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1.
Circ Res ; 134(8): 1006-1022, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38506047

RESUMO

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases , Cardiopatias , Receptores Adrenérgicos , Animais , Feminino , Humanos , Masculino , Camundongos , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cardiopatias/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos/metabolismo , Regulação para Cima
2.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35149557

RESUMO

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via ß2- and ß3-adrenergic receptors (ß-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives ß-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.


Assuntos
Adipócitos/metabolismo , Histona Desacetilases/metabolismo , Lisina/metabolismo , Células 3T3-L1 , Acilação , Animais , Regulação da Expressão Gênica , Histona Desacetilases/genética , Humanos , Lisina/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
FASEB J ; 34(1): 555-570, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914652

RESUMO

Muscle wasting, or muscle atrophy, can occur with age, injury, and disease; it affects the quality of life and complicates treatment. Insulin-like growth factor 1 (IGF1) is a key positive regulator of muscle mass. The IGF1/Igf1 gene encodes multiple protein isoforms that differ in tissue expression, potency, and function, particularly in cellular proliferation and differentiation, as well as in systemic versus localized signaling. Genome engineering is a novel strategy for increasing gene expression and has the potential to recapitulate the diverse biology seen in IGF1 signaling through the overexpression of multiple IGF1 isoforms. Using a CRISPR-Cas9 gene activation approach, we showed that the expression of multiple IGF1 or Igf1 mRNA variants can be increased in human and mouse skeletal muscle myoblast cell lines using a single-guide RNA (sgRNA). We found increased IGF1 protein levels in the cell culture media and increased cellular phosphorylation of AKT1, the main effector of IGF1 signaling. We also showed that the expression of Class 1 or Class 2 mRNA variants can be selectively increased by changing the sgRNA target location. The expression of multiple IGF1 or Igf1 mRNA transcript variants in human and mouse skeletal muscle myoblasts promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro. Our findings suggest that this novel approach for enhancing IGF1 signaling has potential therapeutic applications for treating skeletal muscle atrophy.


Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/citologia , Atrofia Muscular/patologia , Mioblastos/citologia , Ativação Transcricional , Animais , Anti-Inflamatórios/farmacologia , Sequência de Bases , Proliferação de Células , Células Cultivadas , Dexametasona/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosforilação , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência , Transdução de Sinais
4.
J Mol Cell Cardiol ; 138: 12-22, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678351

RESUMO

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.


Assuntos
Relógios Biológicos , Diferenciação Celular , Miocárdio/citologia , Células-Tronco/citologia , Conexinas/metabolismo , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fatores de Transcrição/metabolismo , Transcriptoma/genética
5.
Am J Physiol Heart Circ Physiol ; 317(4): H793-H810, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31441691

RESUMO

Gravin, an A-kinase anchoring protein, is known to play a role in regulating key processes that lead to inflammation and atherosclerosis development, namely, cell migration, proliferation, and apoptosis. We investigated the role of gravin in the development of high-fat diet (HFD)-induced atherosclerosis and hyperlipidemia. Five-week-old male wild-type (WT) and gravin-t/t mice were fed a normal diet or an HFD for 16 wk. Gravin-t/t mice showed significantly lower liver-to-body-weight ratio, cholesterol, triglyceride, and very low-density lipoprotein levels in serum as compared with WT mice on HFD. Furthermore, there was less aortic plaque formation coupled with decreased lipid accumulation and liver damage, as the gravin-t/t mice had lower levels of serum alanine aminotransferase and aspartate aminotransferase. Additionally, gravin-t/t HFD-fed mice had decreased expression of liver 3-hydroxy-3-methyl-glutaryl-CoA reductase, an essential enzyme for cholesterol synthesis and lower fatty acid synthase expression. Gravin-t/t HFD-fed mice also exhibited inhibition of sterol regulatory element binding protein-2 (SREBP-2) expression, a liver transcription factor associated with the regulation of lipid transportation. In response to platelet-derived growth factor receptor treatment, gravin-t/t vascular smooth muscle cells exhibited lower intracellular calcium transients and decreased protein kinase A- and protein kinase C-dependent substrate phosphorylation, notably involving the Erk1/2 signaling pathway. Collectively, these results suggest the involvement of gravin-dependent regulation of lipid metabolism via the reduction of SREBP-2 expression. The absence of gravin-mediated signaling lowers blood pressure, reduces plaque formation in the aorta, and decreases lipid accumulation and damage in the liver of HFD mice. Through these processes, the absence of gravin-mediated signaling complex delays the HFD-induced hyperlipidemia and atherosclerosis.NEW & NOTEWORTHY The gravin scaffolding protein plays a key role in the multiple enzymatic pathways of lipid metabolism. We have shown for the first time the novel role of gravin in regulating the pathways related to the initiation and progression of atherosclerosis. Specifically, an absence of gravin-mediated signaling decreases the lipid levels (cholesterol, triglyceride, and VLDL) that are associated with sterol regulatory element binding protein-2 downregulation.


Assuntos
Proteínas de Ancoragem à Quinase A/deficiência , Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Dieta Hiperlipídica , Hiperlipidemias/prevenção & controle , Lipídeos/sangue , Placa Aterosclerótica , Proteínas de Ancoragem à Quinase A/genética , Animais , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/etiologia , Doenças da Aorta/genética , Aterosclerose/sangue , Aterosclerose/etiologia , Aterosclerose/genética , Proteínas de Ciclo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Hiperlipidemias/genética , Fígado/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
6.
Am J Physiol Heart Circ Physiol ; 315(1): H109-H121, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29600899

RESUMO

One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction, which is regulated by a family of scaffolding proteins, i.e., A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase 4D3 (PDE4D3), among other proteins, and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Single-nucleotide polymorphisms (SNPs) in various proteins have especially been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter the cAMP/PKA pathway, influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease-related patients with the highest predicted deleterious effects, Ser 1653 Arg (S1653R) and Glu 2124 Gly (E2124G). Coimmunoprecipitation data in human embryonic kidney-293T cells showed that the S1653R SNP, present in the PDE4D3-binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and that the E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity, and cytosolic PDE activity compared with the wild type before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be upregulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling, supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions. NEW & NOTEWORTHY We found that single-nucleotide polymorphisms in muscle-specific A-kinase anchoring protein found in human patients with cardiovascular diseases significantly affect the cAMP/PKA signaling pathway. Our results showed, for the first time, that human muscle-specific A-kinase anchoring protein polymorphisms might alter the susceptibility of individuals to develop cardiovascular diseases with known underlying molecular mechanisms.


Assuntos
Proteínas de Ancoragem à Quinase A/genética , Doenças Cardiovasculares/genética , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sítios de Ligação , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Células HEK293 , Humanos , Ligação Proteica
7.
Proc Natl Acad Sci U S A ; 112(29): 9046-51, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26153423

RESUMO

Homozygous cardiac myosin binding protein C-deficient (Mybpc(t/t)) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc(t/t) myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc(t/t) myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc(t/t) mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3(+/-) individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3(-/-) mice is primarily myocyte hyperplasia.


Assuntos
Proteínas de Transporte/metabolismo , Citocinese , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Aurora Quinases/metabolismo , Biomarcadores/metabolismo , Cálcio/metabolismo , Contagem de Células , Diferenciação Celular , Proliferação de Células , Dependovirus/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Histonas/metabolismo , Humanos , Indóis/metabolismo , Camundongos , Modelos Biológicos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Fosforilação , RNA Interferente Pequeno/metabolismo
8.
Heart Surg Forum ; 19(1): E16-22, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26913679

RESUMO

BACKGROUND: Renal dysfunction is a common complication after cardiovascular surgery. Controversial issues have been discussed regarding the role of N-acetyl cysteine in the prevention of postoperative renal dysfunction. The purpose of this meta-analysis is to assess whether N-acetyl cysteine offers any protection against the development of acute renal dysfunction after cardiac surgery. METHODS: Multiple databases were searched for randomized trials comparing the role of N-acetyl cysteine and placebo in human patients undergoing cardiac surgery. End-points studied were: the incidence of acute renal failure, hemodialysis, early mortality, duration of hospital stay, and maximal change in creatinine values. Dichotomous variables were compared using the risk difference (RD) calculated with inverse weighting; continuous data was pooled as (standardized) mean difference. Results are presented with 95% confidence interval (P < .05 is significant); results are presented within 95% confidence interval. RESULTS: Thirteen randomized trials (713 and 707 patients in the N-acetyl cysteine and control groups, respectively) were included in the present analysis; nine dealing with patients at high-risk for acute renal failure. The incidence of postoperative acute renal dysfunction was 23% and 36% in the N-acetyl cysteine and control cohorts, respectively. N-acetyl cysteine therapy did not reduce acute renal dysfunction in the high-risk cohort [RD -0.03 (-0.09 to 0.02); P = .22; I2 = 24%]. Maximal change in creatinine levels after surgery was also comparable [standardized mean difference 0.07 (-0.23, 0.09); P = .39]. Early mortality was 2.9% and 3.7% in the N-acetyl cysteine and control cohorts respectively; [RD 0 (-0.03 to 0.02); P = .63; I2 = 20%]. Hospital stay (mean length of stay 10.4 and 10.1 days in the N-acetyl cysteine and control cohorts, respectively) was also similar in both cohorts [WMD 0.17 (-0.02 to 0.37) days; P = .81]. CONCLUSION: Prophylactic N-acetyl cysteine therapy does not reduce the incidence of renal dysfunction in high-risk patients undergoing cardiac surgery.


Assuntos
Acetilcisteína/uso terapêutico , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/prevenção & controle , Procedimentos Cirúrgicos Cardíacos/mortalidade , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/prevenção & controle , Idoso , Procedimentos Cirúrgicos Cardíacos/estatística & dados numéricos , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/uso terapêutico , Mortalidade Hospitalar , Hospitalização/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Ensaios Clínicos Controlados Aleatórios como Assunto , Fármacos Renais , Fatores de Risco , Taxa de Sobrevida , Falha de Tratamento , Resultado do Tratamento
10.
Int J Mol Sci ; 16(1): 218-29, 2014 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-25547489

RESUMO

The Beta-adrenergic receptors (ß-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure-function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cardiomegalia/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Animais , Humanos , Transporte Proteico
11.
Biomedicines ; 11(3)2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36979895

RESUMO

The COVID-19 pandemic has underscored the urgent need to develop highly potent and safe medications that are complementary to the role of vaccines. Specifically, it has exhibited the need for orally bioavailable broad-spectrum antivirals that are able to be quickly deployed against newly emerging viral pathogens. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and its variants Delta and Omicron are still a major threat to patients of all ages. In this brief report, we describe that the small molecule CD04872SC was able to neutralize SARS-CoV2 infection with a half-maximal effective concentration (EC50) = 248 µM. Serendipitously, we also were able to observe that CD04872SC inhibited the infection of the SARS-CoV-2 variants; Delta (EC50 = 152 µM) and Omicron (EC50 = 308 µM). These properties may define CD04872SC as a potential broad-spectrum candidate lead for the development of treatments for COVID-19.

12.
FASEB Bioadv ; 4(12): 758-774, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36479208

RESUMO

The ß2AR is a prototypical G protein-coupled receptor (GPCR) known to orchestrate different cellular responses by the stimulation of specific signaling pathways. The best-established signaling pathways for the ß2AR are the canonical Gs pathway and the alternative ß arrestin 2 (ßarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the ß2AR with its cognate transducers, Gαs and ßarr2, using short linkers as a novel approach for restricting the conformation of the receptor and preferentially activating one of its two signaling pathways. We characterized the behavior of our fusion proteins ß2AR-Gαs and ß2AR-ßarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacological modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the ß2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by ß2AR ligands. Based on these characteristics, we further explored the possibility of a gain-of-function mechanism in the human lung non-tumorigenic epithelial cell line, BEAS-2B cells. This immortalized human bronchial epithelial cell line has immunomodulatory properties through cytokine release mediated by ß2AR stimulation. Our findings suggest that each signaling pathway of the ß2AR is biased toward either the Th1 or Th2 inflammatory response suggesting a role in regulating the immune phenotype of respiratory diseases. Our data imply that our fusion proteins can be used as tools to isolate the function elicited by a single signaling pathway in physiologically relevant cell types.

13.
J Cardiovasc Aging ; 2(3)2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35891703

RESUMO

Introduction: The adult heart lacks the regenerative capacity to self-repair. Serum response factor (SRF) is essential for heart organogenesis, sarcomerogenesis, and contractility. SRF interacts with co-factors, such as NKX2.5 and GATA4, required for cardiac specified gene activity. ETS factors such as ELK1 interact with SRF and drive cell replication. To weaken SRF interactions with NKX2.5 and GATA4, one mutant, SRF153(A3) named STEMIN, did not bind CArG boxes, yet induced stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. The mutant YAP5SA of the Hippo pathway also promotes cardiomyocyte proliferation and growth. Aim: Infarcted adult mouse hearts were injected with translatable STEMIN and YAP5SA mmRNA to evaluate their clinical potential. Methods and Results: Mice were pulsed one day later with alpha-EDU and then heart sections were DAPI stained. Replicating cells were identified by immuno-staining against members of the DNA replisome pathway that mark entry to S phase of the cell cycle. Echocardiography was used to determine cardiac function following infarcts and mRNA treatment. To monitor cardiac wall repair, microscopic analysis was performed, and the extent of myocardial fibrosis was analyzed for immune cell infiltration. Injections of STEMIN and YAP5SA mmRNA into the left ventricles of infarcted adult mice promoted a greater than 17-fold increase in the DAPI stained and alpha-EDU marked cardiomyocyte nuclei, within a day. We observed de novo expression of phospho-histone H3, ORC2, MCM2, and CLASPIN. Cardiac function was significantly improved by four weeks post-infarct, and fibrosis and immune cell infiltration were diminished in hearts treated with STEMIN and YAP5SA mmRNA than each alone. Conclusion: STEMIN and YAP5SA mmRNA improved cardiac function and myocardial fibrosis in left ventricles of infarcted adult mice. The combinatorial use of mmRNA encoding STEMIN and YAP5SA has the potential to become a powerful clinical strategy to treat human heart disease.

14.
Commun Biol ; 5(1): 212, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260793

RESUMO

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.


Assuntos
Descoberta de Drogas/métodos , Proteínas de Membrana , Transporte Proteico/fisiologia , Espectrometria de Fluorescência/métodos , COVID-19 , Desenvolvimento de Medicamentos/métodos , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Nanotecnologia , Receptores Acoplados a Proteínas G , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Internalização do Vírus
15.
iScience ; 25(11): 105402, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36388958

RESUMO

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

16.
J Endocr Soc ; 6(9): bvac109, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37283844

RESUMO

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

17.
Cardiovasc Drugs Ther ; 25(3): 223-32, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21562974

RESUMO

INTRODUCTION: The present study determined whether late-ischemia/early reperfusion therapy with the ß(1)-adrenergic receptor (AR) blocker esmolol and phosphodiesterase III inhibitor milrinone reduced left ventricular (LV) myocardial infarct size (IS). METHODS AND RESULTS: In an ischemia/reperfusion rat model (30-min ischemia/4-hr reperfusion), esmolol, milrinone or esmolol + milrinone were intravenous (IV) infused over 10 min (from the last 5 min of ischemia to the first 5 min of reperfusion). LV-IS were 48.9 ± 8.9%, 41.5 ± 5.4%, 25.8 ± 7.7% and 16.8 ± 7.3% for saline, esmolol, milrinone, and esmolol + milrinone, respectively (n = 12/group). Esmolol + milrinone further reduced LV-IS compared with esmolol or milrinone alone (p < 0.05). LV-IS-reduction induced by esmolol + milrinone was eliminated in the presence of protein kinase A-(PKA)-inhibitor (Rp-cAMPS) or Akt-inhibitor (AKT 1/2 kinase inhibitor). In mixed rat ventricular cardiomyocyte cultures, intra-ischemic application of esmolol, milrinone or esmolol + milrinone reduced myocyte death rates by 5.5%, 13.3%, and 16.8%, respectively, compared with saline (p < 0.01). This cell protective effect by esmolol + milrinone was abrogated in the presence of PKA-inhibitor or Akt-inhibitor. Esmolol, milrinone or esmolol + milrinone increased myocardial PKA activity by 22%, 28% and 59%, respectively, compared with saline (n = 6, p < 0.01). No non-specific adverse effect of Rp-cAMPS on myocytes was identified in a purified myocyte preparation during hypoxia/re-oxygenation. Antiapoptotic pathways were assessed by measuring myocardial phosphorylated Akt (pAkt) levels combined with terminal dUTP nick-end labelling staining analysis. Ten minutes following infusion of esmolol, milrinone or esmolol + milrinone, there were 1.7-, 2.7-, and 6-fold increase in tissue pAkt levels, respectively. This esmolol + milrinone induced pAkt activation was abolished in the presence of PKA inhibitor. Esmolol, milrinone and esmolol + milrinone reduced myocyte apoptosis rates by 22%, 37% and 60%, respectively, compared with saline (p < 0.01). CONCLUSIONS: Late-ischemia/early reperfusion therapy with esmolol + milrinone additively reduces LV-IS associated with robust activation of myocardial PKA and subsequent Akt-antiapoptotic pathway.


Assuntos
Cardiotônicos/farmacologia , Milrinona/farmacologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Propanolaminas/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1/administração & dosagem , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiotônicos/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Infusões Intravenosas , Milrinona/administração & dosagem , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidores da Fosfodiesterase 3/administração & dosagem , Inibidores da Fosfodiesterase 3/farmacologia , Propanolaminas/administração & dosagem , Ratos
18.
J Cachexia Sarcopenia Muscle ; 12(6): 2174-2186, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34704401

RESUMO

BACKGROUND: Skeletal muscle atrophy is a debilitating complication of many chronic diseases, disuse conditions, and ageing. Genome-wide gene expression analyses have identified that elevated levels of microRNAs encoded by the H19X locus are among the most significant changes in skeletal muscles in a wide scope of human cachectic conditions. We have previously reported that the H19X locus is important for the establishment of striated muscle fate during embryogenesis. However, the role of H19X-encoded microRNAs in regulating skeletal mass in adults is unknown. METHODS: We have created a transgenic mouse strain in which ectopic expression of miR-322/miR-503 is driven by the skeletal muscle-specific muscle creatine kinase promoter. We also used an H19X mutant mouse strain in which transcription from the locus is interrupted by a gene trap. Animal phenotypes were analysed by standard histological methods. Underlying mechanisms were explored by using transcriptome profiling and validated in the two animal models and cultured myotubes. RESULTS: Our results demonstrate that the levels of H19X microRNAs are inversely related to postnatal skeletal muscle growth. Targeted overexpression of miR-322/miR-503 impeded skeletal muscle growth. The weight of gastrocnemius muscles of transgenic mice was only 54.5% of the counterparts of wild-type littermates. By contrast, interruption of transcription from the H19X locus stimulates postnatal muscle growth by 14.4-14.9% and attenuates the loss of skeletal muscle mass in response to starvation by 12.8-21.0%. Impeded muscle growth was not caused by impaired IGF1/AKT/mTOR signalling or a hyperactive ubiquitin-proteasome system, instead accompanied by markedly dropped abundance of translation initiation factors in transgenic mice. miR-322/miR-503 directly targets eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M. CONCLUSIONS: Our study illustrates a novel pathway wherein H19X microRNAs regulate skeletal muscle growth and atrophy through regulating the abundance of translation initiation factors, thereby protein synthesis. The study highlights how translation initiation factors lie at the crux of multiple signalling pathways that control skeletal muscle mass.


Assuntos
MicroRNAs , Atrofia Muscular , Animais , Camundongos , MicroRNAs/genética , Fibras Musculares Esqueléticas , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Fatores de Iniciação de Peptídeos
19.
J Am Heart Assoc ; 9(13): e016615, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32573313

RESUMO

Heart failure is a complex clinical syndrome, represented as an impairment in ventricular filling and myocardial blood ejection. As such, heart failure is one of the leading causes of death in the United States. With a mortality rate of 1 per 8 individuals and a prevalence of 6.2 million Americans, it has been projected that heart failure prevalence will increase by 46% by 2030. Cardiac remodeling (a general determinant of heart failure) is regulated by an extensive network of intertwined intracellular signaling pathways. The ability of signalosomes (molecular signaling complexes) to compartmentalize several cellular pathways has been recently established. These signalosome signaling complexes provide an additional level of specificity to general signaling pathways by regulating the association of upstream signals with downstream effector molecules. In cardiac myocytes, the AKAP12 (A-kinase anchoring protein 12) scaffolds a large signalosome that orchestrates spatiotemporal signaling through stabilizing pools of phosphatases and kinases. Predominantly upon ß-AR (ß2-adrenergic-receptor) stimulation, the AKAP12 signalosome is recruited near the plasma membrane and binds tightly to ß-AR. Thus, one major function of AKAP12 is compartmentalizing PKA (protein kinase A) signaling near the plasma membrane. In addition, it is involved in regulating desensitization, downregulation, and recycling of ß-AR. In this review, the critical roles of AKAP12 as a scaffold protein in mediating signaling downstream GPCRs (G protein-coupled receptor) are discussed with an emphasis on its reported and potential roles in cardiovascular disease initiation and progression.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Insuficiência Cardíaca/enzimologia , Miocárdio/enzimologia , Sistemas do Segundo Mensageiro , Função Ventricular , Animais , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Miocárdio/patologia , Receptores Adrenérgicos beta 2/metabolismo
20.
Fundam Clin Pharmacol ; 33(1): 25-30, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29959870

RESUMO

Inbred mouse strains are the most widely used mammalian model organism in biomedical research owing to ease of genetic manipulation and short lifespan; however, each inbred strain possesses a unique repertoire of deleterious homozygous alleles that can make a specific strain more susceptible to a particular disease. In the current study, we report dystrophic cardiac calcinosis (DCC) in C.B-17 SCID male mice at 10 weeks of age with no significant change in cardiac function. Acquisition of DCC was characterized by myocardial injury, fibrosis, calcification, and necrosis of the tissue. At 10 weeks of age, 38% of the C.B-17 SCID mice from two different commercial colonies exhibited significant calcinosis on the ventricular epicardium, predominantly on the right ventricle. The frequency of calcinosis was more than 50% for mice obtained from Taconic's Cambridge City colony and 25% for mice obtained from Taconic's German Town colony. Interestingly, the DCC phenotype did not affect cardiac function at 10 weeks of age. No differences in echocardiography or electrocardiography were observed between the calcinotic and non-calcinotic mice from either colony. Our findings suggest that C.B-17 SCID mice exhibit DCC as early as 10 weeks of age with no significant impact on cardiac function. This strain of mice should be cautiously considered for the study of cardiac physiology.


Assuntos
Calcinose/patologia , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Pericárdio/patologia , Animais , Ecocardiografia/métodos , Eletrocardiografia/métodos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Fenótipo
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