The aim of this master thesis is to create a simple 3D electro-anatomical model of cardiac tissue that will be able to simulate the electrical activation in both a healthy heart and a heart with arrhytmogenic substrate. The model of electrical activation is realized in the COMSOL Multiphysics, simulation software for modelling using the finite element method. The Fitzhugh-Nagumo equation was used to model the excitatory feature of the myocardium and 2D models of myocardial tissue describing the propagation of action potential in healthy tissue, ischemic tissue, spontaneous action potential formation in the SA node, and spiral wave formation were first developed based on appropriate parameters. Subsequently, simplified 3D models of the heart describing the spread of excitement in a healthy heart, in the presence of accessory pathway and in third-degree atrioventricular block were created. The simplified 3D heart model offers a compromise between computational load and model complexity and can be used as a diagnostic tool for tissue and whole heart adjustment with appropriate equation parameter settings.

Horizontal cells (HCs) are retinal interneurons which provide feedback to photoreceptors to produce visual contrast. They are depolarized by glutamate released from photoreceptors, leading to a constant influx of Ca2+ which would be fatal to most neurons. In addition, HCs present spontaneous Ca2+-based action potentials, which are poorly understood and whose function is unknown. Given these unique Ca2+ dynamics, the present thesis sought to define action potentials (APs) and mechanisms of Ca2+ homeostasis in HCs. APs were observed in isolated goldfish HCs with electrophysiology, Ca2+ imaging, and voltage-sensitive dye imaging. Pharmacological inhibition of ion channels suggests APs required extracellular Ca2+ entry via L-type Ca2+ channels, followed by Ca2+-induced Ca2+ release from ryanodine receptors. Next, we developed a novel system to classify all four HC subtypes in vitro, and validated it with immunocytochemistry for a subtype-specific biomarker. All subtypes presented APs, although frequency and duration varied by subtype. APs were also found in HCs of tissue slices prepared from whole retina, where similar trends were found between subtype, frequency, and duration. This highlights subtype-specific differences in Ca2+ dynamics. Lastly, [Ca2+]i was monitored throughout hypoxia in HCs of the hypoxia-tolerant goldfish and the hypoxia-sensitive rainbow trout. In Ca2+ imaging experiments, hypoxia destabilized [Ca2+]i in HCs of trout; but in goldfish, HCs were resistant to the effects of hypoxia. However, when mitochondrial ATP-dependent K+ (mKATP) channels were inhibited, goldfish HCs lost the ability to maintain [Ca2+]i homeostasis during hypoxia. By contrast, in trout HCs, opening of mKATP stabilized [Ca2+]i during hypoxia. Furthermore, in goldfish, hypoxia protected against increases in [Ca2+]i caused by inhibiting glycolysis, showing that hypoxia is not just tolerated, but is actively protective in goldfish HCs. The present thesis includes the first comprehensive description of spontaneous Ca2+-based APs in HCs, and introduces the first cellular model of intrinsic hypoxic neuroprotection in the vertebrate retina.

Damage to the central nervous system (CNS) leads to the generation of an immune response which culminates with the encapsulation of the damaged area. The encapsulation, known as a glial scar, essentially breaks neural signal pathways and blocks signal transmissions to and from the CNS. The effect is the loss of motor and sensory control for the damaged individual. One method that has been used successfully to treat this problem is the use of a brain-machine interface (BMI) which can intercept signals from the brain and use these signals to control a machine. Although there are many types of BMI devices, implantable devices show the greatest promise with the ability to target specific areas of the CNS, with reduced noise levels and faster signal interception, and the fact that they can also be used to send signals to neurons. The largest problem that has plagued this type of BMI device is that the materials that have been used for their construction are not chemically resilient, elicit a negative biological response, or have difficulty functioning for extended periods of time in the harsh body environment. Many of these implantable devices experience catastrophic failure within weeks to months because of these negative factors. New materials must be examined to advance the future utilization of BMI devices to assist people with CNS damage or disease. We have proposed that two semiconductor materials, cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD), which should provide solutions to the material biocompatibility problems experienced by implantable BMI devices. We have shown in this study that these two materials show chemical resilience to neuronal cellular processes, and we show evidence which indicates that these materials possess good biocompatibility with neural cell lines that, in the worst case, is comparable to celltreated polystyrene and, in most cases, even surpasses polystyrene. We have utilized 3C-SiC within an electrode device and activated the action potential of differentiated PC12 cells. This work details our initial efforts to modify the surfaces of these materials in order to improve cellular interaction and biocompatibility, and we examine our current and future work on improving our implantable BMI devices.

No description available.

A well documented laboratory setup, leech preparation process, and bio-potential data recording process are needed. Repeatability and quality data recordings are essential and thus dictate the requirements of the laboratory setup and processes listed above. Advances in technology have both helped and hindered this development. While very precise equipment is required to record the low voltage bio-potentials, noisy electronic equipment and wires surrounding the work area provide high levels of interference. Proper laboratory setup and data recording processes, however, limit the unwanted interference. Quality data can only be recorded from a properly handled and prepared leech subject. Proper setup and procedures result in quality recordings which lend a clean signal for furthering the understanding of nerve functionality. The electrophysiology lab at California Polytechnic State University in San Luis Obispo is an example of a proven lab setup for high quality signal capture.

Yes / Introduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86.

No / Evaluation of drug candidates in in-vitro assays of action potential duration (APD) is one component of preclinical safety assessment. Current assays are limited by technically-demanding, time-consuming electrophysiological methods. This study aimed to assess whether a voltage-sensitive dye-based assay could be used instead. Methods Optical APs were recorded using di-4-ANEPPS in electrically field stimulated Beagle left ventricular midmyocardial myocytes (LVMMs). Pharmacological properties of di-4-ANEPPS on the main cardiac ion channels that shape the ventricular AP were investigated using IonWorks™ and conventional electrophysiology. Effects of 9 reference drugs (dofetilide, E4031, d-sotalol, ATXII, cisapride, terfenadine, alfuzosin, diltiazem and pinacidil) with known APD-modulating effects were assessed on optically measured APD at 1 Hz. Results Under optimum conditions, 0.1 μM di-4-ANEPPS could be used to monitor APs paced at 1 Hz during nine, 5 s exposures without altering APD. di-4-ANEPPS had no effect on either hIERG, hINa, hIKs and hIto currents in transfected CHO cells (up to 10 µM) or ICa,L current in LVMMs (at 16 µM). di-4-ANEPPS had no effect on APs recorded with microelectrodes at 1 or 0.5 Hz over a period of 30 min di-4-ANEPPS displayed the sensitivity to record changes in optically measured APD in response to altered pacing frequencies and sequential vehicle additions did not affect the optically measured APD. APD data obtained with 9 reference drugs were as expected except (i) d-sotalol-induced increases in duration were smaller than those caused by other IKr blockers and (ii) increases in APD were not detected using low concentrations of terfenadine. Discussion Early in drug discovery, the di-4-ANEPPS-based method can reliably be used to assess drug effects on APD as part of a cardiac risk assessment strategy.

Cardiac arrhythmias are caused by a disruption of the normal initiation or propagation of electrical impulses in the heart. Hundreds of mutations in genes encoding ion channels or ion channel regulatory proteins are linked to congenital arrhythmia syndromes that increase the risk for sudden cardiac death. This dissertation focuses on how mutations in a gene (KCNQ1) that encodes a voltage-gated K+ ion channel (Kv7.1) can disrupt proper channel function and lead to abnormal repolarization of atrial and ventricular cardiomyocytes. In the heart, Kv7.1 coassembles with a regulatory protein to conduct the slowly activating delayed rectifier K+ current (IKs). Loss-of-function KCNQ1 mutations are linked to type 1 long QT syndrome (LQT1), and typically decrease IKs, which can lead to ventricular action potential (AP) prolongation. In patients, LQT1 is often characterized by an abnormally long corrected QT (QTc) interval on an electrocardiogram (ECG), and increases the risk for polymorphic ventricular tachycardias. KCNQ1 mutations are also linked to atrial fibrillation (AF), but cause a gain-of-function phenotype that increases IKs. Surprisingly, patients diagnosed with both LQT1 and AF are increasingly identified as genotype positive for a KCNQ1 mutation. The first aim of this dissertation was to determine a unique functional phenotype of KCNQ1 mutations linked to both arrhythmia syndromes by functional analyses via the whole-cell patch clamp technique in HEK293 cells. A proportion of patients with LQT1-linked KCNQ1 mutations do not have abnormal QTc prolongation known as latent LQT1. Interestingly, exercise can reveal abnormal QTc prolongation in these patients. During exercise, beta-adrenergic activation stimulates PKA to phosphorylate Kv7.1, causing an increase in IKs to prevent ventricular AP prolongation. Therefore, the second aim of this dissertation was to determine a molecular mechanism of latent LQT1 through functional analyses in HEK293 cells while incorporating pharmacological and phosphomimetic approaches to study PKA regulation of mutant Kv7.1 channels. The findings in this dissertation provide new insight into how KCNQ1 mutations disrupt the function of Kv7.1 in a basal condition or during beta-adrenergic activation. Also, this dissertation suggests these approaches will improve patient management by identifying mutation specific risk factors for patients with KCNQ1 mutations.

Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 μm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD90 with a half maximal effective concentration (EC50) of 4.5 μM. The IKr blocker E4031 and neuroleptic drug risperidone prolonged APD90 with EC50 values of 31 nM and 0.67 μM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m ⋅ s-1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Este trabalho apresenta a modelagem matemática e a simulação de potenciais de ação de unidades motoras de músculos de vertebrados visando a posterior simulação do eletromiograma. Para conseguir isso, inicialmente se fez uma compilação de dados existentes para a distribuição das fibras musculares (FBs) nas unidades motoras (MUs) de vários músculos, e as modelagens matemáticas descritos na literatura para o potencial de ação de uma FB (SFAP) e de uma MU (MUAP). Com base nos dados fisiológicos, primeiro se localizou as FBs em um músculo, por meio de uma aproximação de que as FBs estão rodeadas de outras seis no músculo. Para conseguir isto se construiram hexágonos concêntricos por MU, e posteriormente se localizou as FBs nas MUs, cobrindo uma faixa entre 75 e 2000 FBs, o que corresponde a músculos distais de mamíferos. Depois se fez uma aproximação para a distribuição de 170000 FBs nas 272 MUs da cabeça medial do músculo gastrocnêmio (MG) do gato, conseguindo numa primeira simulação localizar cerca de 70% das FBs para cada MU. Com esta localização das FBs no músculo baseados nos dados da literatura se aproximaram os retardos axonais por uma distribuição gaussiana, com média de 2 ms (gato) ou 10 ms (homem) e com desvio padrão de menos de 0,5 ms, desprezando o atraso axonal nas ramificações axonais, que foi estimado no máximo 29 vezes menor. Para a geração do SFAP trabalhou-se com dois modelos, um analítico, o qual resulta em simulações numéricas demoradas, e, outro numérico baseado na convolução da corrente com uma função peso. Para o modelo numérico dobrou-se imaginariamente o comprimento das FBs, para levar em conta o erro computacional de fim de fibra. O modelo numérico resultou em um tempo de simulação 30 vezes menor que o analítico. Adicionalmente, para simular a captação externa (i.e. na pele), fez-se uma aproximação para a função que modela os eletrodos de superfície de secção circular localizados a uma distância maior que 1,79 mm das FBs, mostrando um espectro similar ao reportado na literatura. Finalmente, os MUAPs obtidos resultavam com formas de onda e espectros similares ao descrito na literatura. Além disto, em certos casos, obtiveram-se MUAPs com indentações, seja localizando as junções neuromusculares em bandas da ordem de 1 mm de espessura, seja quando o tempo de atraso axonal foi considerado junto com a velocidade de condução da FB em função da raiz quadrada do diâmetro da FB. Foram feitas simulações para os MG e bíceps braquial do homem. Neste último caso, foram obtidos MUAPs similares aos captados para pessoas saludáveis, e foi observada a freqüência de disparos dos potenciais de ação do motoneurônio no espectro do MUAP. Quanto às formas dos agrupamentos das FBs em uma MU, não se obtiveram diferenças significativas para as FBs posicionadas homogênea e aleatoriamente, exceto uma ligeira variação nas amplitudes. No entanto, ocurreu uma mudança na faixa espectral, quando as FBs estavam concentradas. / This work presents the mathematical model and simulation of motor unit action potentials of vertebrate muscles aiming at after simulation of the electromyogram. To obtain this, initially, it was made a compilation of several data about the distribution of muscle fibers (FBs) in motor units (MUs) of many muscles, and the mathematical models of the action potential of a single FB (SFAP) and MU (MUAP), reported in previous works. On the basis of this physiological data, first, the FB was located in a muscle, using an approximation in which the FBs are encircled with other six FBs in the muscle. To reach this, concentric hexagons were constructed to build the surface of the MU, and later the FBs were situated in the MU, covering a range between 75 and 2000 FBs, corresponding to mammals extremity muscles. Later, a new approximation were was madein order to distribute the 170000 FBs in the 272 MUs of the medial head of muscle medialis gastrocnemius (MG) of the cat, reaching, in a first simulation, the localization of almost 70% of the FBs at each MU. With the FBs lalready allocated in the muscle, and based in data of previous works, their axonal delay were approximated by a gaussian distribution, with mean of 2 ms (cat) or 10 ms (man) and standard deviation of less than 0,5 ms, discarding the axonal delay in the axonal branching, that were estimated to affectup to 29 times less. To SFAP generation, two models were used, the first analytical, resulting in delayed numerical simulations, and the other based on convolution of the second derivate of the current with a weight function, where the length of the FBs was imaginarily duplicated, in order to consider the end fiber effect. Using this, a simulation time 30 times lesser than the analytical one was obtained. Additionally, so as to simulate the external recording (i.e. in the skin), it was made an approximation to the function that models the circular shape surface electrodes located at distances greater than 1,79 mm of the FBs, showing a similar spectrum reported. Finally, the waves and spectrum of the simulated MUAPs resulted similar to the ones reported in the literature. Beyond this, in certain cases, MUAPs were simulated with some tuned, either locating the neuromuscular junctions with thickness bands of 1 mm, or, when the axonal delay and the FB muscle fiber conduction velocity were considered as a function of the square root fiber diameter. This was simulated for MUAPs of MG and biceps brachii muscles of human beings, in the last case it has reached the waveforms and tuned found in heath subjects, and it was visualized the mean frequency of firing rate at the spectrum. In order to know how much affects grouping for the FBs to waves a MU, they were not found significant differences with FBs located homogeneously and randomly, except a little variation in the amplitude of the MUAP. However, they presented a change in the spectral bandwidth when the FBs are more concentrated.