Three Dimensional Panoramic Fast Flourescence Imaging of Cardiac Arryhtymias in the Rabbit Heart

Cardiac high spatio-temporal optical mapping provides a unique opportunity to investigate the dynamics of propagating waves of excitation during ventricular arrhythmia and defibrillation. However, studies using single camera imaging systems are hampered by the inability to monitor electrical activity from the entire surface of the heart. We have developed a three dimensional panoramic imaging system which allows high-resolution and high-dynamic-range optical mapping from the entire surface of the heart. Rabbit hearts (n=4) were Langendorff perfused and imaged by the system during sinus rhythm, epicardial pacing, and arrhythmias. The reconstructed 3D electrical activity provides us with a powerful tool to investigate fundamental mechanisms of arrhythmia and antiarrhythmia therapy in normal and diseased hearts

RHYTHM: An Open Source Imaging Toolkit for Cardiac Panoramic Optical Mapping

Fluorescence optical imaging techniques have revolutionized the field of cardiac electrophysiology and advanced our understanding of complex electrical activities such as arrhythmias. However, traditional monocular optical mapping systems, despite having high spatial resolution, are restricted to a two-dimensional (2D) field of view. Consequently, tracking complex three-dimensional (3D) electrical waves such as during ventricular fibrillation is challenging as the waves rapidly move in and out of the field of view. This problem has been solved by panoramic imaging which uses multiple cameras to measure the electrical activity from the entire epicardial surface. However, the diverse engineering skill set and substantial resource cost required to design and implement this solution have made it largely inaccessible to the biomedical research community at large. To address this barrier to entry, we present an open source toolkit for building panoramic optical mapping systems which includes the 3D printing of perfusion and imaging hardware, as well as software for data processing and analysis. In this paper, we describe the toolkit and demonstrate it on different mammalian hearts: mouse, rat, and rabbit

Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein-nanomaterial hybrids

Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine

Cardiac Remodeling Of Conduction, Repolarization and Excitation-Contraction Coupling: From Animal Model to Failing Human Heart

Heart failure is one of the leading causes of death worldwide, with rising impact with the increasing ageing population. This is in sharp contrast with the limited and non-ideal therapies available. Approximately 50% of deaths from heart failure are sudden and unexpected, and presumably the consequence of lethal ventricular arrhythmias. Despite significant reduction of mortality from sudden cardiac death achieved by ICDs and drugs such as beta-blockers, there remains a large room for improving the survivability of heart failure patients by advancing our understanding of arrhythmogenesis from molecular level to multi-cellular tissue level. Another important aspect of heart failure is abnormal excitation-contraction: EC) coupling and calcium handling, functional changes of which exert great impact on both arrhythmia vulnerability and pump failure. Advancing the understanding the remodeling of EC coupling and calcium handling might provide potential molecular and anatomical targets for clinical intervention. In this dissertation, I first developed two optical imaging systems: both hardware and software) for quantifying the conduction, repolarization and excitation-contraction coupling. The first one is the panoramic imaging system for mapping the entire ventricular epicardium of a rabbit heart. The second one is the dual imaging system for simultaneous measurement of action potential and calcium transient. Using the systems I developed, I conducted two rabbit studies to investigate the role electrical instability and structural heterogeneity in the induction and maintenance of arrhythmias. We first identified the importance of both dynamic instability and effective tissue size in the spontaneous termination of arrhythmia in the normal rabbit heart. We then identified novel mechanism of how healed myocardial infarction promotes the induction of ventricular arrhythmia. Finally, guided by the knowledge from the animal studies, I studied the failing human heart with the aim to advance our understanding of cardiac electrophysiology in human heart failure. We first demonstrated the transmural heterogeneity of EC coupling in nonfailing heart and identified potential mechanisms of electrical and mechanical dysfunction by quantifying the remodeling of EC coupling. We then studied the remodeling of conduction and repolarization with the aim to determine of the role of dispersion of repolarization and electrical instability in the induction of arrhythmia in human heart failure

A novel method to allow noninvasive, longitudinal imaging of the murine immune system in vivo

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior

Advanced microscopy to elucidate cardiovascular injury and regeneration: 4D light-sheet imaging

The advent of 4-dimensional (4D) light-sheet fluorescence microscopy (LSFM) has provided an entry point for rapid image acquisition to uncover real-time cardiovascular structure and function with high axial resolution and minimal photo-bleaching/-toxicity. We hereby review the fundamental principles of our LSFM system to investigate cardiovascular morphogenesis and regeneration after injury. LSFM enables us to reveal the micro-circulation of blood cells in the zebrafish embryo and assess cardiac ventricular remodeling in response to chemotherapy-induced injury using an automated segmentation approach. Next, we review two distinct mechanisms underlying zebrafish vascular regeneration following tail amputation. We elucidate the role of endothelial Notch signaling to restore vascular regeneration after exposure to the redox active ultrafine particles (UFP) in air pollutants. By manipulating the blood viscosity and subsequently, endothelial wall shear stress, we demonstrate the mechanism whereby hemodynamic shear forces impart both mechanical and metabolic effects to modulate vascular regeneration. Overall, the implementation of 4D LSFM allows for the elucidation of mechanisms governing cardiovascular injury and regeneration with high spatiotemporal resolution

Evaluation of sCD40L in heart regeneration after cryoinjury-induced myocardial infarction in zebrafish: methodological approach

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016O enfarte agudo do miocárdio, ou ataque cardíaco, constitui uma das maiores causas de mortalidade e morbilidade no ser humano. De um modo geral, o enfarte tem como origem uma obstrução das artérias coronárias, em consequência da qual o tecido a jusante fica em isquemia, havendo necrose do mesmo. Como o coração humano é incapaz de regeneração, o miocárdio afectado cicatriza, num processo que envolve inflamação e reparação, o que origina a perda de contractilidade do miocárdio, podendo levar a insuficiência cardíaca. O peixe-zebra (Danio rerio) não possui a limitação do potencial regenerativo, referida anteriormente, tendo já sido evidenciada a capacidade regenerativa de até 25% do ventrículo criocauterizado. Este organismo é um modelo adequado para estudos de desenvolvimento cardíaco e doenças humanas relacionadas, já que detém uma homologia com o ser humano na programação da expressão génica essencial, bem como na actividade eléctrica do coração. Estudos anteriores do grupo de investigação onde esta tese foi realizada mostram níveis do ligando solúvel de CD40 (sCD40L), uma glicoproteína transmembranar, em pacientes de enfarte agudo do miocárdio está associado a disfunção endotelial persistente e a reparação cardíaca pós-enfarte comprometida. A associação da sCD40L à reparação e remodelação do miocárdio pós-enfarte não foi ainda explorada, pelo que é um tópico que carece de esclarecimentos. Para tal, estudos usando o modelo de peixe-zebra são necessários de forma a compreender os mecanismos associados ao papel de sCD40L na recuperação cardíaca após o enfarte do miocárdio. Assim, o objectivo principal deste projecto foi a implementação de protocolos que permitiram avaliar o papel do sCD40L na regeneração do miocárdio de peixe-zebra após enfarte agudo do miocárdio induzido por uma sonda a frio. Para atingir o objectivo, foram estabelecidos vários protocolos como a criolesão, a colheita de sangue, metodologias histológicas e de microscopia. O projecto também visou a implementação do protocolo de um Modelo de Regeneração Comprometida, contribuindo para o objectivo principal proposto. Prospectivamente, esta dissertação pode contribuir para estudos futuros em peixe-zebra, que porventura providenciarão translação para investigação clínica envolvendo reparação cardíaca pós-enfarte, em pacientes humanos. Para cumprir o objectivo proposto, os peixes-zebra adultos, foram sujeitos a criolesão de modo a induzir um enfarte do miocárdio e, por conseguinte, monitorizados em vários pontos temporais. Um modelo de regeneração comprometida foi também estudado, através da administração de glucocorticóides imunossupressores aos peixes. A sCD40L foi avaliada no sangue recorrendo a Enzyme-Linked Immunosorbent Assay (ELISA). Técnicas de histologia e microscopia de campo claro e Light sheet foram comparadas. A microscopia de fluorescência Light Sheet foi usada para comparação histológica do tecido cardíaco de diferentes linhas transgénicas e entre modelos de regeneração. Com o presente trabalho, foram estabelecidas as metodologias inovadoras propostas, não implementadas anteriormente no Instituto de Medicina Molecular (iMM). Obtiveram-se também resultados preliminares que demonstram diferenças na morfologia do enfarte entre modelos de reparação bem como concentrações elevadas de sCD40L no modelo de reparação cardíaca exposto a imunossupressores.Myocardial Infarction (MI), or heart attack, is an important clinical condition. After a MI, the human heart is unable to regenerate and further complications leading to heart failure can occur. Zebrafish (Danio rerio) has the ability to regenerate up to 25% of cyocauterized ventricle. The fish retains a high homology with humans, in terms of gene expression and electric heart activity, thus being a suitable model for heart repair studies. Previous work from research group were the present thesis was conducted shown levels of soluble CD40 ligand (sCD40L), a transmembrane glycoprotein, associate with persistent endothelial dysfunction and compromised cardiac repair after-MI. Further mechanistic studies are mandatory to better understand association of sCD40L to heart repair, which can be achieved using zebrafish model. This thesis objective was to establish protocols for evaluation of longitudinal variations of sCD40L after cryoinjury induced-MI alongside with myocardial repair in zebrafish. To achieve this objective, protocols of cryoinjury, blood collection, histology and microscopy were compared and optimized. Furthermore, the project also intended to implement a Compromised Regeneration/Immunosuppression Model. Prospectively, this dissertation can contribute to further studies in zebrafish that would provide translation for clinical research involving cardiac repair post-MI in human patients. In this project, several innovative techniques were established. Also, preliminary results were obtained showing altered cardiac tissue histology and higher concentrations of sCD40L on fish of the Compromised Regeneration Model (with zebrafish exposed to immunosuppressors)

Advanced microscopy to elucidate cardiovascular injury and regeneration: 4D light-sheet imaging

The advent of 4-dimensional (4D) light-sheet fluorescence microscopy (LSFM) has provided an entry point for rapid image acquisition to uncover real-time cardiovascular structure and function with high axial resolution and minimal photo-bleaching/-toxicity. We hereby review the fundamental principles of our LSFM system to investigate cardiovascular morphogenesis and regeneration after injury. LSFM enables us to reveal the micro-circulation of blood cells in the zebrafish embryo and assess cardiac ventricular remodeling in response to chemotherapy-induced injury using an automated segmentation approach. Next, we review two distinct mechanisms underlying zebrafish vascular regeneration following tail amputation. We elucidate the role of endothelial Notch signaling to restore vascular regeneration after exposure to the redox active ultrafine particles (UFP) in air pollutants. By manipulating the blood viscosity and subsequently, endothelial wall shear stress, we demonstrate the mechanism whereby hemodynamic shear forces impart both mechanical and metabolic effects to modulate vascular regeneration. Overall, the implementation of 4D LSFM allows for the elucidation of mechanisms governing cardiovascular injury and regeneration with high spatiotemporal resolution