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During neuronal activity mitochondria alter cytosolic Ca2+ signaling by buffering then releasing Ca2+ in the cytosol. This calcium transport by mitochondria affects the amplitude, duration, and spacial profile of the Ca2+ signal in the cytosol of neurons. This buffering by mitochondria has been shown to affect a variety of neuronal functions including: neurotransmission, gene expression, cell excitability, and cell death. Recently, researchers discovered that the protein CCDC109A (mitochondrial Ca2+ uniporter) was the protein responsible for mitochondrial Ca2+ uptake. Using a genetic knockout (KO) mouse model for the mitochondrial Ca2+ uniporter (MCU) my research investigated the role of MCU in neuronal function. In cultured central and peripheral neurons, MCU-KO significantly reduced mitochondrial Ca2+ uptake while significantly increasing the amplitude of the cytosolic Ca2+ signal amplitude. Behaviorally, MCU-KO mice show a small but significant impairment in memory tasks: fear conditioning and Barnes maze. Using a maximal electroshock seizure threshold model of in vivo seizure activity my research found that MCU-KO significantly increases the threshold for maximal seizure activity in mice and significantly reduces seizure severity. In addition to mitochondrial Ca2+ uptake, my research also investigated the mechanisms involved in mitochondrial Ca2+ extrusion. The protein SLC8B1 (SLC24A6, NCLX) is the putative transporter responsible for the Na+/Ca2+ exchange, mitochondrial calcium extrusion. Using genetic NCLX-KO mice, our research found that in neurons NCLX contributes to cytosolic Ca2+ extrusion, but does seem to directly affect mitochondrial Ca2+ extrusion.

Mitochondria are dynamic organelles known primarily for their roles in oxidative metabolism and programmed cell death. Both of these processes are regulated by the mitochondrial matrix calcium concentration. Little is known about how mitochondrial calcium is regulated: no plant mitochondrial Ca2+-ATPase pumps or no mitochondrial Ca2+ channels have been identified to date. In addition, little is known concerning any physical interactions between mitochondria and endoplasmic reticulum (ER), an important cellular calcium store, and how these modulate cellular calcium fluxes. In this work stable transgenic Arabidopsis lines expressing fluorescent marker proteins were generated to allow visualisation of mitochondria and the ER in the same cells, and to measure mitochondrial calcium fluxes using aequorin. According to my results, there is a physical association between mitochondria and ER and this association cannot be disrupted by chemical treatments (latrunculin B, methyl viologen and antimycin A). As part of this work I identified an Arabidopsis gene, Mitochondrial Calcium Uptake 1 (MCU1), which encodes a protein with features that suggest a role in mitochondrial calcium dynamics. Fluorescent protein fusions of this protein demonstrated that it localizes to mitochondria. An Arabidopsis T-DNA line was identified with an insertion in MCU1. However, little effect of the insertion on transcript abundance of MCU1 was observed.

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The high metabolic demand of the heart makes it essential that an efficient and tightly controlled system be in place to regulate energy production. Contractility is mediated by a variable flux in intracellular calcium (iCa2+), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover, mitochondrial Ca2+ (mCa2+)-overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. However, the true function of cardiac mCa2+ in physiology remains unknown. Recent studies have reported that the Mcu gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for mCa2+ uptake (Baughman et al., 2011; De Stefani, Raffaello, Teardo, Szabo, & Rizzuto, 2011). To examine the role of mCa2+ in the heart, we generated a conditional, cardiac-specific knockout model and deleted Mcu in adult mice (Mcu-cKO). Loss of Mcu protected against myocardial ischemia-reperfusion (IR) (40 min occlusion of the left coronary artery (LCA) followed by 24h reperfusion) injury by preventing the activation of the MPTP. We observed a 45% reduction in infarct size per area-at-risk and a 65% reduction in cardiac troponin-I serum levels from 24h post-IR. In addition, while we found no baseline phenotype or change in baseline mCa2+ content, Mcu-cKO mice lacked contractile responsiveness to β-adrenergic receptor stimulation (isoproterenol infusion) as assessed by invasive hemodynamics, and, in parallel, were unable to activate mitochondrial dehydrogenases, thereby decreasing tricarboxylic acid (TCA) cycle flux and cardiac NADH. We found that Mcu-cKO mice had a 3-fold increase in pyruvate dehydrogenase (PDH) phosphorylation and a 50% decrease in PDH activity post-isoproterenol infusion. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in oxidative phosphorylation capacity) supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca2+-dependent metabolism during the ‘fight or flight’ response. However, questions still remain on how basal mCa2+ levels are regulated and if it contributes to cardiac disease. The mitochondrial sodium/calcium exchanger (mNCX) is hypothesized as the primary mechanism of mCa2+ efflux, but to date no study has confirmed its identity or function in an in vivo system (Palty et al., 2010). To investigate the role of mNCX in the heart, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene, Slc8b1, and crossed them with a tamoxifen-inducible, cardiomyocyte-specific, αMHC-Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Biophysical study of cardiomyocytes isolated from mNCX-cKO mice revealed a significant reduction in mCa2+ efflux rate. Tamoxifen-induced deletion of Slc8b1 in adult hearts caused sudden death with less than 15% of mice surviving after 10 days. Echocardiographic evaluation of mNCX-cKO hearts 3d post-tamoxifen revealed significant left ventricular (LV) remodeling, characterized by significant dilation and a substantial decrease in function. In addition, mNCX-cKO hearts exhibited increased reactive oxygen species generation when assessed by DHE imaging of live myocardial tissue and mitoSOX Red imaging in isolated adult cardiomyocytes. Using an Evan’s blue dye exclusion technique, we found that mNCX-cKO hearts displayed significant sarcolemmal rupture (~8% of all myocytes at a single time point 3d post-tamoxifen), indicative of cellular necrosis. To rescue the sudden death phenotype and acute loss of cells, we crossed our mNCX-cKO mice with the cyclophilin d (a mediator of MPTP-opening) knockout mice. mNCX-cKO x CypD-KO mice had a significant improvement in survival and LV-function. In addition, loss of MPTP activation also rescued mitochondrial pathology on the subcellular level. Since deletion of mNCX was detrimental on cardiac function, we thought that increasing mNCX could protect cardiomyocytes by reducing mCa2+-overload during cardiac disease. To test this, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to assess if increasing mCa2+ efflux would prevent cardiac injury in multiple pathological surgical models. mNCX-Tg and controls were subjected to in vivo IR injury followed by 24h reperfusion and myocardial infarction (MI) (permanent LCA ligation). mNCX-Tg mice displayed reduced cell death (a 43% reduction in infarct size 24h post-IR and a 33% reduction in scar size 4w post-MI), preserved LV function, a reduction in ROS generation, and a decrease in numerous HF indices. For the first time, we showed that mNCX is essential for maintenance of the mCa2+ microdomain in cardiomyocytes and that mNCX represents a novel therapeutic target in HF. / Biomedical Sciences

Orientadores: Tiago Rezende Figueira, Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T03:45:16Z (GMT). No. of bitstreams: 1 Chweih_Hanan_M.pdf: 1684363 bytes, checksum: edae156378f90e7315bca30c16544071 (MD5) Previous issue date: 2015 / Resumo: Algumas das características das mitocôndrias, incluindo as suas funções de transporte de Ca2+, podem apresentar dimorfismo sexual e especificidades teciduais. No entanto, as mensurações do transporte de Ca2+ em mitocôndrias isoladas estão sujeitas a artefatos secundários a abertura do poro de transição de permeabilidade mitocondrial (PTP) induzido pelo acúmulo excessivo de Ca2+ nesta organela. Neste estudo, o objetivo inicial foi avaliar se a inibição do PTP pela ciclosporina A (CsA) afeta a mensuração de diversas variáveis que descrevem o transporte de Ca2+por mitocôndrias isoladas de fígado de rato. Os resultados obtidos indicam que as concentrações de estado estável do Ca2+ externo a mitocôndria e as taxas deefluxo mitocondrial de Ca2+através de trocadores seletivos foram superestimados em até 4 vezes quando o PTP não foi inibido farmacologicamente pela CsA. O objetivo subsequente foi analisar o transporte de Ca2+ em mitocôndrias isoladas de fígado, de músculo esquelético, de coração e de cérebro de ratos machos e fêmeas sob condições experimentais específicas (i.e. meio de incubação contendo inibidores TPM, substratos energéticos ligados a NAD e níveis relevantes de Ca2+, Mg2+e Na+). Os dados indicaram que a taxa de influxo de Ca2+em mitocôndrias de fígado foi ~4 vezes superior a dos outros tecidos, as quais foram semelhantes entre si. Em contrapartida, as taxas de efluxo de Ca2+ apresentaram uma maior diversidade entre tecidos, especialmente na presença de Na+. Curiosamente, o efluxo de Ca2+na ausência de Na+foi significativamente mais elevado nas mitocôndrias cardíacas (~4nmol/mg/min) em relação às taxas observadas nos outros tecidos, contrariando a concepção de que o efluxo de Ca2+de mitocôndrias de coração é dependente, quase que exclusivamente, de um trocador que requer Na+. A especificidade em relação ao sexo só foi observada em dois índices relacionados a homeostase mitocondrial de Ca2+(i.e. cinética geral normalizada da captação de Ca2+ e a concentração de estado estável do Ca2+ externo a mitocôndria) em mitocôndrias isoladas de coração (mais lentos ou maiores na fêmea) e na respiração estimulada por ADP em mitocôndrias de fígado (~20% maior na fêmea). O presente estudo demonstrou a importância metodológica de se prevenir a abertura do PTP para a análise das propriedades e da variabilidade fisiológica do transporte de Ca2+por mitocôndrias isoladas. Adicionalmente, concluímos que sob as condições experimentais aqui utilizadas, o efluxo de Ca2+ mitocondrial apresenta grandes especificidades teciduais e que alguns achados desafiam conceitos estabelecidos em estudos anteriores sob condições arguivelmente menos controladas / Abstract: The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sex dimorphism. Because the measurements of the Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluates the extent to which MPT inhibition by cyclosporine-A affects the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to 4-fold if MPT pore opening is not prevented. Then, we analyzed the Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats under incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. Except for the liver mitochondria displaying values4-fold higher, the Ca2+ influx rates were similar among the other tissues. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (~4 nmol/mg/min), thus challenging the view that heart mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was only observed in two kinetic indexes (i.e. the normalized overall kinetics of Ca2+ uptake and the steady-state levels of external Ca2+) of heart mitochondrial Ca2+ homeostasis (slower or higher in female)and in the ADP-stimulated respiration of liver mitochondria (~20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria. Moreover, we conclude that mitochondrial Ca2+ efflux exhibits great tissue specificity under our conditions, which may challenge some concepts raised in previous studies that employed experimental conditions that are arguably not well controlled / Mestrado / Fisiopatologia Médica / Mestra em Ciências

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