PROBING TEMPORAL CHANGES IN MITOCHONDRIAL MEMBRANE POTENTIAL WITH IMPEDANCE SPECTROSCOPY

The electrical properties of mitochondria provide fundamental insights into metabolic processes in health and disease. This research studies electrical impedance spectroscopy as a non-invasive, sensitive, and relatively low cost technique to monitor biological processes, such as those involving changes in mitochondrial membrane potential. Our experimental strategy first involves treating suspensions of live mitochondria with the substrate succinate to stimulate activity of succinate dehydrogenase, or more simply Complex II. This triggers electron flux through Complex II and the remaining complexes of the electron transport chain, enabling them to pump protons across the inner membrane and build up a membrane potential. Subsequent variability is introduced by adding various concentrations of the uncoupler trifluorocarbonylcyanide phenylhydrazone (FCCP) and the neurotransmitter dopamine (DA) to mitochondrial suspensions, and measuring changes in impedance. Our results show that adding succinate decreases impedance, consistent with an increase in dielectric response and membrane potential. Overall, our investigation establishes real-time impedance spectroscopy as a non-destructive, potentially powerful method for membrane potential studies of mitochondria.Physics, Department o

Regulation of guard cell anion channels

Stomata account for much of the 70% of global water usage associated with agriculture, and have a profound impact on the water and carbon cycles of the world. Anion channels at the plasma membrane of the guard cell are thought to comprise a major pathway for anion efflux essential for driving stomatal closure. The activity of these channels is therefore tightly linked to abscisic acid (ABA)-dependent stomatal movements. Both the inorganic anion Cl- and the organic acid anion malate (Mal) are transported during stomatal movements. The metabolism of organic acids, primarily Mal plays an especially important role in these processes. However, little is known of the regulation of anion channel current (ICl) or its connection with cytosolic Mal and its immediate metabolite oxaloacetate (OAA). Work described here focuses on the relation of ICl and its connections with metabolism and signals controlling other transport at the guard cell plasma membrane. Thus the impact of Mal, OAA and of acetate in Vicia guard cells was examined, and results shown that all three organic acids affect ICl with different characteristics. Most prominent, the suppression of ICl by OAA within the physiological range in vivo indicates a capacity for OAA to co-ordinate organic acid metabolism with ICl through a direct effect of organic acid pool size. In a second set of studies, the ABA pathway that elevates cytosolic free Ca2+ ([Ca2+]i) in order to activate ICl was explored. These studies build on the discovery of PYR/PRL ABA receptors. Previous analysis of the pyr1/pyl1/pyl2/pyl4 mutant suggested that [Ca2+]i increases are suppressed. However, direct evidence had not been forthcoming. Thus a combination of voltage clamp and fluorescent ratio analysis with the Ca2+-sensitive dye Fura2 was used to show that the activity of Ca2+ channels (ICa) at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant is not activated in ABA, with the effect that [Ca2+]i increases were suppressed. Further studies showed that the normal action of ABA in promoting reactive oxygen species (ROS) was impaired, although adding H2O2 was sufficient to activate the ICa and trigger stomatal closure in the mutant. These results offer direct evidence that the PYR/PYL receptor proteins contribute to the activation by ABA of ICa through ROS, thus affecting [Ca2+]i and its regulation of osmotic solute flux for stomatal closure. Finally, the connection between ICl and the K+ channel currents in the slac1 mutant of Arabidopsis was studied. These studies employed systems dynamic modelling to explain the paradoxical suppression of the inward-rectifying K+ channel current, IK,in and slowing of stomatal opening, by mutation that eliminated the SLAC1 anion channel. Experimental results confirmed the model predictions that the abnormal cytosolic pH (pHi) and [Ca2+]i suppresses IK,in in the slac1 mutant, through measuring pHi and [Ca2+]i in vivo, and experimental manipulation of pHi and [Ca2+]i is sufficient to recover IK,in and stomatal opening. These data uncover a previously unrecognised signaling network that minimises the effects of the slac1 mutant on transpiration, and they underscore the importance of H+-coupled anion transport for pHi homeostasis

Cardiac Adaptation To Chronic Blockade Of Voltage-Gated, L-Type Calcium Channels In The Sarcolemma

L-type Ca2+ channels (dihydropyridine receptors, DHPRs) in the sarcolemma are essential to cardiac excitation-contraction (E-C) coupling. Thus, Ca2+ influx through DHPRs upon cardiomyocyte excitation triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) to initiate myofilament activation and muscle contraction. Muscle relaxation occurs upon sequestration of Ca2+ back into the SR lumen by sarco/endoplasmic reticulum calcium-ATPase (SERCA) in the SR. As a treatment option for hypertension, long-term use of DHPR blockers is associated with increased risk of heart failure; underlying mechanisms are unknown. This research used male Wistar rats treated with verapamil (subcutaneously, 625 µg/h/kg for 4 weeks) to determine the impact of chronic DHPR blockade in vivo, on E-C coupling events and heart function at all levels ranging from molecules to whole organism. The results presented in chapter 2 demonstrate that chronic DHPR blockade caused functional remodeling of RyRs and spatio-temporal dyssynchrony of E-C coupling events, resulting in systolic dysfunction and enhanced susceptibility to arrhythmia. Findings in chapter 3 reveal that chronic DHPR blockade was accompanied by depressed SERCA function, abnormal cardiomyocyte Ca2+ handling, and diastolic dysfunction. Results in chapter 4 reveal adaptational changes in protein phosphorylation-dependent regulation of SR/cardiomyocyte Ca2+ cycling due to chronic DHPR blockade. These include over-expression of Ca2+/calmodulin-dependent protein kinases II (CaMKII), hyper-phosphorylation of SR Ca2+ cycling proteins by CaMKII and cAMP-dependent protein kinase (PKA), paradoxically diminished SR Ca2+ content and contractile reserve, and blunted inotropic response to beta-adrenergic stimulation. The above adaptations to chronic DHPR blockade occurred in the absence of cardiac hypertrophy or fibrosis. Thus, molecular remodeling may invoke cardiac pathology and heart failure without microscopic structural changes in cardiomyocytes. The findings from this thesis reveal, for the first time, integrated mechanisms underlying the increased risk of heart failure associated with chronic DHPR blockade. In addition to urging caution in the conventional clinical use of DHPR blockers, the novel mechanistic events and molecular remodeling revealed here imply that manipulation of the stoichiometry of molecular players in E-C coupling demand critical attention and careful scrutiny in the design and deployment of therapeutic approaches for heart diseases

Calcium dynamics and related alterations in pulmonary hypertension associated with heart failure

Congestive heart failure (CHF) represents an important Canadian health problem. Most patients with CHF develop pulmonary hypertension (PH), which is an important marker that signals progression of the disease and its poor outcome. Significant advances have been made for the treatment of heart failure (HF). Nevertheless, the morbidity and mortality among patients with advanced heart HF, who have developed PH remains high. Increased pulmonary vascular pressure (PVP) observed in PH leads to increased vascular tone and vascular remodelling associated with altered vasodilatory responses. It is noteworthy that a decrease in vasodilatory responses has been observed in PH. At the core of vasodilatory alterations lies endothelial dysfunction. This hallmark of most cardiovascular diseases is associated with alterations in calcium (Ca2+) homeostasis. Although global Ca2+ plays a role in a wide range of cellular functions, this thesis work focused on the impact of local Ca2+ signalling in endothelial cells (ECs). Among the different types of local Ca2+ signals, Ca2+ pulsars were identified. Ca2+ pulsars are local endothelial Ca2+ signals whose activity is finely regulated by physiological agents that modulate intracellular levels of inositol 1,4,5-triphosphate (IP3) and Ca2+. Ca2+ pulsars have been shown to have an effect on several important cellular functions. In mesenteric arteries, Ca2+ pulsars induce endotheliuminduced relaxation of vascular smooth muscle cells. Up until now, the regulatory mechanisms of Ca2+ pulsars remain to be uncovered. The spatio-temporal characteristics of Ca2+ pulsars suggest that they could play a role in the control of pulmonary vascular tone, potentially involving more transmembrane ion channels, as well as regulatory proteins. Transient receptor potential vanilloid 4 (TRPV4) channels are non-selective mechanosensitive osmo-regulated cation channels broadly expressed in a number of tissues. Activation of TRPV4 channels allows Ca2+ entry into the cell. A number of studies have shown the implication of TRPV4 as well as other channels from the TRP family in PH. Endothelial Ca2+-related pathophysiological mechanisms modulating pulmonary vascular tone and leading to the development of group II PH are poorly defined. In addition, the scarcity of studies exploring the pathophysiology and therapies of group II PH resides in the lack of validated small animal models with an adequate determination of the presence and severity of PH. The work in this thesis identified and characterized for the first time intracellular Ca2+ pulsars in pulmonary endothelium and their alterations in a clinically relevant mouse model of group II PH that was developed. In addition, this work revealed the implication of endothelial TRPV4 channels in Ca2+ pulsars dysregulation in group II-PH.L'insuffisance cardiaque (IC) représente un problème de santé important au Canada. La plupart des patients atteints d'IC développent une hypertension pulmonaire (HP), qui est un marqueur de la progression de la maladie et de son mauvais pronostic. Des progrès significatifs ont été réalisés pour le traitement de l'IC. Néanmoins, la morbidité et la mortalité chez les patients atteints d'IC avancée, qui ont développé l’HP reste élevée. L'augmentation de la pression vasculaire pulmonaire (PVP) observée en HP entraîne une augmentation du tonus vasculaire et un remodelage vasculaire associés à des réponses vasodilatatrices altérées. En effet, une diminution des réponses vasodilatatrices a été observée dans l'HP. La dysfonction endothéliale est au coeur des altérations vasodilatatrices. Cette caractéristique de la plupart des maladies cardiovasculaires est associée à des altérations de l'homéostasie du calcium (Ca2+). Bien que le Ca2+ global joue un rôle dans un grand nombre de fonctions cellulaires, la présente thèse est concentrée sur l'impact de la signalisation calcique locale dans les cellules endothéliales (CE). Parmi les différents types de signaux calciques locaux, les pulsars ont été identifiés. Les pulsars calciques sont des évènements endothéliaux locaux dont l'activité est finement régulée par des agents physiologiques qui modulent les niveaux intracellulaires d'inositol 1,4,5-triphosphate (IP3) et de Ca2+. Les pulsars ont un effet sur plusieurs fonctions cellulaires importantes. Dans les artères mésentériques, les pulsars induisent une relaxation des cellules musculaires lisses vasculaires. Jusqu'à présent, les mécanismes de régulation des pulsars Ca2+ restent à découvrir. Les caractéristiques spatio-temporelles des pulsars suggèrent qu'ils pourraient jouer un rôle dans le contrôle du tonus vasculaire pulmonaire, impliquant potentiellement plus de canaux ioniques transmembranaires, ainsi que des protéines régulatrices. Les canaux TRP de la famille vanilloïde 4 (TRPV4) sont des canaux cationiques méchanosensitifs, non sélectifs, largement exprimés dans un nombre de tissus. L'activation des canaux TRPV4 permet l'entrée de Ca2+ dans la cellule. Des études ont montré l'implication de TRPV4 ainsi que d'autres canaux de la famille TRP dans l’HP. Les mécanismes physiopathologiques liés au Ca2+ endothélial modulant le tonus vasculaire pulmonaire et conduisant au développement de l’HP du groupe II sont mal définis. En outre, la rareté des études explorant la physiopathologie et les thérapies de l’HP du groupe II réside dans l'absence de modèles animaux validés pour l’étude de l’HP du groupe II, avec une détermination adéquate de la présence et de la sévérité de l'HP. Les travaux issus de cette thèse ont identifié et caractérisé pour la première fois des pulsars Ca2+ intracellulaires dans l'endothélium pulmonaire et leurs altérations dans un modèle de souris cliniquement significatif de l’HP de groupe II qui a été développé. En outre, ce travail a révélé l'implication des canaux TRPV4 endothéliaux dans la dérégulation des pulsars Ca2+ dans l’HP du groupe II

UMBRELLA CELL MECHANOTRANSDUCTION AND STRETCH-REGULATED EXOCYTOSIS/ENDOCYTOSIS

Cells interact with mechanical environments by mechanotransduction, a cellular process that converts mechanical signals into biochemical signals. Umbrella cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are coordinated and the mechanotransduction pathways involved are not well understood. By manipulating different forces and force parameters applied on umbrella cells, the responses of electrophysiological parameters (TEV, TER, and Isc) and apical membrane capacitance (1µF 1cm2 membrane surface area) are monitored through the modified Ussing chamber system. Stretch of the umbrella cells result in an acute change of electrical parameters, but not hydrostatic pressure. Further, the stretched response is sensitive to force direction, indicating that stretch of apical membrane causes umbrella cell TEV hyperpolarization, TER decrease, Isc increase, and apical membrane exocytosis, while stretch of basolateral membrane causes opposite effects, and this observation can be modeled mathematically. Stretch speed, which is defined by the filling rate, is further defined to play the key role in modulating the degree and time course of stretched umbrella cell responses, suggesting a mechnosensory function of umbrella cells. Use of channel blockers and openers established that the stretch of apical membrane is likely dependent on cation transport pathway, while stretch of basolateral membrane is dependent on K+ transport at the basolateral surface of the cells, indicating distinctive apical and basolateral membrane requirements for umbrella cell mechanotransduction. These results indicate that mechanotransduction in umbrella cells depends on the sequential activity of its distinct apical and basolateral membrane domains, which act in a collaborative manner to regulate apical membrane dynamics

Electrophysiological and microfluorimetric studies of mouse dorsal root ganglion cells

Primary sensory neurones consist of discrete functional groups, previously defined in terms of cell size and action potential shape. Whole cell patch clamp recordings were made from cells isolated from mouse dorsal root ganglia (DRGs) to define the ionic basis for these characteristics. Three groups emerged, expressing different arrays of voltage-activated currents. These correlated well with cell size, measured as a function of membrane capacitance. Capsaicin was used to identify a subset of small nociceptive cells. The properties of the major currents underlying the differences in electrophysiological behaviour of the cells were characterised. The cells were used to investigate the basis for altered neuronal function during hypoxia/anoxia. Microfluorimetric techniques were used to monitor: (i) intracellular calcium ([Ca2+]i) using indo-1 (ii) mitochondrial membrane potential (ψm) using Rhodamine 123 and (iii) the autofluorescence of mitochondrial NADH. Anoxia increased [Ca2+]i), depolarised ψm and increased autofluorescence. Blockade of mitochondrial electron transport by cyanide had equivalent effects. The pO2 sensitivity of these effects was defined. The baseline redox status of cells was estimated through measurement of maximal (blockade of oxygen consumption with CN/anoxia) and minimal autofluorescence (maximising oxygen consumption with uncoupler, FCCP). The contribution of the F1-F0 ATPase to ψm was studied using oligomycin. This revealed the reversal of the ATPase with anoxia, slowing the rate of depolarisation of ψm. The actions of the convulsant barbiturate CHEB were examined. It was found to inhibit electron transport at Complex I, and was more potent than other barbiturates. In a proportion of cells which did not correlate clearly with the classification described above, CHEB raised [Ca2+l]i dramatically. This was distinct from its metabolic effects, resulting from the opening of a non-selective cation conductance. This action could underlie the convulsant effect of the drug

Murine Electrophysiological Models of Cardiac Arrhythmogenesis

Cardiac arrhythmias can follow disruption of the normal cellular electrophysiological processes underlying excitable activity and their tissue propagation as coherent wavefronts from the primary sinoatrial node pacemaker, through the atria, conducting structures and ventricular myocardium. These physiological events are driven by interacting, voltage-dependent, processes of activation, inactivation, and recovery in the ion channels present in cardiomyocyte membranes. Generation and conduction of these events are further modulated by intracellular Ca$^{2+}$ homeostasis, and metabolic and structural change. This review describes experimental studies on murine models for known clinical arrhythmic conditions in which these mechanisms were modified by genetic, physiological, or pharmacological manipulation. These exemplars yielded molecular, physiological, and structural phenotypes often directly translatable to their corresponding clinical conditions, which could be investigated at the molecular, cellular, tissue, organ, and whole animal levels. Arrhythmogenesis could be explored during normal pacing activity, regular stimulation, following imposed extra-stimuli, or during progressively incremented steady pacing frequencies. Arrhythmic substrate was identified with temporal and spatial functional heterogeneities predisposing to reentrant excitation phenomena. These could arise from abnormalities in cardiac pacing function, tissue electrical connectivity, and cellular excitation and recovery. Triggering events during or following recovery from action potential excitation could thereby lead to sustained arrhythmia. These surface membrane processes were modified by alterations in cellular Ca$^{2+}$ homeostasis and energetics, as well as cellular and tissue structural change. Study of murine systems thus offers major insights into both our understanding of normal cardiac activity and its propagation, and their relationship to mechanisms generating clinical arrhythmias.Medical Research Council, Wellcome Trust, McVeigh Benefaction, British Heart Foundation, Helen Kirkland Trust, Sudden Arrhythmic Death Syndrome - SADS U