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The sympathetic nervous system is essential for maintenance of cardiac function via activation of post-junctional adrenergic receptors. Prolonged adrenergic receptor activation, however, has deleterious long-term effects leading to hypertrophy and the development of heart failure. Here we investigate the effect of chronic adrenergic receptors activation on excitation-contraction coupling (ECC) in ventricular cardiomyocytes from a previously characterized mouse model of chronic sympathetic hyperactivity, which are genetically deficient in the adrenoceptor α2A and α2C genes (ARDKO). When compared to wild-type (WT) cardiomyocytes, ARDKO displayed reduced fractional shortening (~33%) and slower relaxation (~20%). Furthermore, ARDKO cells exhibited several electrophysiological changes such as action potential (AP) prolongation (~50%), reduced L-type calcium channel (LCC) current (~33%), reduced outward potassium (K+) currents (~30%), and increased sodium/calcium exchanger (NCX) activity (~52%). Consistent with reduced contractility and calcium (Ca2+) currents, the cytosolic Ca2+ ([Ca2+]i) transient from ARDKO animals was smaller and decayed slower. Importantly, no changes were observed in membrane resting potential, AP amplitude, or the inward K+ current. Finally, we modified our existing cardiac ECC computational model to account for changes in the ARDKO heart. Simulations suggest that cellular changes in the ARDKO heart resulted in variable and dyssynchronous Ca2+-induced Ca2+ release therefore altering [Ca2+]i transient dynamics and reducing force generation. In conclusion, chronic sympathetic hyperactivity impairs ECC by changing the density of several ionic currents (and thus AP repolarization) causing altered Ca2+ dynamics and contractile activity. This demonstrates the important role of ECC remodeling in the cardiac dysfunction secondary to chronic sympathetic activity.

Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O advento das células-tronco humanas pluripotentes induzidas (hiPSC ou iPSC's) causou uma grande expectativa no campo científico e clínico, pois são derivadas de células somáticas, crescem indefinidamente in vitro sem perder sua "pluripotenticidade" e tem enorme potencial de diferenciação, podendo produzir diversos tipos celulares tais como miócitos cardíacos. Apesar dos imensos esforços para desenvolver uma terapia baseada em células tronco, os cardiomiócitos derivados de iPSC's (iPSC’s-CM’s) possuem função basal ainda aquém de células adultas do tecido cardíaco. Nesse trabalho propomos que o protocolo de maturação que consiste da simples suplementação do meio de cultura com hormônio triiodotironina (T3), fator de crescimento semelhante à insulina tipo 1 (IGF-1) e dexametasona, aqui chamado de TDI seja suficiente para o amadurecimento celular. O tratamento com TDI induziu um aumento na área celular, alongamento das células e melhor organização dos sarcômeros. Além disso, foi observado um aumento na expressão de mRNA para alguns marcadores de maturação como MYH7, FKBP5 e PPARα. Quando comparadas com células sem o tratamento, as células expostas ao TDI tinham um transiente de cálcio de maior amplitude e um decaimento mais rápido do mesmo, além de um potencial de ação mais prolongado. Foi observado que essas células também tinham maior carga de cálcio no reticulo sarcoplasmático. Isso pode estar associado ao aumento nos níveis de mRNA para a SERCA2A que foi observado nas iPSC’s-CM’s tratadas com o TDI. Essas células expostas ao TDI foram usadas para construção de microtecidos cardíaco, junto com células endoteliais e fibroblastos. Esses tecidos apresentaram maior força passiva e também maior força durante a contração desse tecido, corroborando com os dados obtidos da morfologia celular e manejo de cálcio. Em conclusão, o uso do TDI se provou um simples e robusto método de maturação de iPSC’s-CM’s e pode ser uma alternativa para obtenção de tecidos in vitro com maiores chances de integração funcional após sua enxertia em coração de pacientes. The advent of induced pluripotent human stem cells (hiPSC or iPSCs) has caused great expectations in the scientific and clinical field. Since they are derived from somatic cells, they can grow indefinitely in vitro without losing their pluripotency and have enormous potential of differentiation, being able to differentiate in many cell types such as cardiac myocytes. Despite the vast efforts to develop therapies based in stem cells, iPSC-derived cardiomyocytes (iPSCs-CMs) have impaired basal function compared with the adult cells in cardiac tissue. In this work, we propose a maturation protocol consisting of the simple supplementation of the culture medium with triiodothyronine (T3) hormone, insulin-like growth factor type 1 (IGF-1) and dexamethasone, here called TDI, is sufficient for cell maturation. Treatment with TDI induced an increase in cell area, cell elongation and better sarcomere organization. In addition, was observed an increase in mRNA expression for some maturation markers such as MYH7, FKBP5 and PPARα. When compared to non-treated cells, the cells exposed to TDI had a calcium transient larger in amplitude and with faster decay kinetics, with prolonged action potential as well. Also, these cells had larger calcium content on the sarcoplasmic reticulum. This may be associated with the increase in mRNA levels for SERCA2A that was observed in iPSC's-CM's treated with TDI. Furthermore, TDI-treated cells were used to mount cardiac microtissues (CMT), along with endothelial cells and fibroblasts. These tissues had enhanced static force and increase in dynamic force during the contraction of this tissue, corroborating with the data obtained from their cellular morphology and calcium handling. In conclusion, use of TDI has proved a simple and robust method of maturation of iPSC's-CM's and may be an alternative to obtaining cardiac tissues in vitro with better chance to successful engraftment in patients' hearts.

The monoterpenoid carvacrol (1) is present in many essential oils of plants and has attracted attention because of its beneficial biological activities, especially analgesic activity. However, the mechanism of action of 1 remains unknown. The present study aimed to explore the mechanisms whereby 1 produces its effects on the peripheral nervous system. Carvacrol reversibly blocked the excitability of the rat sciatic nerve in a concentration-dependent manner with an IC(50) value of 0.50 ± 0.04 mM. At 0.6 mM, 1 increased the rheobase from 3.30 ± 0.06 V to 4.16 ± 0.14 V and the chronaxy from 59.6 ± 1.22 μs to 75.0 ± 1.82 μs. Also, 1 blocked the generation of action potentials (IC(50) 0.36 ± 0.14 mM) of the intact dorsal root ganglion (DRG) neurons without altering the resting potential and input resistance. Carvacrol reduced the voltage-gated sodium current of dissociated DRG neurons (IC(50) 0.37 ± 0.05 mM). In this study it has been demonstrated that 1 blocks neuronal excitability by a direct inhibition of the voltage-gated sodium current, which suggests that this compound acts as a local anesthetic. The present findings add valuable information to help understand the mechanisms implicated in the analgesic activity of carvacrol.

1 RESUMO Este trabalho teve por objetivo estudar os efeitos da aplicacao de agua (CEa = 0,5 dS m-1) e/ou solucao salina (CEa = 5,0 dS m-1), alternadamente, nas duas fases iniciais de desenvolvimento do feijao de corda cv. Epace 10. O experimento foi realizado, em condicoes de ambiente protegido (telado), na Embrapa Agroindustria Tropical. As sementes foram colocadas em vasos com capacidade para 15 L dispostos em um delineamento de blocos ao acaso, com 4 tratamentos e quatro repeticoes. As plantas foram submetidas aos tratamentos: 1. irrigacao com agua ate o final da fase II; 2. irrigacao com solucao salina ate o final da fase II, 3. irrigacao com agua ate o final da fase I e solucao salina ate o final da fase II e 4. Irrigacao com solucao salina ate o final da fase I e agua ate o final da fase II, momento em que os dados foram coletados. O crescimento nao foi afetado pela aplicacao de solucao salina, independente da fase aplicada. Nos solutos compativeis verificou-se reducao nos teores de proteinas. PALAVRAS-CHAVE: Vigna unguiculata L. estresse salino, crescimento CALVET, A. S. F.; PINTO, C. de M.; LIMA, R. E. M.; MAIA-JOCA, R. P. M.; BEZERRA, M. A. GROWTH AND SOLUTE ACCUMULATION IN COWPEA IRRIGATED WITH WATERS RISING SALINITY IN DIFFERENT STAGES OF DEVELOPMENT 2 ABSTRACT This work aimed to study the effects of application of water (CEw=0,5 dS m-1) and saline solution (CEw = 5,0 dS m-1) on the two initial stages of cowpea (cv. Epace 10) development. The experiment was performed in greenhouse environmental conditions at EMBRAPA Agroindustria Tropical. The seeds were placed in vessels with 15 L capacity and arranged in randomized blocks design with four treatments and four replications. The plants were subjected to treatments: 1. Irrigation with water until the end of phase II; 2. Irrigation with saline solution until the end of phase II; 3. Irrigation with water until the end of phase I and saline solution until the end of phase II; 4. Irrigation with saline solution until the end of Phase I and water until the end of phase II. The plant growth was not affected by application of saline solution, regardless the applied phase. In the compatible solutes it was found a decrease in protein levels. KEYWORDS: Vigna unguiculata L. saline stress, growth