Doctor of Philosophy

dissertationDoes strain induce changes in the electrical properties of the heart? Does strain affect the microstructure of cardiac myocytes? Others have considered these questions, but have been limited in their findings. I addressed the first question by measuring conduction velocity in papillary muscles in rest conditions and during applied strain. I also applied streptomycin, a nonselective stretch ion channel blocker, in the above conditions. In control, conduction velocity increased with strain before conduction block occurred. When streptomycin was applied conduction velocity peaked at a higher strain, but conduction block remained unchanged. Changes in electrical properties of papillary muscle allowed for changes in conduction velocity. Although streptomycin did not alter the strain at which conduction block occurred, it did shift the peak conduction velocity to a higher strain. The second question was addressed by imaging isolated cardiac ventricular myocytes in varying degrees of contraction and strain using confocal microscopy. The length of transverse tubules (t-tubules), along with cross-section ellipticity, and orientation in myocytes were analyzed for cells in 16% contraction, rest, and 16% strain. Cells in contraction showed an increase in length of t-tubules with less elliptical cross-sections compared to cells in rest. Strained cells showed a decrease in length of t-tubules with less elliptical cross-sections than cells at rest. The orientation of t-tubule cross-sections changed in a similar manner when comparing contracted and strained cells with cells at rest. The transfer of strain to the t-tubule system supports the hypothesis that the motion of t-tubules during contraction and stretch may constitute a mechanism for pumping extracellular fluid

Dynamic regulation of subcellular calcium handling in the atria:modifying effects of stretch and adrenergic stimulation

Atrial fibrillation is the fast and irregular heart rate that occurs when the upper chambers of the heart experience chaotic electrical activation. Three main factors contribute to the development of this disease: triggers, substrate and modifying factors. An arrhythmia is thus like a fire that needs a spark (Trigger) to ignite a pile of wood (Substrate) and depends on the humidity or accelerants (modifying factors) to burn faster or slower. This body of work takes a closer look at such modifying factors. The major finding of this thesis is that stretching atrial heart muscle cells releases Calcium ions from storage spaces within each cell. If these Calcium release events get frequent enough they can act as triggers for the arrhythmia. The thickness of the atrial muscle is heterogeneous, thus filling the atrium with blood distends thinner parts stronger than ticker portions. The varying degree of stretch might stimulate Calcium release predominantly from myocytes in thinner regions of the atria. This heterogeneity in spontaneous Calcium release can modify also the substrate. A comparable effect of stretch was previously described in the heart’s main chambers. However, it appears that the in the atria it depends on another mechanism, which could serve as a treatment target that mainly acts on the atria without negatively affecting the ventricle

Doctor of Philosophy

dissertationTreatment and management of heart disease is challenging due to the heart's limited ability to self-repair. Although current approaches to manage heart disease, such as pharmacotherapy, medical devices, lifestyle changes, and heart transplantation, have improved and extended the quality of life for millions of individuals, they have inherent shortcomings. Future strategies to manage heart disease will likely be based upon a combination of biological and engineering approaches through cell therapy and tissue engineering strategies, both of which have the potential to regenerate the myocardium and improve cardiac function. However, a key hurdle in applying biological approaches is our limited ability to produce reliable tissue to study disease progression and tissue development, therapeutic intervention, drug discovery, or tissue replacement. Establishing hallmarks of the native myocardium in engineered cardiac tissue is a central goal and appears to be required for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. The objective of this research was to apply an engineering approach to develop tools and methods to produce engineered cardiac tissue and characterize both native and engineered cardiac tissue. Three phases of research included: 1) the development and utilization of a framework to characterize microstructure in living cardiac tissue using confocal microscopy and local dye delivery, 2) the development a next-generation bioreactor capable of continuously monitoring force-displacement in engineered tissue, and 3) the application of confocal imaging and image analysis to quantitatively describe features of the native myocardium, focusing on myocyte geometry and spatial distribution of a major gap junction protein connexin-43, in both engineered tissue and native tissue

Modelling pathological effects in intracellular calcium dynamics leading to atrial fibrillation

The heart beating is produced by the synchronization of the cardiac cells' contraction. A dysregulation in this mechanism may produce episodes of abnormal heart contraction. The origin of these abnormalities often lies at the subcellular level where calcium is the most important ion that controls the cell contraction. The regulation of calcium concentration is determined by the ryanodine receptors (RyR), the calcium channels that connect the cytosol and the sarcoplasmic reticulum. RyRs open and close stochastically with calcium-dependent rates. The fundamental calcium release event is known as calcium spark, which refers to a local release of calcium through one or more RyRs. Thus, a deep knowledge on both the spatio-temporal characteristics of the calcium patterns and the role of the RyRs is crucial to understand the transition between healthy to unhealthy cells. The aim of this Thesis has been to figure out these changes at the submicron scale, which may induce the transition to Atrial Fibrillation (AF) in advanced stages. To address this issue, I have developed, and validated, a subcellular mathematical model of an atrial myocyte which includes the electro-physiological currents as well as the fundamental intracellular structures. The high resolution of the model has allowed me to study the spatio-temporal calcium features that arise from both the cell stimulation and the resting conditions. Simulations show the relevance of the assembly of RyRs into clusters, leading to the formation of macro-sparks for heterogeneous distributions. These macro-sparks may produce ectopic beats under pathophysiological conditions. The incorporation of RyR-modulators into the model produces a nonuniform spatial distribution of calcium sparks, a situation observed during AF. In this sense, calsequestrin (CSQ) has emerged as a key calcium buffer that modifies the calcium handling. The lack of CSQ produces an increase in the spark frequency and, during calcium overload, it also promotes the appearance of global calcium oscillations. Finally, I have also characterized the effect of detubulation, a common issue in cells with AF and heart failure. Thus, the present work represents a step forward in the understanding of the mechanisms leading to AF, with the development of computational models that, in the future, can be used to complement in vitro or in vivo studies, helping find therapeutic targets for this disease

Characterizing Nav1.5 expression, organization, and electrical behavior in cardiomyocyte domains

Proper function of the heart depends on the function of voltage-gated ion channels. These channels open and close in a tightly regulated way. The resulting ion currents change the membrane potential and shape the action potential, which initiates cardiac muscle contraction. The sodium channel Nav1.5 is especially important as it generates the initial upstroke of the action potential. In cardiomyocytes, it is expressed in different membrane domains, including the intercalated disc, where two cardiomyocytes are mechanically and electrically coupled; the lateral membrane; and possibly at the T-tubules, which are invaginations of the lateral membrane. Many different proteins and molecules bind Nav1.5, together forming a macromolecular complex, and modulate Nav1.5 expression and/or function. Mutations in the Nav1.5-encoding gene SCN5A can confer a loss or gain of channel function, and are associated with several heart rhythm disorders, including Brugada syndrome, long-QT syndrome type 3 (LQTS3), and sick-sinus syndrome. The mechanisms that lead to this phenotypic variability remain unknown. Since Nav1.5 occurs at different cardiomyocyte membrane domains and several interacting proteins are specific to a domain, we hypothesize that the effects of a mutation depend on the subcellular location of Nav1.5 and the composition of the macromolecular complex. This thesis aims to (1) determine Nav1.5 cluster organization in cardiomyocyte membrane domains from mice with different genetic backgrounds using single-molecule localization techniques; (2) contribute to the fundamental understanding of cardiomyocyte excitability by assessing voltage-gated ion channel expression with next-generation RNA sequencing; and (3) assess passive electrical properties of T-tubules and the effects of a large T-tubular sodium current with an in silico model. In addition, this thesis contains two thorough literature reviews on the cardiac intercalated disc and T-tubules. Firstly, we show that Nav1.5 is expressed in T-tubules of wild-type cells using single-molecule localization microscopy and computational modeling techniques. We observed that Nav1.5 cluster organization and density partly depend on the presence of the large scaffolding protein dystrophin and on the three C-terminal amino acids of Nav1.5, Ser-Ile-Val. Cardiomyocytes expressing C-terminally truncated Nav1.5 (ΔSIV) display a loss of Nav1.5 expression at the lateral membrane and particularly at the lateral membrane groove compared to wild-type cells. Dystrophin-deficient cardiomyocytes also display a reduction of Nav1.5 expression at the lateral membrane, but no groove-specific reduction, and most notably an increase of T-tubular Nav1.5 expression. Nav1.5 cluster shapes are less complex in dystrophin-deficient cells at the lateral membrane and inside the cell compared to wild type. ΔSIV cells show this effect only inside the cells, not at the lateral membrane. Secondly, we show in murine cardiomyocytes of Black/6J mice that of the voltage-gated sodium channels, mRNAs are expressed encoding mainly Nav1.5 and Nav1.4, and a small amount of Nav2.1-encoding mRNA. No other isoforms were detected. Of the β-subunits, only β1- and β4-encoding mRNA are found. Thirdly, we assessed electrical properties of T-tubules. We compared the depolarization delay of a deep T-tubular segment to the mouth of a T-tubule upon a large depolarizing voltage step reminiscent of the upstroke of the cardiac action potential. We chose to compare the time to reach the activation threshold of voltage-gated calcium channels as these channels are highly expressed in T-tubules and crucial for initiating cardiomyocyte contraction. Deep inside the T-tubule, the activation threshold of voltage-gated calcium channels was reached only 10 microseconds later than at the mouth of the T-tubule. This delay increased 10-20 times when we introduced constrictions. Then, we introduced a large sodium current to the model. We show that the sodium current is smaller deep inside the T-tubule than at the mouth due to the positive extracellular potential, which decreases the driving force of the channels. In the constricted tubules, we observed a stronger sodium current self-attenuation, but an increase of peak sodium current in the first constriction due to an increase in open probability and driving force. In conclusion, these studies contribute to the fundamental understanding of voltage-gated sodium channel composition, organization, and function in cardiomyocytes, with a focus on Nav1.5. Exciting subjects of further study include the functional implications of the changes in Nav1.5 cluster organization in ΔSIV and dystrophin-deficient mice, the functional contributions of Nav1.4, and β1- and β4-subunits to murine cardiomyocyte function, and the composition of voltage-gated ion channels in human cardiomyocytes

La bioénergétique systémique moléculaire des cellules cardiaques (la relation structure-fonction dans la régulation du métabolisme énergétique compartmentalisé)

An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: a-actinin and b-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of b-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, a-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-a-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cells.Un élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'a-actinine et les isoformes des b-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de b-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, a-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-a-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Computational Approaches to Understanding Structure-Function Relationships at the Intersection of Cellular Organization, Mechanics, and Electrophysiology

The heart is a complex mechanical and electrical environment and small changes at the cellular and subcellular scale can have profound impacts at the tissue, organ, and organ system levels. The goal of this research is to better understand structure-function relationships at these cellular and subcellular levels of the cardiac environment. This improved understanding may prove increasingly important as medicine begins shifting toward engineered replacement tissues and organs. Specifically, we work towards this goal by presenting a framework to automatically create finite element models of cells based on optical images. This framework can be customized to model the effects of subcellular structure and organization on mechanical and electrophysiological properties at the cellular level and has the potential for extension to the tissue level and beyond. In part one of this work, we present a novel algorithm is presented that can generate physiologically relevant distributions of myofibrils within adult cardiomyocytes from confocal microscopy images. This is achieved by modelling these distributions as directed acyclic graphs, assigning a cost to each node based on observations of cardiac structure and function, and determining to minimum-cost flow through the network. This resulting flow represents the optimal distribution of myofibrils within the cell. In part two, these generated geometries are used as inputs to a finite element model (FEM) to determine the role the myofibrillar organization plays in the axal and transverse mechanics of the whole cell. The cardiomyocytes are modeled as a composite of fiber trusses within an elastic solid matrix. The behavior of the model is validated by comparison to data from combined Atomic Force Microscopy (AFM) and Carbon Fiber manipulation. Recommendations for extending the FEM framework are also explored. A secondary goal, discussed in part three of this work, is to make computational models and simulation tools more accessible to novice learners. Doing so allows active learning of complicated course materials to take place. Working towards this goal, we present CellSpark: a simulation tool developed for teaching cellular electrophysiology and modelling to undergraduate bioengineering students. We discuss the details of its implementation and implications for improved student learning outcomes when used as part of a discovery learning assignment

Novel optics-based approaches for cardiac electrophysiology: a review

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research

STED microscopy of cardiac membrane nanodomains

Heart muscle cells (cardiomyocytes) have to fulfill a demanding task. They have to ensure continuous and regular heartbeat and maintain their integrity despite undergoing constant mechanical stress during muscle contraction. To accomplish this, they feature a characteristic membrane architecture: the Transverse-Axial Tubular System (TATS), a network of membrane invaginations – the Transverse Tubules (TT) – which allows the fast translation of an electrical stimulus into a mechanical response. Further, specialized membrane protein complexes ensure membrane flexibility and stability during cycles of contraction and relaxation. Structural alterations of the TATS and the membrane protein complexes are linked to cardiac pathologies. This thesis presents the subdiffraction image based investigations of the cardiac membrane architecture and membrane nanodomains using STimulated Emission Depletion (STED) microscopy of mouse ventricular cardiomyocytes (VM). Experimental and analytical methods comprising single- and multicolor, one- and two-photon-excitation STED microscopy are developed and applied. Special focus is laid on the three-dimensional (3D) TATS topology, on the membrane lipids Cholesterol (Chol) and Ganglioside GM1 (GM1), and on the membrane associated proteins Caveolin-3 (Cav-3) and Dystrophin (Dyst). Novel fluorescent Chol analogs are characterized and established as a class of membrane labels with superior properties for STED microscopy of living VM. These dye compounds allow the visualization of the TATS with an unprecedented lateral resolution of below 35 nm and can be used for both membrane bulk staining and labeling of nanoscopic membrane compartments. Using a custom-built two-photon-excitation-STED (2P-Exc-STED) microscope, the new Chol dyes enable the acquisition of 3D subdiffraction images of the TATS of living VM. These 3D images reveal that TT bud from Chol rich membrane domains and that these Chol rich domains can also form shallow membrane invaginations which are hypothesized to be caveolae. The signal patterns of the caveolae-associated protein Cav-3 and of Chol are comparatively investigated and their similarities quantitatively evaluated. The dramatic effect of membrane Chol depletion on the nanoscopic Cav-3 signal distribution is assessed. The correlation between the Cav-3 and Chol membrane patterns is further supported by two-color STED microscopy of VM labeled for Cav-3 and GM1, and Chol and GM1. Finally, the spatial association between Cav-3 and the cytoskeletal protein Dyst is studied in detail. For this, two- and three-color STED imaging protocols and image analysis procedures are developed. To determine the molecular orientation of the Dyst protein with respect to Cav-3 and with respect to the cardiac membrane, a multicolor “intra-protein” labeling protocol is developed that is based on immunofluorescence staining using different primary antibodies that target specific epitopes along the Dyst protein. A cardiac membrane nanodomain model summarizing the presented observations and findings is derived, validated, and discussed in detail