Influência da via mTOR no processo de remodelação cardíaca induzida pelo ácido retinóico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Focal adhesion kinase governs cardiac concentric hypertrophic growth by activating the AKT and mTOR pathways

The heart responds to sustained overload by hypertrophic growth in which the myocytes distinctly thicken or elongate on increases in systolic or diastolic stress. Though potentially adaptive, hypertrophy itself may predispose to cardiac dysfunction in pathological settings. The mechanisms underlying the diverse morphology and outcomes of hypertrophy are uncertain. Here we used a focal adhesion kinase (FAK) cardiac-specific transgenic mice model (FAK-Tg) to explore the function of this non-receptor tyrosine kinase on the regulation of myocyte growth. FAK-Tg mice displayed a phenocopy of concentric cardiac hypertrophy, reflecting the relative thickening of the individual myocytes. Moreover, FAK-Tg mice showed structural, functional and molecular features of a compensated hypertrophic growth, and preserved responses to chronic pressure overload. Mechanistically, FAK overexpression resulted in enhanced myocardial FAK activity, which was proven by treatment with a selective FAK inhibitor to be required for the cardiac hypertrophy in this model. Our results indicate that upregulation of FAK does not affect the activity of Src/ERK1/2 pathway, but stimulated signaling by a cascade that encompasses PI3K, AKT, mTOR, S6K and rpS6. Moreover, inhibition of the mTOR complex by rapamycin extinguished the cardiac hypertrophy of the transgenic FAK mice. These findings uncover a unique role for FAK in regulating the signaling mechanisms that governs the selective myocyte growth in width, likely controlling the activity of PI3K/AKT/mTOR pathway, and suggest that FAK activation could be important for the adaptive response to increases in cardiac afterload. This article is part of a Special Issue entitled "Local Signaling in Myocytes". (C) 2011 Elsevier Ltd. All rights reserved.

T-3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T-3) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T-3 was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T-3 effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. Methods: Rats were surgically thyroidectomized and treated with T-3 (0.3 to 100 mu g/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T-3 treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T-3 treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T-3 exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Myocardial Remodeling after Large Infarcts in Rat Converts Post Rest-Potentiation in Force Decay

Background: Post-rest contraction (PRC) of cardiac muscle provides indirect information about the intracellular calcium handling. Objective: Our aim was to study the behavior of PRC, and its underlying mechanisms, in rats with myocardial infarction. Methods: Six weeks after coronary occlusion, the contractility of papillary muscles (PM) obtained from sham-operated (C, n = 17), moderate infarcted (MMI, n = 10) and large infarcted (LMI, n = 14) rats was evaluated, following rest intervals of 10 to 60 seconds before and after incubation with lithium chloride (Li+) substituting sodium chloride or ryanodine (Ry). Protein expression of SR Ca(2+)-ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLB) and phospho-Ser(16)-PLB were analyzed by Western blotting. Results: MMI exhibited reduced PRC potentiation when compared to C. Opposing the normal potentiation for C, post-rest decays of force were observed in LMI muscles. In addition, Ry blocked PRC decay or potentiation observed in LMI and C; Li+ inhibited NCX and converted PRC decay to potentiation in LMI. Although MMI and LMI presented decreased SERCA2 (72 +/- 7% and 47 +/- 9% of Control, respectively) and phospho-Ser(16)-PLB (75 +/- 5% and 46 +/- 11%, respectively) protein expression, overexpression of NCX (175 +/- 20%) was only observed in LMI muscles. Conclusion: Our results showed, for the first time ever, that myocardial remodeling after MI in rats may change the regular potentiation to post-rest decay by affecting myocyte Ca(2+) handling proteins. (Arq Bras Cardiol 2012;98(3):243-251)

Insulin alone or with captopril: effects on signaling pathways (AKT and AMPK) and oxidative balance after ischemia-reperfusion in isolated hearts

Insulin and the inhibition of the reninangiotensin system have independent benefits for ischemiareperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemiareperfusion. Isolated hearts were perfused (Langendorff technique) with KrebsHenseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was similar to 31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (similar to 2.2 times) in all groups vs. group KH and was similar to 35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was similar to 28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects.

Chronic Stress Improves the Myocardial Function without Altering L-type Ca+2 Channel Activity in Rats

Background: Chronic stress is associated with cardiac remodeling; however the mechanisms have yet to be clarified. Objective: The purpose of this study was test the hypothesis that chronic stress promotes cardiac dysfunction associated to L-type calcium Ca2+ channel activity depression. Methods: Thirty-day-old male Wistar rats (70 - 100 g) were distributed into two groups: control (C) and chronic stress (St). The stress was consistently maintained at immobilization during 15 weeks, 5 times per week, 1h per day. The cardiac function was evaluated by left ventricular performance through echocardiography and by ventricular isolated papillary muscle. The myocardial papillary muscle activity was assessed at baseline conditions and with inotropic maneuvers such as: post-rest contraction and increases in extracellular Ca2+ concentration, in presence or absence of specific blockers L-type calcium channels. Results: The stress was characterized for adrenal glands hypertrophy, increase of systemic corticosterone level and arterial hypertension. The chronic stress provided left ventricular hypertrophy. The left ventricular and baseline myocardial function did not change with chronic stress. However, it improved the response of the papillary muscle in relation to positive inotropic stimulation. This function improvement was not associated with the L-type Ca2+ channel. Conclusion: Chronic stress produced cardiac hypertrophy; however, in the study of papillary muscle, the positive inotropic maneuvers potentiated cardiac function in stressed rats, without involvement of L-type Ca2+ channel. Thus, the responsible mechanisms remain unclear with respect to Ca2+ influx alterations. (Arq Bras Cardiol 2012;99(4):907-914)

Mechanisms underlying skeletal muscle insulin resistance induced by fatty acids: importance of the mitochondrial function

Insulin resistance condition is associated to the development of several syndromes, such as obesity, type 2 diabetes mellitus and metabolic syndrome. Although the factors linking insulin resistance to these syndromes are not precisely defined yet, evidence suggests that the elevated plasma free fatty acid (FFA) level plays an important role in the development of skeletal muscle insulin resistance. Accordantly, in vivo and in vitro exposure of skeletal muscle and myocytes to physiological concentrations of saturated fatty acids is associated with insulin resistance condition. Several mechanisms have been postulated to account for fatty acids-induced muscle insulin resistance, including Randle cycle, oxidative stress, inflammation and mitochondrial dysfunction. Here we reviewed experimental evidence supporting the involvement of each of these propositions in the development of skeletal muscle insulin resistance induced by saturated fatty acids and propose an integrative model placing mitochondrial dysfunction as an important and common factor to the other mechanisms.

Acute inhibition of excessive mitochondrial fission after myocardial infarction prevents long-term cardiac dysfunction

BACKGROUND: Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats. METHODS AND RESULTS: We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28 ± 2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions. CONCLUSIONS: Together, we show that excessive mitochondrial fission at reperfusion contributes to long-term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long-term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.

Cell therapy prevents structural, functional and molecular remodeling of remote non-infarcted myocardium

Background/objectives: Therapy using bone marrow (BM) cells has been tested experimentally and clinically due to the potential ability to restore cardiac function by regenerating lost myocytes or increasing the survival of tissues at risk after myocardial infarction (MI). In this study we aimed to evaluate whether BM-derived mononuclear cell (MNC) implantation can positively influence the post-MI structural remodeling, contractility and Ca(2 +)-handling proteins of the remote non-infarcted tissue in rats. Methods and results: After 48 h of MI induction, saline or BM-MNC were injected. Six weeks later, MI scars were slightly smaller and thicker, and cardiac dilatation was just partially prevented by cell therapy. However, the cardiac performance under hemodynamic stress was totally preserved in the BM-MNC treated group if compared to the untreated group, associated with normal contractility of remote myocardium as analyzed in vitro. The impaired post-rest potentiation of contractile force, associated with decreased protein expression of the sarcoplasmic reticulum Ca2 +-ATPase and phosphorylated-phospholamban and overexpression of Na(+)/Ca(2 +) exchanger, were prevented by BM-MNC, indicating preservation of the Ca(2 +) handling. Finally, pathological changes on remodeled remote tissue such as myocyte hypertrophy, interstitial fibrosis and capillary rarefaction were also mitigated by cell therapy. Conclusions: BM-MNC therapy was able to prevent cardiac structural and molecular remodeling after MI, avoiding pathological changes on Ca(2 +)-handling proteins and preserving contractile behavior of the viable myocardium, which could be the major contributor to the improvements of global cardiac performance after cell transplantation despite that scar tissue still exists.

Untersuchungen zur Zell-Transistor-Kopplung mittels der Voltage-Clamp-Technik

Untersuchungen zur Zell-Transistor Kopplung mittels der Voltage-Clamp TechnikIn der vorliegenden Arbeit wird die extrazelluläre Einkopplung elektrischer Signale von Zellen in Transistoren hinsichtlich der an der Kopplung beteiligten Parameter untersucht. Dafür werden Zellen aus Primärkulturen und von Zell-Linien direkt auf den aktiven Sensorflächen der hergestellten Chips kultiviert. Für die Experimente werden n- und p-Kanal Feldeffekttransistoren (FET) sowie Extended-Gate-Elektroden (EGE) mit Gold- und Titanoberflächen entwickelt.Zur Untersuchung der Kopplungseigenschaften werden die neuronale Zell-Linie SH-SY5Y, die humane Endothel Zell-Linie EA.hy-926 sowie als Primärzellen hippocampale Neuronen und Kardiomyozyten embryonaler und neonataler Ratten eingesetzt. Die Voltage-Clamp Technik erlaubt die Untersuchung spannungsgesteuerter Ionenkanäle in der Zellmembran. Maßgebend für den Signalverlauf des extrazellulär eingekoppelten Signals ist der Ionenstrom von Na+, K+ und Ca2+ durch die Membran im Kontaktbereich zwischen Zelle und Sensor.Die Kopplung kann elektrisch mithilfe eines Ersatzschaltkreises beschrieben werden, der alle beteiligten elektrischen Größen der Membran und der Ionenströme, sowie die Parameter des Kontaktbereichs und des Sensors enthält.Die Simulation der extrazellulären Signale zeigt, dass die beobachteten Signalformen nur durch eine Erhöhung der Ionenkanaldichten und dadurch einer deutlich vergrößerten Leitfähigkeit der Ionenarten im Kontaktbereich gegenüber der freien Membran erklärt werden können.

Characterisation of the cell-transistor coupling

Die Verbindung von elektrisch aktiven, lebenden Zellen zu extrazellulären Sensorsystemen eröffnet vielfälige Möglichkeiten im Bereich der Biosensorik. Die vorliegende Arbeit leistet einen Beitrag zum tieferen Verständnis der elektrischen Kopplungsmechanismen zwischen den biologischen und elektronischen Teilen solcher Hybridsysteme. Es wurden dazu drei Hauptbereiche bearbeitet:Ein System zur extrazellulären Signalableitung an lebenden Zellen bestehend aus einem Sensorchip, einem Vorverstärkerkopf und einem Hauptverstärker wurde weiterentwickelt.Als Sensoren wurden entweder Metallmikroelektroden-Chips mit 64 Kanälen oder Feldeffekt Transistoren-Chips mit 16 Kanälen (FET) eingesetzt. Es wurden zusätzlich spezielle FET Sensoren mit Rückseitenkontakten hergestellt und eingesetzt.Die elektrische Kopplung von einzelnen Nervenzellen der neuronalen Zell-Linien SH-SY5Y und TR14 oder primär kultivierten Neuronen aus dem Hirnstamm oder dem Hippocampus von embryonalen Ratten mit den extrazellulären Sensoren wurde untersucht. In der 'whole-cell' Patch-Clamp Technik wurden die Beiträge der spannungsgesteuerten Na+- und K+-Ionenkanäle zur extrazellulären Signalform identifiziert. Die Simulation der Signale mit einem Ersatzschaltkreis (Punkt-Kontakt Modell), der in PSPICE implementiert wurde, deutet auf eine starke Abhängigkeit der Signalformen in bezug auf Konzentrationsänderungen von Na+- und K+-Ionen im Volumenbereich zwischen Zelle und den ionensensitiven Transistoren hin. Ein empirisch erweitertes Punkt-Kontakt Modell wurde daraufhin vorgestellt.Im dritten Teil der Arbeit wurden Zellschichten von Kardiomyocyten embryonaler Ratten auf den extrazellulären Sensoren kultiviert. Die Eignung eines solchen Hybridsensors als Modellherz fuer das pharmazeutische Screeing wurde durch Messungen mit Herzstimulanzien und -relaktanzien bestätigt.

The effects of hypocalcemia on spatial alternans and ventricular fibrillation studied with the optical mapping technique

The heart is a wonderful but complex organ: it uses electrochemical mechanisms in order to produce mechanical energy to pump the blood throughout the body and allow the life of humans and animals. This organ can be subject to several diseases and sudden cardiac death (SCD) is the most catastrophic manifestation of these diseases, responsible for the death of a large number of people throughout the world. It is estimated that 325000 Americans annually die for SCD. SCD most commonly occurs as a result of reentrant tachyarrhythmias (ventricular tachycardia (VT) and ventricular fibrillation (VF)) and the identification of those patients at higher risk for the development of SCD has been a difficult clinical challenge. Nowadays, a particular electrocardiogram (ECG) abnormality, “T-wave alternans” (TWA), is considered a precursor of lethal cardiac arrhythmias and sudden death, a sensitive indicator of risk for SCD. TWA is defined as a beat-to-beat alternation in the shape, amplitude, or timing of the T-wave on the ECG, indicative of the underlying repolarization of cardiac cells [5]. In other words TWA is the macroscopic effect of subcellular and celluar mechanisms involving ionic kinetics and the consequent depolarization and repolarization of the myocytes. Experimental activities have shown that TWA on the ECG is a manifestation of an underlying alternation of long and short action potential durations (APDs), the so called APD-alternans, of cardiac myocytes in the myocardium. Understanding the mechanism of APDs-alternans is the first step for preventing them to occur. In order to investigate these mechanisms it’s very important to understand that the biological systems are complex systems and their macroscopic properties arise from the nonlinear interactions among the parts. The whole is greater than the sum of the parts, and it cannot be understood only by studying the single parts. In this sense the heart is a complex nonlinear system and its way of working follows nonlinear dynamics; alternans also, they are a manifestation of a phenomenon typical in nonlinear dynamical systems, called “period-dubling bifurcation”. Over the past decade, it has been demonstrated that electrical alternans in cardiac tissue is an important marker for the development of ventricular fibrillation and a significant predictor for mortality. It has been observed that acute exposure to low concentration of calcium does not decrease the magnitude of alternans and sustained ventricular Fibrillation (VF) is still easily induced under these condition. However with prolonged exposure to low concentration of calcium, alternans disappears, but VF is still inducible. This work is based on this observation and tries to make it clearer. The aim of this thesis is investigate the effect of hypocalcemia spatial alternans and VF doing experiments with canine hearts and perfusing them with a solution with physiological ionic concentration and with a solution with low calcium concentration (hypocalcemia); in order to investigate the so called memory effect, the experimental activity was modified during the way. The experiments were performed with the optical mapping technique, using voltage-sensitive dye, and a custom made Java code was used in post-processing. Finding the Nolasco and Dahlen’s criterion [8] inadequate for the prediction of alternans, and takin into account the experimental results, another criterion, which consider the memory effect, has been implemented. The implementation of this criterion could be the first step in the creation of a method, AP-based, discriminating who is at risk if developing VF. This work is divided into four chapters: the first is a brief presentation of the physiology of the heart; the second is a review of the major theories and discovers in the study of cardiac dynamics; the third chapter presents an overview on the experimental activity and the optical mapping technique; the forth chapter contains the presentation of the results and the conclusions.

Computational Modeling of Cardiac Excitation-Contraction Coupling in Physiological and Pathological Conditions

The cardiomyocyte is a complex biological system where many mechanisms interact non-linearly to regulate the coupling between electrical excitation and mechanical contraction. For this reason, the development of mathematical models is fundamental in the field of cardiac electrophysiology, where the use of computational tools has become complementary to the classical experimentation. My doctoral research has been focusing on the development of such models for investigating the regulation of ventricular excitation-contraction coupling at the single cell level. In particular, the following researches are presented in this thesis: 1) Study of the unexpected deleterious effect of a Na channel blocker on a long QT syndrome type 3 patient. Experimental results were used to tune a Na current model that recapitulates the effect of the mutation and the treatment, in order to investigate how these influence the human action potential. Our research suggested that the analysis of the clinical phenotype is not sufficient for recommending drugs to patients carrying mutations with undefined electrophysiological properties. 2) Development of a model of L-type Ca channel inactivation in rabbit myocytes to faithfully reproduce the relative roles of voltage- and Ca-dependent inactivation. The model was applied to the analysis of Ca current inactivation kinetics during normal and abnormal repolarization, and predicts arrhythmogenic activity when inhibiting Ca-dependent inactivation, which is the predominant mechanism in physiological conditions. 3) Analysis of the arrhythmogenic consequences of the crosstalk between β-adrenergic and Ca-calmodulin dependent protein kinase signaling pathways. The descriptions of the two regulatory mechanisms, both enhanced in heart failure, were integrated into a novel murine action potential model to investigate how they concur to the development of cardiac arrhythmias. These studies show how mathematical modeling is suitable to provide new insights into the mechanisms underlying cardiac excitation-contraction coupling and arrhythmogenesis.

Synthese und Evaluierung von 68Ga-Schiff'schen Basen zur Diagnostik der koronaren Herzerkrankung

ZusammenfassungrnDie häufigsten Todesfälle weltweit sind auf Herzerkrankungen zurückzuführen. Bei der koronaren Herzkrankheit (KHK) sammeln sich über Jahre arteriosklerotische Ablagerungen in den Herzkranzgefäßen an und führen so zu einer verminderten Durchblutung und Versorgung des Herzmuskelgewebes mit Sauerstoff und Nährstoffen. Zur nuklearmedizinischen Bildgebung finden am häufigsten das SPECT-Nuklid 201Tl sowie die beiden 99mTc-Radiopharmaka Sestamibi und Tetrofosmin Anwendung. Die PET-Technik ist der SPECT-Technik in Bezug auf absolute Quantifizierung sowie Auflösung überlegen. Ziel der vorliegenden Arbeit war es, ein mögliches PET-Radiopharmakon zur Diagnostik der KHK zu entwickeln. Um eine dem 99mTc-Nuklid vergleichbare Verfügbarkeit im klinischen Alltag zu erreichen, sollte als Basis des neuen Radiopharmakons das mittels Radionuklid-Generator verfügbare 68Ga dienen. Schiff’sche Basen-Verbindungen zeigten nach Komplexierung mit 67/68Ga eine deutliche Aufnahme in die Herzmuskelzellen. Auf dieser Grundlage wurden verschiedene Schiff’sche Basen-Strukturen synthetisiert. Diese unterscheiden sich einerseits durch das Substitutionsmuster der verwendeten Aldehyde und andererseits durch das verwendete Rückgrat. Alle synthetisierten Chelatoren wurden erfolgreich mit 68Ga radioaktiv markiert und konnten anschließend aufgereinigt werden. Die Evaluierung dieser Substanzen in vitro zeigte, dass sie in unterschiedlichen Medien stabil ist. Die Lipophilie der 68Ga-Verbindungen (log D) lag zwischen 0,87±0,24 und 2,72±0,14. Die Ladung der Verbindungen wurde mittels Papierelektrophorese bei pH= 7 als kationisch bestimmt. Zusätzlich fanden in vitro-Untersuchungen zur Bestimmung der Aufnahme der Komplexe in HL-1 Herzzellen statt. Um den Einfluss des Zellmembranpotentials bzw. des Mitochondrienmembranpotentials zu untersuchen, wurde ein Teil der Zellen dafür mit Valinomycin (Ionophor, zerstört das Potential) behandelt. Mittels ex vivo-Biodistributionen wurde die Organverteilung von zwei Schiff’schen Basen (68Ga-BADED-2 und 68Ga-BAPDMEN-2) mit dem routinemäßig in der Klinik eingesetzten Derivat 99mTc-Sestamibi sowie dem 18F-Flurpiridaz in Ratten verglichen. Alle Verbindungen zeigten dabei eine deutliche Herzaufnahme von mehr als 2 % der injizierten Dosis pro Gramm Gewebe. Durch in vivo-PET-Aufnahmen wurden die Zeit-Aktivitätskurven der 68Ga-Verbindungen sowie zum Vergleich des 18F-Flurpiridaz bestimmt. Die Aufnahmen lagen im Bereich von 0,63±0,15 für 68Ga-BAPEN-3 bis 2,72±0,86 für 68Ga-BADED-8.In dem zweiten Teil der Arbeit wurden die Vorteile des hochaffinen Herztracers Flurpiridaz mit dem lipophilen, positiv-geladenen Ga-Schiff’sche Base-Chelator kombiniert. Hierzu wurde zunächst das Insektizid Flurpiridaz synthetisiert und mit dem BAPEN-Rückgrat gekoppelt. Die entstandene Verbindung wurde erstmals mit 68Ga radioaktiv markiert und muss in weiterführenden Arbeiten evaluiert werden.

Closed-loop investigation of the dynamical properties of cardiomyocyte electrophysiology by means of Dynamic clamp and Mathematical modelling

The research field of my PhD concerns mathematical modeling and numerical simulation, applied to the cardiac electrophysiology analysis at a single cell level. This is possible thanks to the development of mathematical descriptions of single cellular components, ionic channels, pumps, exchangers and subcellular compartments. Due to the difficulties of vivo experiments on human cells, most of the measurements are acquired in vitro using animal models (e.g. guinea pig, dog, rabbit). Moreover, to study the cardiac action potential and all its features, it is necessary to acquire more specific knowledge about single ionic currents that contribute to the cardiac activity. Electrophysiological models of the heart have become very accurate in recent years giving rise to extremely complicated systems of differential equations. Although describing the behavior of cardiac cells quite well, the models are computationally demanding for numerical simulations and are very difficult to analyze from a mathematical (dynamical-systems) viewpoint. Simplified mathematical models that capture the underlying dynamics to a certain extent are therefore frequently used. The results presented in this thesis have confirmed that a close integration of computational modeling and experimental recordings in real myocytes, as performed by dynamic clamp, is a useful tool in enhancing our understanding of various components of normal cardiac electrophysiology, but also arrhythmogenic mechanisms in a pathological condition, especially when fully integrated with experimental data.