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.

A 2-dimensional computational model to analyze the effects of cellular heterogeinity on cardiac pacemaking

The mechanical action of the heart is made possible in response to electrical events that involve the cardiac cells, a property that classifies the heart tissue between the excitable tissues. At the cellular level, the electrical event is the signal that triggers the mechanical contraction, inducing a transient increase in intracellular calcium which, in turn, carries the message of contraction to the contractile proteins of the cell. The primary goal of my project was to implement in CUDA (Compute Unified Device Architecture, an hardware architecture for parallel processing created by NVIDIA) a tissue model of the rabbit sinoatrial node to evaluate the heterogeneity of its structure and how that variability influences the behavior of the cells. In particular, each cell has an intrinsic discharge frequency, thus different from that of every other cell of the tissue and it is interesting to study the process of synchronization of the cells and look at the value of the last discharge frequency if they synchronized.

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.