ContHeart : software for monitoring isolated cardiomyocyte shortening

ContHeart is a software designed to analyze cardiomyocyte contractile dynamics using Canny’s method for edge detection in recorded videos. It is a tool that allows researchers (users) to post-analyze video usually captured in their work routine, without special capture apparatus. The software has a user-friendly graphical interface in which the user can apply filters and modify parameters to optimize edge detection and shortening measurement. Therefore, the software quickly generates reliable data on the variation of cell dimensions over time, which can be interpreted by the researcher. We believe researchers will find here a powerful tool to enhance the reach of their basic cardiovascular research, allowing them to include the cardiomyocytes shortening analysis on their work. That might increase the scope of knowledge within the field, as the effect of different pathophysiological conditions may be analyzed on cardiomyocyte contraction

Development of a Novel Dataset and Tools for Non-Invasive Fetal Electrocardiography Research

This PhD thesis presents the development of a novel open multi-modal dataset for advanced studies on fetal cardiological assessment, along with a set of signal processing tools for its exploitation. The Non-Invasive Fetal Electrocardiography (ECG) Analysis (NInFEA) dataset features multi-channel electrophysiological recordings characterized by high sampling frequency and digital resolution, maternal respiration signal, synchronized fetal trans-abdominal pulsed-wave Doppler (PWD) recordings and clinical annotations provided by expert clinicians at the time of the signal collection. To the best of our knowledge, there are no similar dataset available. The signal processing tools targeted both the PWD and the non-invasive fetal ECG, exploiting the recorded dataset. About the former, the study focuses on the processing aimed at the preparation of the signal for the automatic measurement of relevant morphological features, already adopted in the clinical practice for cardiac assessment. To this aim, a relevant step is the automatic identification of the complete and measurable cardiac cycles in the PWD videos: a rigorous methodology was deployed for the analysis of the different processing steps involved in the automatic delineation of the PWD envelope, then implementing different approaches for the supervised classification of the cardiac cycles, discriminating between complete and measurable vs. malformed or incomplete ones. Finally, preliminary measurement algorithms were also developed in order to extract clinically relevant parameters from the PWD. About the fetal ECG, this thesis concentrated on the systematic analysis of the adaptive filters performance for non-invasive fetal ECG extraction processing, identified as the reference tool throughout the thesis. Then, two studies are reported: one on the wavelet-based denoising of the extracted fetal ECG and another one on the fetal ECG quality assessment from the analysis of the raw abdominal recordings. Overall, the thesis represents an important milestone in the field, by promoting the open-data approach and introducing automated analysis tools that could be easily integrated in future medical devices

An apparatus for high throughput muscle cell experimentation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.MIT Science Library copy: printed in pages versus leaves.Also issued in pages.Includes bibliographical references (leaves 183-197).The cardiac ventricular muscle cell (myocyte) is a key experimental system for exploring the mechanical properties of the diseased and healthy heart. The myocyte experimental model provides a higher level of physiological relevance than molecular or myofibril studies while avoiding problems inherent to multicellular preparations including heterogeneity of cell types and diffusion limited extracellular spaces. Millions of primary myocytes that remain viable for four to six hours can be readily isolated from animal models. However, the mechanical properties of only a few physically loaded myocytes can be explored in this time period using current, bulky and expensive instrumentation. In this thesis, a prototype instrument is described that is modular and inexpensive and could form the basis of an array of devices for probing the mechanical properties of single mammnalian myocytes in parallel. This would greatly increase the throughput of scientific experimentation and could be applied as a high content screening instrument in the pharmaceutical industry providing information at the level of a critical cellular phenotype, myocyte mechanical properties, for drug development and toxicology studies.(cont.) The design, development and experimental verification of the modular instrument are presented here. The mathematical, mechanical and electrical characteristics of the novel force sensor and actuator system, Ho control implementation and data processing methodology are discussed. Finally, the functionality of the instrument is demonstrated by implementing novel methodologies for loading and attaching healthy, single mammalian ventricular myocytes to the force sensor and actuator and measuring their isometric twitch force and passive dynamic stiffness at varied sarcomere lengths.by Michael G. Garcia-Webb.Ph.D

Digital capture of the histological microarchitecture in the myometrium and its implications for the propagation of electrophysiological excitation.

Coordination of uterine contractions during labour is critical for successful delivery. The mechanisms underlying this coordination are not fully understood. Propagation of contraction signals has previously been observed to occur through transmission of electrical excitation waves. This thesis aims to examine the histological microarchitecture of the muscular layer of the uterus (myometrium) and determine how this structure affects the propagation of excitation by means of in silico three-dimensional reconstruction of the myometrium and numerical simulations of a spatially structured excitation-relaxation model. A key aim of the in silico reconstruction of the smooth muscle architecture of the myometrium is to identify structural features that correspond to the control of excitation behaviour in the myometrium. This examination is aided by analysis of excitation patterns observed in multi-electrode array recordings. The reconstruction is subsequently used as a basis for simulating electrical activity in the myometrium. Novel structural features are identified here that are located at the initiation points of electrical activity and are proposed to be the pacemaker sites in rat myometrium. Furthermore, boundary of low connectivity across the mesometrial border was observed in the rat, which corresponds to the termination of excitation waves observed in multielectrode array recordings. In addition, bridges of smooth muscle cells connecting the inner and outer layers of the myometrium were observed in both rat and human myometrium. Taken together these three features suggest a novel mechanism for control of contraction in the rat myometrium; an analogous mechanism is proposed for the human myometrium. The results presented in this thesis could provide an explanation for the patterns of excitation propagation observed in human and rat uteri. Further refinements of the methods used here are outlined and expected to generate a more detailed visualisation of the structures underpinning these mechanisms

Optical imaging of cardiac atrial activation and repolarisation in genetically altered models

A method for developing an optical mapping system to quantify electrical activation and repolarisation in murine left atria was created. The spread of activation is important in understanding the mechanisms for the rhythm of the heart in healthy and diseased states as cardiovascular disease is the leading cause of death worldwide. The activation spread was recorded using a novel 2nd generation high resolution (128x2048 pixels) CMOS camera with the voltage sensitive dye di-4-ANEPPS. Algorithms for automatic quantification of action potential duration and conduction velocities were implemented in MATLAB. Optical mapping results were validated against monophasic action potentials and microelectrode measurements showing comparable duration measurements. A genetic mouse model of atrial fibrillation was used (Pitx2c$^+$$^/$$^-$) and was found to have a shorter action potential duration in the left atrium compared to wild-type mice. The results showed a preferential antiarrhythmic effect of the sodium channel blocker, flecainide, to the left atrium of Pitx2c$^+$$^/$$^-$ mice. A second mouse model was used to mimic arrhythmogenic right ventricular cardiomyopathy (plako$^+$$^/$$^-$). No significant changes were witnessed in young sedentary cohorts at baseline and flecainide slowed conduction in both WT and plako$^+$$^/$$^-$. In endurance trained mice, a prolongation of the effective refractory period was seen after flecainide treatment. Plako$^+$$^/$$^-$ sedentary mice treated with dihydrotestosterone showed a prolongation in action potential duration

The second generation of the CCCM system for in-vitro cardiac tissue engineering.

Cardiovascular disease is the leading cause of death worldwide. When a myocardial infarction occurs, scar tissue compensates the damaged myocardial tissue. This scar tissue increases the stiffness of the heart tissue, reduces the heart’s function, and finally leads to the heart failure (HF) disease. To have the tissue engraftment, in-vitro cardiac tissue should have the same properties as the native mature cardiac tissue. However, current in-vitro cell culture technologies fail to accurately recreate the in-vivo like mechanically physiological environment for in-vitro cardiac tissue culture, and therefore, fail to regenerate the in-vivo like mature cardiac tissue. Hence, a microfluidic cardiac cell culture model (CCCM) system was developed to better recreate the cellular environment and advance cardiac regeneration. CCCM system replicates the hemodynamic loading and unloading conditions occurring inside the left ventricle of a heart. With this system, different pressures of human heart conditions may be replicated for a variety of clinical and physiologic conditions. For proof-of-concept, embryonic chick cardiac cells with normal heart condition were applied. Compared to the tissue cultured in a static condition, tissues stimulated in the CCCM system achieved an in-vivo like cardiac matured phenotype, had higher proliferating rate, showed more maturity, and expressed more contractile proteins. These results demonstrated that the CCCM system can be used to study the behavior of cardiomyocytes in different mechanical heart conditions and to create mature cardiac tissue which will benefit cardiac tissue transplant for HF

Modulation of cardiac muscle contractility by phosphorylation, HCM and DCM causing mutations and small molecules

Mutations in sarcomeric proteins that cause familial hypertrophic cardiomyopathy and dilated cardiomyopathy have been shown to abolish the coupled relationship between troponin I phosphorylation and myofilament Ca2+-sensitivity, a phenomenon referred to as uncoupling. In normal heart, PKA phosphorylation of troponin I Ser22 and 23 leads to a 2-fold decrease in Ca2+-sensitivity and a corresponding increase in the rate of Ca2+ release from TnC and is essential for the lusitropic response to adrenergic stimulation. Therefore, uncoupling results in a blunted response to β1-adrenergic activation and has been demonstrated in animal models with hypertrophic cardiomyopathy and dilated cardiomyopathy mutations at a cellular, tissue and whole animal level. However, the molecular mechanisms and physiological relevance of uncoupling as a common phenomenon in cardiomyopathy-associated sarcomeric mutations are not well-understood. In this study, I have employed a multidisciplinary approach to probe for the presence of troponin uncoupling in mutation-containing cardiomyopathy models at an atomistic, molecular and cellular level. I have employed molecular dynamics simulation to elucidate how the structure and dynamics of troponin can give rise to physiological properties of cardiomyopathy. Additionally, I have investigated small molecules analogues of EGCG and silybin for recoupling properties that can restore the abolished relationship between troponin I phosphorylation and myofilament Ca2+-sensitivity in vitro, identifying a number of promising recoupling agents via in vitro motility assay. Moreover, I have demonstrated uncoupling at the cellular level as a blunting of the time to relaxation in intact cardiomyocytes following β-adrenergic stimulation and have developed a methodology that is capable of distinguishing cellularly-active recoupling molecules from candidates that are toxic. I have identified two promising recoupling agents, silybin B and resveratrol, using the contractile study presented herein, demonstrated by a reversal of the blunted phenotype. My investigation has demonstrated the feasibility of small molecules as recoupling agents and their therapeutic potential.Open Acces

Multiscale image analysis of calcium dynamics in cardiac myocytes

Cardiac myocytes constitute a unique physiological system. They are the muscle cells that build up heart tissue and provide the force to pump blood by synchronously contracting at every beat. This contraction is regulated by calcium concentration, among other ions, which exhibits a very complex behaviour, rich in dynamical states at the molecular, cellular and tissue levels. Details of such dynamical patterns are closely related to the mechanisms responsible for cardiac function and also cardiac disease, which is the first cause of death in the modern world. The emerging field of translational cardiology focuses on the study of how such mechanisms connect and influence each other across spatial and temporal scales finally yielding to a certain clinical condition. In order to study such patterns, we benefit from the recent and very important advances in the field of experimental cell physiology. In particular, fluorescence microscopy allows us to observe the distribution of calcium in the cell with a spatial resolution below the micron and a frame rate around the millisecond, thus providing a very accurate monitoring of calcium fluxes in the cell. This thesis is the result of over five years' work on biological signal and digital image processing of cardiac cells. During this period of time the aim has been to develop computational techniques for extracting quantitative data of physiological relevance from microscopy images at different scales. The two main subjects covered in the thesis are image segmentation and classification methods applied to fluorescence microscopy imaging of cardiac myocytes. These methods are applied to a variety of problems involving different space and time scales such as the localisation of molecular receptors, the detection and characterisation of spontaneous calcium-release events and the propagation of calcium waves across a culture of cardiac cells. The experimental images and data have been provided by four internationally renowned collaborators in the field. It is thanks to them and their teams that this thesis has been possible. They are Dr. Leif Hove-Madsen from the Institut de Ciències Cardiovasculars de Catalunya in Barcelona, Prof. S. R. Wayne Chen from the Department of Physiology and Pharmacology in the Libin Cardiovascular Institute of Alberta, University of Calgary, Dr. Peter P. Jones from the Department of Physiology in the University of Otago, and Prof. Glen Tibbits from the Department of Biomedical Physiology & Kinesiology at the Simon Fraser University in Vancouver. The work belongs to the biomedical engineering discipline, focusing on the engineering perspective by applying physics and mathematics to solve biomedical problems. Specifically, we frame our contributions in the field of computational translational cardiology, attempting to connect molecular mechanisms in cardiac cells up to cardiac disease by developing signal and image-processing methods and machine-learning methods that are scalable through the different scales. This computational approach allows for a quantitative, robust and reproducible analysis of the experimental data and allows us to obtain results that otherwise would not be possible by means of traditional manual methods. The results of the thesis provide specific insight into different cell mechanisms that have a non-negligible impact at the clinical level. In particular, we gain a deeper knowledge of cell mechanisms related to cardiac arrhythmia, fibrillation phenomena, the emergence of alternans and anomalies in calcium handling due to cell ageing.Els cardiomiòcits constitueixen un sistema fisiològic únic. Són les cèl·lules muscular que formen el cor i proporcionen la força per bombar la sang fent una contracció a cada batec. La regulació d'aquesta contracció es fa mitjançant concentració de calci (entre d'altres ions) i presenta una dinàmica molt complexa tant a l'escala molecular, cel·lular i de teixit. Detalls d'aquesta dinàmica estan fortament relacionats amb la funció cardíaca i per sobre de tot amb patologies cardíaques. La disciplina emergent de la cardiologia translacional es centra en l'estudi de com aquests mecanismes es connecten i s'influencien entre sí a través de diferents escales temporals i espacials finalment donant lloc a condicions clíniques. Per estudiar aquests patrons ens beneficiem dels recents avenços en fisiologia i biologia cel·lular. En particular, la microscòpia de fluorescència ens permet observar la distribució de calci dins una cèl·lula amb una resolució espacial per sota de la micra i temporal per sota del mil·lisegon, permetent un monitoratge acurat dels fluxos de calci en la cèl·lula cardíaca. Aquesta tesi és el resultat de més de cinc anys de feina en processament de senyal i imatge de cardiomiòcits humans. Durant aquest període de temps l'objectiu principal ha estat desenvolupar tècniques computacionals per extraure dades d'imatges de microscòpia amb rellevància fisiològica. Els dos temes principals coberts a la tesi són segmentació d'imatges i classificadors, aplicats a imatges de microscòpia de fluorescència de cardiomiòcits. Els mètodes s'apliquen a diferents problemes involucrant diverses escales espacials i temporals, des de determinar la posició de receptors a l’escala molecular passant detectar i caracteritzar alliberament espontani de calci intracel·lular fins a la propagació d'ones de calci en un cultiu de cèl·lules cardíaques. Les dades experimentals han estat proporcionades per quatre col·laboradors de renom internacional. És gràcies a ells i els seus equips que aquesta tesi ha estat possible. Són el Dr. Leif Hove-Madsen de l'Institut de Ciències Cardiovasculars de Catalunya a Barcelona, el Dr. S.R. Wayne Chen del Department of Physiology and Pharmacology al Libin Cardiovascular Institute of Alberta, University of Calgary, el Dr. Peter P. Jones del Department of Physiology a la University of Otago, i el Dr. Glen Tibbits del Department of Biomedical Physiology & Kinesiology de la Simon Fraser University a Vancouver. El treball pertany a la disciplina de la enginyeria biomèdica, fent èmfasi a la perspectiva de l'enginyeria, aplicant física i matemàtiques per solucionar problemes de la biomedicina. Específicament, s'emmarca en la cardiologia translacional computacional, mirant de connectar mecanismes a l’escala molecular amb patologies cardíaques mitjançant tècniques de processament de dades i aprenentatge automàtic que són escalables a les diferents escales d’aplicació. Aquest enfocament computacional permet una anàlisi quantitatiu, robust i reproduïble de les dades experimentals i ens permet d'obtenir resultats que serien impossibles d'assolir mitjançant els tradicionals mètodes manuals. Els resultats que proporciona la tesi han permès aprofundir en l'enteniment de diferents mecanismes fisiològics amb impacte en l'àmbit clínic. Particularment hem permès d’assolir coneixements relacionats amb l'arítmia cardíaca, la fibril·lació, processos d'alternança i anomalies relacionades amb l’envelliment

Automated deep phenotyping of the cardiovascular system using magnetic resonance imaging

Across a lifetime, the cardiovascular system must adapt to a great range of demands from the body. The individual changes in the cardiovascular system that occur in response to loading conditions are influenced by genetic susceptibility, and the pattern and extent of these changes have prognostic value. Brachial blood pressure (BP) and left ventricular ejection fraction (LVEF) are important biomarkers that capture this response, and their measurements are made at high resolution. Relatively, clinical analysis is crude, and may result in lost information and the introduction of noise. Digital information storage enables efficient extraction of information from a dataset, and this strategy may provide more precise and deeper measures to breakdown current phenotypes into their component parts. The aim of this thesis was to develop automated analysis of cardiovascular magnetic resonance (CMR) imaging for more detailed phenotyping, and apply these techniques for new biological insights into the cardiovascular response to different loading conditions. I therefore tested the feasibility and clinical utility of computational approaches for image and waveform analysis, recruiting and acquiring additional patient cohorts where necessary, and then applied these approaches prospectively to participants before and after six-months of exercise training for a first-time marathon. First, a multi-centre, multi-vendor, multi-field strength, multi-disease CMR resource of 110 patients undergoing repeat imaging in a short time-frame was assembled. The resource was used to assess whether automated analysis of LV structure and function is feasible on real-world data, and if it can improve upon human precision. This showed that clinicians can be confident in detecting a 9% change in EF or a 20g change in LV mass. This will be difficult to improve by clinicians because the greatest source of human error was attributable to the observer rather than modifiable factors. Having understood these errors, a convolutional neural network was trained on separate multi-centre data for automated analysis and was successfully generalizable to the real-world CMR data. Precision was similar to human analysis, and performance was 186 times faster. This real-world benchmarking resource has been made freely available (thevolumesresource.com). Precise automated segmentations were then used as a platform to delve further into the LV phenotype. Global LVEFs measured from CMR imaging in 116 patients with severe aortic stenosis were broken down into ~10 million regional measurements of structure and function, represented by computational three-dimensional LV models for each individual. A cardiac atlas approach was used to compile, label, segment and represent these data. Models were compared with healthy matched controls, and co-registered with follow-up one year after aortic valve replacement (AVR). This showed that there is a tendency to asymmetric septal hypertrophy in all patients with severe aortic stenosis (AS), rather than a characteristic specific to predisposed patients. This response to AS was more unfavourable in males than females (associated with higher NT-proBNP, and lower blood pressure), but was more modifiable with AVR. This was not detected using conventional analysis. Because cardiac function is coupled with the vasculature, a novel integrated assessment of the cardiovascular system was developed. Wave intensity theory was used to combine central blood pressure and CMR aortic blood flow-velocity waveforms to represent the interaction of the heart with the vessels in terms of traveling energy waves. This was performed and then validated in 206 individuals (the largest cohort to date), demonstrating inefficient ventriculo-arterial coupling in female sex and healthy ageing. CMR imaging was performed in 236 individuals before training for a first-time marathon and 138 individuals were followed-up after marathon completion. After training, systolic/diastolic blood pressure reduced by 4/3mmHg, descending aortic stiffness decreased by 16%, and ventriculo-arterial coupling improved by 14%. LV mass increased slightly, with a tendency to more symmetrical hypertrophy. The reduction in aortic stiffness was equivalent to a 4-year reduction in estimated biological aortic age, and the benefit was greater in older, male, and slower individuals. In conclusion, this thesis demonstrates that automating analysis of clinical cardiovascular phenotypes is precise with significant time-saving. Complex data that is usually discarded can be used efficiently to identify new biology. Deeper phenotypes developed in this work inform risk reduction behaviour in healthy individuals, and demonstrably deliver a more sensitive marker of LV remodelling, potentially enhancing risk prediction in severe aortic stenosis