the activation of the beta2 adrenergic receptor (ß2-AR) that is known to mediate the effect of HG on antagonizing CMs apoptosis. CONCLUSION: Our findings suggest that HG ameliorates pathological cardiac fibrosis and dysfunction at least partially by suppressing TGF-ß1/Smad signaling and CFs activation.


Asunto(s)
Alcaloides/farmacología , Fibrinolíticos/farmacología , Fibroblastos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/antagonistas & inhibidores , Tetrahidroisoquinolinas/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Actinas/antagonistas & inhibidores , Agonistas Adrenérgicos beta , Animales , Aorta/efectos de los fármacos , Apoptosis/efectos de los fármacos , Cardiomegalia/inducido químicamente , Cardiomegalia/prevención & control , Fibrosis/prevención & control , Cardiopatías/inducido químicamente , Cardiopatías/prevención & control , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Isoproterenol , Ratones , Ratas , Ratas Sprague-Dawley
9.
Talanta ; 209: 120507, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-31892003

RESUMEN

To overcome the poor conductivities and promote the application in the biosensors of metal-organic frameworks (MOFs), a simple approach was employed to improve their overall conductivity by adjusting the metal centers of MOFs and coating conductive polypyrrole (PPy) in the work. An unprecedented molybdenum oxide-based three-dimensional MOFs with helical channels (CuTRZMoO4) was synthesized based on MoO4-, Cu2+ ions and 1,2,3-trz for the first time, then combined with PPy to fabricate hybrid composites (CuTRZMoO4@PPy-n) with both advantages. The CuTRZMoO4 modified glassy carbon electrode show high sensitivity for detecting the neurotransmitter dopamine (DA), and the CuTRZMoO4@PPy-2 modified glassy carbon electrode has the highest catalytical activity to DA with the linear detection range from 1 µM to 100 µM and the detection limit of 80 nM (S/N = 3) by differential pulse voltammetry (DPV). Moreover, the developed biosensor has good selectivity, reproducibility and stability. The concept behinds the new architecture to modify electrodes should promote the further development of MOF-based biosensors.


Asunto(s)
Dopamina/sangre , Estructuras Metalorgánicas/química , Molibdeno/química , Nanocompuestos/química , Óxidos/química , Polímeros/química , Pirroles/química , Técnicas Biosensibles/métodos , Carbono/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Humanos , Límite de Detección , Estructuras Metalorgánicas/síntesis química
10.
Circulation ; 141(3): 217-233, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31801360

RESUMEN

BACKGROUND: Heart failure is a leading cause of death worldwide. Cyclic nucleotide phosphodiesterases (PDEs), through degradation of cyclic nucleotides, play critical roles in cardiovascular biology and disease. Our preliminary screening studies have revealed PDE10A upregulation in the diseased heart. However, the roles of PDE10A in cardiovascular biology and disease are largely uncharacterized. The current study is aimed to investigate the regulation and function of PDE10A in cardiac cells and in the progression of cardiac remodeling and dysfunction. METHODS: We used isolated adult mouse cardiac myocytes and fibroblasts, as well as preclinical mouse models of hypertrophy and heart failure. The PDE10A selective inhibitor TP-10, and global PDE10A knock out mice were used. RESULTS: We found that PDE10A expression remains relatively low in normal and exercised heart tissues. However, PDE10A is significantly upregulated in mouse and human failing hearts. In vitro, PDE10A deficiency or inhibiting PDE10A with selective inhibitor TP-10, attenuated cardiac myocyte pathological hypertrophy induced by Angiotensin II, phenylephrine, and isoproterenol, but did not affect cardiac myocyte physiological hypertrophy induced by IGF-1 (insulin-like growth factor 1). TP-10 also reduced TGF-ß (transforming growth factor-ß)-stimulated cardiac fibroblast activation, proliferation, migration and extracellular matrix synthesis. TP-10 treatment elevated both cAMP and cGMP levels in cardiac myocytes and cardiac fibroblasts, consistent with PDE10A as a cAMP/cGMP dual-specific PDE. In vivo, global PDE10A deficiency significantly attenuated myocardial hypertrophy, cardiac fibrosis, and dysfunction induced by chronic pressure overload via transverse aorta constriction or chronic neurohormonal stimulation via Angiotensin II infusion. Importantly, we demonstrated that the pharmacological effect of TP-10 is specifically through PDE10A inhibition. In addition, TP-10 is able to reverse pre-established cardiac hypertrophy and dysfunction. RNA-Sequencing and bioinformatics analysis further identified a PDE10A-regualted transcriptome involved in cardiac hypertrophy, fibrosis, and cardiomyopathy. CONCLUSIONS: Taken together, our study elucidates a novel role for PDE10A in the regulation of pathological cardiac remodeling and development of heart failure. Given that PDE10A has been proven to be a safe drug target, PDE10A inhibition may represent a novel therapeutic strategy for preventing and treating cardiac diseases associated with cardiac remodeling.


Asunto(s)
Cardiomegalia/enzimología , Fibroblastos/enzimología , Miocitos Cardíacos/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Remodelación Ventricular , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Modelos Animales de Enfermedad , Fibroblastos/patología , Ratones , Ratones Noqueados , Miocitos Cardíacos/patología , Hidrolasas Diéster Fosfóricas/genética , Transcriptoma
11.
World J Microbiol Biotechnol ; 35(2): 36, 2019 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-30712096

RESUMEN

In order to define the role of oxalic acid (OA) in the invasion of Botrytis cinerea in tomato plants, the OA induction of resistance related to oxalate oxidase (O×O) and germin was examined. In greenhouse experiments, OA at 3 mmol/L significantly induced resistance in tomato plants against B. cinerea strains B05.10 and T4, reducing lesion size of 37.55% and 24.91% by compared with distilled water control, respectively, while 20 mmol/L OA increasing by 36.14% and 41.48%. OA contents were 98 and 46 µg/mL when tomato plants were infected by B. cinerea strains B05.10 and T4, respectively. To define the molecular-genetic mechanisms, we compared the gene expression under four different conditions: 3 mmol/L OA-treated plants, 20 mmol/L OA-treated plants, B. cinerea strain B05.10-infected plants (B05.10 Inf plants) and B. cinerea strain T4-infected plants (T4 Inf plants). In 3 mmol/L OA-treated plants, the expressions of O×O and Germin peaked at 48 h after spraying, with approximate threefold and 18-fold increase compared with the control expression, respectively. In T4 Inf plants, the expression (mRNA accumulation) of O×O and Germin reached the highest levels at 24 h after inoculation, with 3- and 13-times that immediately after inoculation, respectively. In total, these findings suggest that elevated levels of OA correlated with increased fungal invasion and lower OA induced resistance in tomato plants by increasing expressions of O×O and Germin.


Asunto(s)
Botrytis/fisiología , Ácido Oxálico/inmunología , Enfermedades de las Plantas/microbiología , Solanum lycopersicum/inmunología , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Oxidorreductasas/genética , Oxidorreductasas/inmunología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología
12.
Circulation ; 138(18): 1988-2002, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-29871977

RESUMEN

BACKGROUND: cAMP plays a critical role in regulating cardiomyocyte survival. Various cAMP signaling pathways behave distinctly or in opposition. We have previously reported that activation of cAMP hydrolysis by cyclic nucleotide phosphodiesterase 1C (PDE1C) promotes cardiomyocytes death/apoptosis, yet the underlying molecular mechanism remains unknown. In this study, we aimed to identify the specific cAMP signaling pathway modulated by PDE1C and determine the mechanism by which Ca2+/calmodulin-stimulated PDE1C is activated. METHODS: To study cardiomyocyte death/apoptosis, we used both isolated mouse adult cardiomyocytes in vitro and doxorubicin-induced cardiotoxicity in vivo. We used a variety of pharmacological activators and inhibitors as well as genetically engineered molecular tools to manipulate the expression and activity of proteins of interest. RESULTS: We found that the protective effect of PDE1C inhibition/deficiency on Ang II or doxorubicin-induced cardiomyocyte death/apoptosis is dependent on cAMP-generating adenosine A2 receptors (A2Rs), suggesting that PDE1C's cAMP-hydrolyzing activity selectively modulates A2R-cAMP signaling in cardiomyocytes. In addition, we found that the effects of PDE1C activation on Ang II-mediated cAMP reduction and cardiomyocyte death are dependent on transient receptor potential-canonical (TRPC) channels, in particular TRPC3. We also observed synergistic protective effects on cardiomyocyte survival from the combination of A2R stimulation together with PDE1 or TRPC inhibition. Coimmunostaining and coimmunoprecipitation studies showed that PDE1C is localized in proximity with A2R and TRPC3 in the plasma membrane and perhaps T tubules. It is important to note that we found that doxorubicin-induced cardiac toxicity and dysfunction in mice are attenuated by the PDE1 inhibitor IC86340 or in PDE1C knockout mice, and this protective effect is significantly diminished by A2R antagonism. CONCLUSIONS: We have characterized a novel multiprotein complex comprised of A2R, PDE1C, and TRPC3, in which PDE1C is activated by TRPC3-derived Ca2+, thereby antagonizing A2R-cAMP signaling and promoting cardiomyocyte death/apoptosis. Targeting these molecules individually or in combination may represent a compelling therapeutic strategy for potentiating cardiomyocyte survival.


Asunto(s)
AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Receptores de Adenosina A2/metabolismo , Canales Catiónicos TRPC/metabolismo , Antagonistas del Receptor de Adenosina A2/farmacología , Angiotensina II/toxicidad , Animales , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Doxorrubicina/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Adenosina A2/química , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética
13.
Eur J Pharmacol ; 822: 1-12, 2018 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-29337194

RESUMEN

Fasudil is a potent Rho-kinase (ROCK) inhibitor and can relax smooth muscle or cardiac muscle contraction through decreasing the phosphorylation level of myosin regulatory light chain (p-MLC20 or p-MLC2v), while p-MLC2v can function as a transcription factor to promote the NADPH oxidase 2 (NOX2) expression in rat hearts subjected to ischemia/reperfusion (I/R). This study aims to explore whether fasudil can protect the rat hearts against I/R oxidative injury through suppressing NOX2 expression via reduction of p-MLC2v level. The SD rat hearts were subjected to 1h-ischemia plus 3h-reperfusion, which showed myocardial injuries (myocardial fiber loss and disarray, increase of creatine kinase release and myocardial infarction/apoptosis), increase in ROCK activity and nuclear p-MLC2v level concomitant with up-regulation of NOX2 and H2O2 production; these phenomena were attenuated by fasudil in a dose-dependent manner. Next, we verified the cardioprotective effect of fasudil and the underlying mechanisms in hypoxia-reoxygenation (H/R) -treated H9c2 cells. Consistent with the results in vivo, the H/R-treated H9c2 cells showed cellular injury (increase in apoptotic ratio), elevation in ROCK activity and nuclear p-MLC2v level, accompanied by up-regulation of NOX2 and H2O2 production; these effects were blocked in the presence of fasudil in a dose-dependent way. Based on these observations, we conclude that beneficial effect of fasudil against myocardial I/R or H/R oxidative injury is related to the suppression of NOX2 expression through decrease of the p-MLC2v level. Our findings also highlight that intervention of MLC2v phosphorylation by drugs may provide a novel strategy to protect heart from I/R oxidative injury.


Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Corazón/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , NADPH Oxidasa 2/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Miosinas Cardíacas/metabolismo , Línea Celular , Masculino , Miocardio/patología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Quinasas Asociadas a rho/antagonistas & inhibidores
14.
Eur J Pharmacol ; 819: 30-34, 2018 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-29183836

RESUMEN

Vinpocetine, a derivative of the alkaloid vincamine, has been clinically used in many countries for treatment of cerebrovascular disorders such as stroke and dementia for more than 30 years. Currently, vinpocetine is also available in the market as a dietary supplement to enhance cognition and memory. Due to its excellent safety profile, increasing efforts have been put into exploring the novel therapeutic effects and mechanism of actions of vinpocetine in various cell types and disease models. Recent studies have revealed a number of novel functions of vinpocetine, including anti-inflammation, antagonizing injury-induced vascular remodeling and high-fat-diet-induced atherosclerosis, as well as attenuating pathological cardiac remodeling. These novel findings may facilitate the repositioning of vinpocetine for preventing or treating relevant disorders in humans.


Asunto(s)
Alcaloides de la Vinca , Animales , Cardiopatías/tratamiento farmacológico , Humanos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades Vasculares/tratamiento farmacológico , Alcaloides de la Vinca/farmacología , Alcaloides de la Vinca/uso terapéutico
15.
Cardiovasc Drugs Ther ; 31(2): 157-166, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28321644

RESUMEN

PURPOSE: Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). METHODS: Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. RESULTS: We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFß-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). CONCLUSIONS: Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.


Asunto(s)
Cardiomegalia/prevención & control , Cardiomiopatías/prevención & control , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Remodelación Ventricular/efectos de los fármacos , Alcaloides de la Vinca/farmacología , Angiotensina II , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/enzimología , Cardiomegalia/patología , Cardiomiopatías/inducido químicamente , Cardiomiopatías/enzimología , Cardiomiopatías/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Transducción de Señal/efectos de los fármacos
16.
Proc Natl Acad Sci U S A ; 113(45): E7116-E7125, 2016 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-27791092

RESUMEN

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-ß-stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.

17.
Pharmacol Res ; 104: 115-23, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26746354

RESUMEN

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (ß2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by ß2-AR but not ß1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a ß2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the ß2-AR/PI3K/AKT cascade.


Asunto(s)
Alcaloides/farmacología , Cardiotónicos/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Tetrahidroisoquinolinas/farmacología , Alcaloides/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Cardiotónicos/uso terapéutico , Caspasa 3/metabolismo , Células Cultivadas , Peróxido de Hidrógeno , Masculino , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/efectos de los fármacos , Tetrahidroisoquinolinas/uso terapéutico
18.
Basic Res Cardiol ; 110(4): 38, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25982880

RESUMEN

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.


Asunto(s)
Miosinas Cardíacas/fisiología , Glicoproteínas de Membrana/genética , Miocardio/metabolismo , Cadenas Ligeras de Miosina/fisiología , NADPH Oxidasas/genética , Animales , Miosinas Cardíacas/análisis , Núcleo Celular/química , Células Cultivadas , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Cadenas Ligeras de Miosina/análisis , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , NADPH Oxidasa 2 , Estrés Oxidativo , Fosforilación , Ratas , Ratas Sprague-Dawley
19.
Free Radic Biol Med ; 83: 115-28, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25701432

RESUMEN

Nuclear myosin regulates gene transcription and this novel function might be modulated through phosphorylation of the myosin regulatory light chain (p-MLC20). Nonmuscle MLC20 (nmMLC20) is also present in the nuclei of cardiomyocytes and a potential nmMLC20 binding sequence has been identified in the promoter of the xanthine oxidase (XO) gene. Thus, we investigated its function in the regulation of XO transcription after myocardial ischemia/reperfusion (IR). In a rat model of myocardial IR and a cardiomyocyte model of hypoxia/reoxygenation (HR) injury, the cardiac or cell injury, myosin light chain kinase (MLCK) content, XO expression and activity, XO-derived products, and level of nuclear p-nmMLC20 were detected. Coimmunoprecipitation (co-IP), chromatin immunoprecipitation, DNA pull-down, and luciferase reporter gene assays were used to decipher the molecular mechanisms through which nmMLC20 promotes XO expression. IR or HR treatment dramatically elevated nuclear p-nmMLC20 level, accompanied by increased XO expression, activity, and products (H2O2 and uric acid), as well as the IR or HR injury; these effects were ameliorated by inhibition of MLCK or knockdown of nmMLC20. Our findings from these experiments demonstrated that nuclear p-nmMLC20 binds to the consensus sequence GTCGCC in the XO gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and hence activates XO gene transcription. These results suggest that nuclear p-nmMLC20 plays an important role in IR/HR injury by transcriptionally upregulating XO gene expression to increase oxidative stress in myocardium. Our findings demonstrate nuclear nmMLC20 as a potential new therapeutic target to combat cardiac IR injury.


Asunto(s)
Núcleo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Isquemia Miocárdica/patología , Daño por Reperfusión Miocárdica/patología , Cadenas Ligeras de Miosina/metabolismo , Xantina Oxidasa/genética , Animales , Apoptosis , Western Blotting , Núcleo Celular/genética , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Peróxido de Hidrógeno/metabolismo , Inmunoprecipitación , Masculino , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Cadenas Ligeras de Miosina/genética , Estrés Oxidativo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xantina Oxidasa/metabolismo
20.
Can J Physiol Pharmacol ; 90(7): 851-62, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22702833

RESUMEN

Myeloperoxidase (MPO) is involved in myocardial ischemia-reperfusion (IR) injury and vascular peroxidase (VPO) is a newly identified isoform of MPO. This study was conducted to explore whether VPO is involved in IR-induced cardiac dysfunction and apoptosis. In a rat Langendorff model of myocardial IR, the cardiac function parameters (left ventricular pressure and the maximum derivatives of left ventricular pressure and coronary flow), creatine kinase (CK) activity, apoptosis, VPO1 activity were measured. In a cell (rat-heart-derived H9c2 cells) model of hypoxia-reoxygenation (HR), apoptosis, VPO activity, and VPO1 mRNA expression were examined. In isolated heart, IR caused a marked decrease in cardiac function and a significant increase in apoptosis, CK, and VPO activity. These effects were attenuated by pharmacologic inhibition of VPO. In vitro, pharmacologic inhibition of VPO activity or silencing of VPO1 expression significantly suppressed HR-induced cellular apoptosis. Our results suggest that increased VPO activity contributes to IR-induced cardiac dysfunction and inhibition of VPO activity may have the potential clinical value in protecting the myocardium against IR injury.


Asunto(s)
Apoptosis/fisiología , Cardiopatías/enzimología , Corazón/fisiopatología , Hemoproteínas/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/enzimología , Peroxidasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Catalasa/metabolismo , Células Cultivadas , Creatina Quinasa/antagonistas & inhibidores , Creatina Quinasa/metabolismo , Corazón/efectos de los fármacos , Cardiopatías/genética , Cardiopatías/patología , Hemoproteínas/genética , Hemoproteínas/metabolismo , Masculino , Reperfusión Miocárdica/métodos , Miocardio/enzimología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Peroxidasa/metabolismo , Peroxidasas/genética , Peroxidasas/metabolismo , Floroglucinol/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley
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