Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-19T11:34:49Z (GMT). No. of bitstreams: 1 Monteiro_MarinaCarneiro_M.pdf: 5677929 bytes, checksum: 5c42afe705a43b66a4505a6ecded7b7c (MD5) Previous issue date: 2011 / Resumo: A fração do conteúdo de Ca2+ do retículo sarcoplasmático (RS) liberada a cada contração (Fractional Release - FR) em miócitos cardíacos é regulada pela corrente de entrada de Ca2+ através da membrana celular pelos canais de Ca2+ tipo-L (ICa,L) e pelo conteúdo de Ca2+ do RS ([Ca2+]RS). Em trabalho anterior foi desenvolvido, no nosso laboratório, um modelo experimental denominado de modelo do RS funcionalmente isolado (MRSFI). Neste modelo, cardiomiócitos são perfundidos em solução sem Na+ e sem Ca2+, o que torna as suas membranas eletricamente inexcitáveis e inibe o transporte do íon pelo trocador Na+/Ca2+. As variações (transientes) da concentração intracelular de Ca2+ ([Ca2+]i) medidas com o indicador fluorescente Fluo-3 AM (5 ?M, 20 min, 24ºC) são evocadas por aplicação de pulsos rápidos (100 ms) de cafeína (10 mM). No presente trabalho, o MRSFI foi usado para estudo da relação entre FR e [Ca2+]RS na ausência do gatilho fisiológico (ICa,L) para liberação reticular de Ca2.... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (Fractional Release - FR) in cardiac myocytes is regulated by the transmembrane inward Ca2+ current through the L-type Ca2+ channel (ICa,L) and by the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR model (FISRM), previously developed in this laboratory, cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells electrically unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Variations in the intracellular Ca2+ concentration ([Ca2+]i) was measured with the Ca2+ indicator fluo-3 and Ca2+ transients due to SR release are evoked by pulse-like (100 ms duration) application of 10 mM caffeine. In the present work, the FISRM was used to study the relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for the release of Ca2+ from the SR.... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Abstract Background – The heart cell is a prime example of a system, in which numerous interconnected regulatory mechanisms affect the dynamic balance of cellular function. The function of the system emerges from the interactions of its components rather than from their individual properties. Thus, it is a challenging task to understand the causal relations within such a system, based on the analysis of experimental results. Facing this complexity, the systems biological approach has gained interest during recent years, since with using it we can make an effort to observe, quantitatively and simultaneously, multiple components and their interdependencies in biological networks. Methods and aims – One of the most important tools in systems biology is mathematical modelling. In this thesis, novel model components have been developed and existing components integrated to describe mathematically the calcium dynamics in cardiac myocytes with improved physiological accuracy. Special attention was paid to both the activity-dependent and automatic regulation of the dynamics. This enabled the quantitative analysis of the regulation’s role in both physiological and pathophysiological conditions. Results – Validation of the novel model components that describe the calcium transport mechanisms indicates that the developed schemes are accurate and applicable also beyond the normal physiological state of the cardiac myocyte. Results also highlight the importance of autoregulation of calcium dynamics in the excitation-contraction coupling. Furthermore, the analysis indicates that the CaMK-dependent regulation of the calcium uptake to and release from the sarcoplasmic reticulum calcium stores could have substantial roles as downstream effectors in beta-adrenergic stimulation. Conclusions – Results emphasize mathematical modelling as a valuable complement to experiments in understanding causal relations within complex biological systems such as the cardiac myocytes. That is, rigorous data integration with mathematical models can provide significant insight to the quantitative role of both the individual model components and the interconnected regulatory loops. This is especially true for the analysis of genetically engineered animal models, in which the intended modification is always accompanied by compensatory changes that can mask to a varying degree the actual phenomenon of interest.

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that β-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs). β-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. β-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ∼50% below basal levels at 3 h after β-AR stimulation. This decline in eIF-2a phosphorylation was prevented by β1-AR, not by β2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited β-ARstimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited β-AR-stimulated and thapsigargininduced apoptosis. In vivo, β-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased β-AR-stimulated apoptosis in the heart. Thus, β-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role.

Mutagenic heterocyclic amines are produced during the ordinary cooking of meats and fish. When metabolically activated, heterocyclic amines will form covalent adducts with DNA, which, if not repaired, may affect the flow of genetic information in a cell. It has been proposed previously that heterocyclic amine mutagens contribute to the incidence of dietary-related cancers because they cause somatic cell mutations and induce tumors in rodents and nonhuman primates. Recent work has shown that some cooked food mutagens preferentially produce DNA damage in heart cells, DNA adduct levels are directly related to dose and duration of mutagen exposure, the dietary damage persists for long intervals in cardiac tissue, and mitochondrial DNA is more vulnerable than nuclear DNA to these mutagens. Because cardiac myocytes are terminally differentiated cells that have lost their ability to divide, the capacity to repair DNA damage is a critical factor in the proper function of cardiomyocytes, and cardiac myocytes seem to have limited DNA repair capabilities. DNA damage formed by dietary components, such as heterocyclic amines, might accumulate with time because of inefficient repair and thereby affect heart cell function or viability. The possibility that dietary habits play a role in idiopathic cardiomyopathies and congestive heart disease should be explored in greater depth.

Aims: β-Adrenergic receptor (β-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3β (GSK-3β) and mitochondrial pathways. However, β-AR stimulation induces apoptosis only in a fraction (∼15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis. Methods and results: Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with β-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after β-AR stimulation. Inhibition of β1-AR and β2-AR subtypes inhibited β-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of β-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after β-AR stimulation (1.9 ± 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited β-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited β-AR-stimulated activation of GSK-3β and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination. Conclusion: β-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in β-AR-stimulated apoptosis, possibly via the inactivation of GSK-3β/JNK and mitochondrial pathways.

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Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 μm okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nm and a nearly full effect during metabolic inhibition at 1 μm concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

Exogenous ubiquitin (UB) plays a protective role in β-adrenergic receptor-stimulated and ischemia/reperfusion (I/R)-induced myocardial remodeling. Here, we report that UB treatment inhibits hypoxia/reoxygenation (H/R)-induced apoptosis in adult rat ventricular myocytes (ARVMs). The activation of Akt was elevated, whereas the activation of glycogen synthase kinase-3β was reduced in UB-treated cells post-H/R. The level of oxidative stress was lower, whereas the number of ARVMs with polarized mitochondria was significantly greater in the UB-treated samples. ARVMs express CXCR4 with majority of CXCR4 localized in the membrane fraction. CXCR4 antagonism using AMD3100, and siRNA-mediated knockdown of CXCR4 negated the protective effects of UB. Two mutated UB proteins (unable to bind CXCR4) had no effect on H/R-induced apoptosis, activation of Akt and GSK-3β, or oxidative stress. UB treatment enhanced mitochondrial biogenesis, and inhibition of mitochondrial fission using mdivi1 inhibited H/R-induced apoptosis. Ex vivo, UB treatment significantly decreased infarct size and improved functional recovery of the heart following global I/R. Activation of caspase-9, a key player of the mitochondrial death pathway, was significantly lower in UB-treated hearts post-I/R. UB, most likely acting via CXCR4, plays a protective role in H/R-induced myocyte apoptosis and myocardial I/R injury via modulation of mitochondrial homeostasis and the mitochondrial death pathway of apoptosis.