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1.
Biomolecules ; 13(2)2023 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-36830621

RESUMO

Several population studies have observed lower serum bilirubin levels in patients with non-alcoholic fatty liver disease (NAFLD). Yet, treatments to target this metabolic phenotype have not been explored. Therefore, we designed an N-Acetylgalactosamine (GalNAc) labeled RNAi to target the enzyme that clears bilirubin from the blood, the UGT1A1 glucuronyl enzyme (GNUR). In this study, male C57BL/6J mice were fed a high-fat diet (HFD, 60%) for 30 weeks to induce NAFLD and were treated subcutaneously with GNUR or sham (CTRL) once weekly for six weeks while continuing the HFD. The results show that GNUR treatments significantly raised plasma bilirubin levels and reduced plasma levels of the bilirubin catabolized product, urobilin. We show that GNUR decreased liver fat content and ceramide production via lipidomics and lowered fasting blood glucose and insulin levels. We performed extensive kinase activity analyses using our PamGene PamStation kinome technology and found a reorganization of the kinase pathways and a significant decrease in inflammatory mediators with GNUR versus CTRL treatments. These results demonstrate that GNUR increases plasma bilirubin and reduces plasma urobilin, reducing NAFLD and inflammation and improving overall liver health. These data indicate that UGT1A1 antagonism might serve as a treatment for NAFLD and may improve obesity-associated comorbidities.


Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Urobilina/metabolismo , Bilirrubina , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Transdução de Sinais , Lipídeos , Resistência à Insulina/genética
2.
Cureus ; 14(8): e27702, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36081977

RESUMO

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by venous or arterial thrombosis and/or pregnancy morbidity in the presence of persistent laboratory evidence of antiphospholipid antibodies (APL). APS can occur as a primary condition but can also occur in the presence of systemic lupus erythematosus (SLE) or other systemic autoimmune diseases such as rheumatoid arthritis (RA) or Sjogren's Syndrome. Our case focuses on a 21-year-old female with a history of "going numb and having no ability to speak" with a total of approximately 20 such episodes, with no known triggers for these episodes. A hypercoagulable profile was performed and indicated an elevation in lupus anticoagulant (LA), which was also positive at repeat testing after 12 weeks, meeting the criteria for APS. Oral contraceptive pills (OCP) were stopped immediately, and she was started on daily aspirin. When hematology was consulted and evaluated, the patient reported a history of possible transient ischemic attacks (TIA); however, there was no history of deep vein thrombosis (DVT), pulmonary embolism (PE), or miscarriages. Recommendations from hematology were to continue the daily aspirin but did not recommend the addition of anticoagulation therapy. Additional recommendations included avoiding risk factors for thrombosis such as the use of birth control pills, smoking, and a sedentary lifestyle or obesity. Given the young age of our patient, as well as multiple TIA associated with APS secondary to LA, the patient was started on anticoagulation contrary to hematology's recommendations.

3.
Nat Biotechnol ; 40(10): 1500-1508, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35654979

RESUMO

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.


Assuntos
Precursor de Proteína beta-Amiloide , Terapêutica com RNAi , Animais , Camundongos , Primatas/genética , Primatas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico
5.
Chem Commun (Camb) ; 55(35): 5139-5142, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30977478

RESUMO

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.


Assuntos
Morfolinos/química , RNA Interferente Pequeno/química , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Proteínas Argonautas/genética , Inativação Gênica , Humanos , Camundongos , Modelos Moleculares , Morfolinos/síntese química , Morfolinos/genética , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética
6.
Nucleic Acids Res ; 46(16): 8090-8104, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30107495

RESUMO

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cß-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and ß-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'ß-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cß-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cß-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'ß-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cß-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.


Assuntos
Oligonucleotídeos/química , Estabilidade de RNA/genética , RNA Interferente Pequeno/química , Termodinâmica , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Fosfatos/química , Interferência de RNA , Ribonucleases/química , Ribose/química , Uridina/química , Uridina/genética
7.
Mol Ther ; 26(3): 708-717, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29456020

RESUMO

Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.


Assuntos
Acetilgalactosamina , Interferência de RNA , RNA Interferente Pequeno , Acetilgalactosamina/química , Animais , Proteínas Argonautas/genética , Regulação da Expressão Gênica , Inativação Gênica , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética
8.
J Med Chem ; 61(3): 734-744, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29376650

RESUMO

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.


Assuntos
Organofosfonatos/química , Organofosfonatos/síntese química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/química , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Sequência de Bases , Técnicas de Química Sintética , Inativação Gênica , Camundongos , Modelos Moleculares , Domínios Proteicos , RNA Interferente Pequeno/genética , Estereoisomerismo
9.
J Am Chem Soc ; 139(41): 14542-14555, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-28937776

RESUMO

We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.


Assuntos
Inativação Gênica , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Ribonucleotídeos/genética , Termodinâmica , Uridina/química , Uridina/metabolismo
10.
J Am Chem Soc ; 139(25): 8537-8546, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28570818

RESUMO

Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.


Assuntos
Glicóis/química , Ácidos Nucleicos/química , RNA Interferente Pequeno/química , Animais , Cristalografia por Raios X , Inativação Gênica/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Modelos Biológicos , Ácidos Nucleicos Heteroduplexes/química , RNA Interferente Pequeno/farmacologia , Receptores de Albumina/efeitos dos fármacos , Temperatura
11.
Amyloid ; 23(2): 109-18, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27033334

RESUMO

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.


Assuntos
Neuropatias Amiloides Familiares/terapia , Fígado/metabolismo , Pré-Albumina/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Benzoxazóis/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica , Humanos , Fígado/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Pré-Albumina/genética , Pré-Albumina/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética
12.
Chembiochem ; 17(11): 985-9, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27121751

RESUMO

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.


Assuntos
Acetilgalactosamina/química , Organofosfonatos/química , Interferência de RNA , RNA Interferente Pequeno/química , Compostos de Vinila/química , Animais , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Cultivadas , Fator IX/antagonistas & inibidores , Fator IX/genética , Fator IX/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipoproteínas LDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Organofosfonatos/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de Transcrição/metabolismo , Compostos de Vinila/farmacologia
13.
Physiol Rep ; 4(3)2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26869681

RESUMO

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this in vitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.


Assuntos
Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Técnicas de Cultura de Órgãos/métodos , Músculos Papilares/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Eletrofisiologia/métodos , Ratos
14.
ACS Chem Biol ; 11(4): 953-60, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26675211

RESUMO

5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.


Assuntos
Inativação Gênica , Malonatos/química , RNA Interferente Pequeno/genética , Animais , Células Cultivadas , Camundongos , Fosforilação
15.
Int J Cardiol ; 203: 145-55, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26512830

RESUMO

Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.


Assuntos
Fosfatase 1 de Especificidade Dupla/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Ratos , Ratos Sprague-Dawley
16.
J Clin Exp Cardiolog ; 5(6): 314, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25485172

RESUMO

The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. However, mechanical overload is a pathogenesis or comorbidity existing in a variety of heart diseases, such as hypertension, aortic regurgitation and myocardial infarction. Physical stimuli sensed by cells are transmitted through intracellular signal transduction pathways resulting in altered physiological responses or pathological conditions. Emerging evidence from experimental studies indicate that ß1-integrin and the angiotensin II type I (AT1) receptor play critical roles as mechanosensors in the regulation of heart contraction, growth and leading to heart failure. Integrin link the extracellular matrix and the intracellular cytoskeleton to initiate the mechanical signalling, whereas, the AT1 receptor could be activated by mechanical stress through an angiotensin-II-independent mechanism. Recent studies show that both Integrin and AT1 receptor and their downstream signalling factors including MAPKs, AKT, FAK, ILK and GTPase regulate heart function in cardiac myocytes. In this review we describe the role of mechanical sensors residing within the plasma membrane, mechanical sensor induced downstream signalling factors and its potential roles in cardiac contraction and growth.

17.
Brain Imaging Behav ; 8(3): 335-45, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23070748

RESUMO

Restoration of motor function following stroke involves reorganization of motor output through intact pathways, with compensatory brain activity likely variable by task. One class of motor tasks, those involved in self-care, is particularly important in stroke rehabilitation. Identifying the brain areas that are engaged in self-care and how they reorganize after stroke may enable development of more effective rehabilitation strategies. We piloted a paradigm for functional MRI assessment of self-care activity. In two groups, young adults and older adults, two self-care tasks (buttoning and zipping) produce activation similar to a bimanual tapping task, with bilateral activation of primary and secondary motor cortices, primary sensory cortex, and cerebellum. Quantitative differences include more activation of sensorimotor cortex and cerebellum in buttoning than bimanual tapping. Pilot subjects with stroke showed greater superior parietal activity across tasks than controls, potentially representing an increased need for sensorimotor integration to perform motor tasks.


Assuntos
Atividades Cotidianas , Encéfalo/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Destreza Motora/fisiologia , Reabilitação do Acidente Vascular Cerebral , Acidente Vascular Cerebral/fisiopatologia , Adulto , Idoso , Envelhecimento/fisiologia , Mapeamento Encefálico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto Jovem
18.
Methods Mol Biol ; 1066: 45-56, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23955732

RESUMO

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.


Assuntos
Cálcio/análise , Fibroblastos/citologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Centrifugação com Gradiente de Concentração , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Coração/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
19.
Methods Mol Biol ; 1066: 57-66, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23955733

RESUMO

Mechanical stretch is a major factor for myocardial hypertrophy and heart failure. Stretch activates mechanical sensors from cardiac myocytes, leading to a series of signal transduction cascades, which can result in cell malfunction and remodeling. It is well known that mechanical stretch also induces the release of paracrine factors from cardiac fibroblasts, as well as myocytes. Due to complicated circumstance of heart tissue, it is difficult to fully investigate the characteristics of these factors in situ. Here we describe static stretch and conditioned medium experiments as methods to examine the function of paracrine factors between primary cultured cardiac myocytes and fibroblasts.


Assuntos
Fibroblastos/fisiologia , Miócitos Cardíacos/fisiologia , Comunicação Parácrina/fisiologia , Estresse Mecânico , Animais , Cardiomegalia , Células Cultivadas , Centrifugação com Gradiente de Concentração , Matriz Extracelular/metabolismo , Coração/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Musculares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley
20.
Int J Cardiol ; 168(4): 3884-95, 2013 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-23907041

RESUMO

BACKGROUND: Anthrax lethal toxin (LT), secreted by Bacillus anthracis, causes severe cardiac dysfunction by unknown mechanisms. LT specifically cleaves the docking domains of MAPKK (MEKs); thus, we hypothesized that LT directly impairs cardiac function through dysregulation of MAPK signaling mechanisms. METHODS AND RESULTS: In a time-course study of LT toxicity, echocardiography revealed acute diastolic heart failure accompanied by pulmonary regurgitation and left atrial dilation in adult Sprague-Dawley rats at time points corresponding to dysregulated JNK, phospholamban (PLB) and protein phosphatase 2A (PP2A) myocardial signaling. Using isolated rat ventricular myocytes, we identified the MEK7-JNK1-PP2A-PLB signaling axis to be important for regulation of intracellular calcium (Ca(2+)(i)) handling, PP2A activation and targeting of PP2A-B56α to Ca(2+)(i) handling proteins, such as PLB. Through a combination of gain-of-function and loss-of-function studies, we demonstrated that over-expression of MEK7 protects against LT-induced PP2A activation and Ca(2+)(i) dysregulation through activation of JNK1. Moreover, targeted phosphorylation of PLB-Thr(17) by Akt improved sarcoplasmic reticulum Ca(2+)(i) release and reuptake during LT toxicity. Co-immunoprecipitation experiments further revealed the pivotal role of MEK7-JNK-Akt complex formation for phosphorylation of PLB-Thr(17) during acute LT toxicity. CONCLUSIONS: Our findings support a cardiogenic mechanism of LT-induced diastolic dysfunction, by which LT disrupts JNK1 signaling and results in Ca(2+)(i) dysregulation through diminished phosphorylation of PLB by Akt and increased dephosphorylation of PLB by PP2A. Integration of the MEK7-JNK1 signaling module with Akt represents an important stress-activated signalosome that may confer protection to sustain cardiac contractility and maintain normal levels of Ca(2+)(i) through PLB-T(17) phosphorylation.


Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca Diastólica/induzido quimicamente , Insuficiência Cardíaca Diastólica/metabolismo , Transdução de Sinais/fisiologia , Doença Aguda , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Células Cultivadas , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
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