Investigating the delivery of IGF-1 with in vitro and in vivo model systems of myocardial infarction

Myocardial infarction (MI) is characterised by the irreversible death of cardiac muscle with loss of up to 1 billion cardiomyocytes (CM). Despite survival post-MI dramatically improving in the last two decades, more than 20% of patients suffering MI will still develop heart failure (HF), an incurable condition where the heart is no longer able to meet the body’s needs for blood supply. Amongst novel therapeutic avenues currently being explored, intramyocardial delivery of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) holds great promise to replace the lost functional tissue. However, the effects of the ischemic microenvironment on these cells still need to be investigated, and protective strategies need to be developed. This thesis examines the delivery of the pro-survival growth factor Insulin like Growth Factor-1 (IGF-1) in the settings of hiPSC-CMs exposed to acidic pH and through a hydrogel-based approach in an in vivo model of MI. Following MI, the heart switches from aerobic metabolism to anaerobic glycolysis, causing a pH drop to 6.5-6.8. The aim of the first part of this thesis was to mitigate the effects of acidic pH on hiPSC-CMs using the pro-survival growth factor IGF-1. It was shown that acidic pH negatively affects hiPSC-CMs in terms of viability, metabolic activity, cardiac gene expression and CMs yield obtained through differentiation. IGF-1 was able to recover the effects of acidic pH, and it could, therefore, be used as a protective strategy for in vivo cell therapy approaches. Another promising strategy for preventing HF progression following MI is the minimally invasive delivery of injectable hydrogels, which can provide mechanical support to damaged tissue and deliver bioactive factors with pro-survival actions. Here, a thermoresponsive injectable hydrogel composed of a triblock copolymer of polyethylene glycol (PEG) and polycaprolactone (PCL) was synthesised and characterised in vitro and in vivo. The hydrogel was prepared with or without insulin-like growth factor-1 (IGF-1) and injected intramyocardially in a mouse MI model. Echocardiography, strain analysis and histological assessments showed that the injection of the biodegradable thermoresponsive hydrogel was effective in ameliorating pathological remodelling, improving overall cardiac function and myocardial mechanics. In the future, implementing novel therapeutic approaches like the ones presented in this thesis could prevent the progression to HF, improving the quality of life of patients affected by myocardial infarction and limiting the socio-economic burden of the disease.Open Acces

Mechanics of the tricuspid valve: from clinical diagnosis/treatment, in vivo and in vitro investigations, to patient-specific biomechanical modeling

Proper tricuspid valve (TV) function is essential to unidirectional blood flow through the right side of the heart. Alterations to the tricuspid valvular components, such as the TV annulus, may lead to functional tricuspid regurgitation (FTR), where the valve is unable to prevent undesired backflow of blood from the right ventricle into the right atrium during systole. Various treatment options are currently available for FTR; however, research for the tricuspid heart valve, functional tricuspid regurgitation, and the relevant treatment methodologies are limited due to the pervasive expectation among cardiac surgeons and cardiologists that FTR will naturally regress after repair of left-sided heart valve lesions. Recent studies have focused on (i) understanding the function of the TV and the initiation or progression of FTR using both in-vivo and in-vitro methods, (ii) quantifying the biomechanical properties of the tricuspid valve apparatus as well as its surrounding heart tissue, and (iii) performing computational modeling of the TV to provide new insight into its biomechanical and physiological function. This review paper focuses on these advances and summarizes recent research relevant to the TV within the scope of FTR. Moreover, this review also provides future perspectives and extensions critical to enhancing the current understanding of the functioning and remodeling tricuspid valve in both the healthy and pathophysiological states

Folding and Assembly of Cytoskeletal Proteins Under Force - From Single Molecule Studies of Dystrophin to Studies of Intact Cells

Changes in tertiary and quaternary structure of proteins within the actin cytoskeletal network are a likely way cells read mechanical signals from their environment. However, showing that these conformational changes occur as a result of mechanical stress and that such changes are important to the function of the cell is a major challenge. This thesis seeks to address these questions using a cohort of molecular biophysical and cell biological methods applied in increasingly complex contexts. First, the importance of force-driven unfolding to function and how changes in unfolding pathway correlate with diseased states was determined with single molecule Atomic Force Microscopy on nano-constructs of wild-type and mutant forms of dystrophin. Biophysical studies showed that the ability to fold into mechanically stable, spectrin-type helical bundle domains and the preservation of cooperative unfolding were common characteristics of functional truncated dystrophins. Second, a newly developed in-cell cysteine labeling technique demonstrated stress-enhanced repeat unfolding within spectrin in wild-type red blood cells under shear stress versus static conditions, thus demonstrating that forced unfolding is not just an in vitro phenomena. The importance of the cytoskeletal network to spectrin function was also demonstrated in mutant, 4.1R-null red blood cells, where the intrinsic properties of spectrin remain intact but the network integrity is compromised by absence of 4.1R. Loss of network integrity was evident in a decrease in spectrin unfolding under stress. Repeat unfolding was accompanied by changes in associations of spectrin with its binding partners in a time- and stress- dependent manner, indicating that the erythrocyte cytoskeleton exhibits a graded response to stress. Lastly, with cardiomyocytes derived from embryonic stem cells, the importance of stress to quaternary structure of actinin within the sarcomeric cytoskeleton and its effects on cell-wide function was tested in cells adhered to elastic substrates. Substrate stiffness sets the load on these spontaneously contracting cells, and differences in load lead to cytoskeletal reorganization with significant effects on cardiogenic development. Taken together, these findings present evidence of various cytoskeletal proteins – especially in the spectrin superfamily – as mediators of mechanical signaling within cell

Motion tracking tMRI datasets to quantify abnormal left ventricle motion using finite element modelling

According to `The Atlas of Heart Disease and Stroke'[MMMG04] published by the World Health Organization, heart disease accounts for nearly half the deaths in both the developed and developing countries and is the world's single biggest killer. However, early detection of a diseased heart condition can prevent many of these fatalities. Regional wall motion abnormalities of the heart precede both ECG abnormalities and chest pain as an indicator of myocardial ischaemia and are an excellent indicator of coronary stenosis [GZM97]. These motion abnormalities of the heart muscle are difficult to observe and track, because the heart is a relatively smooth organ with few landmarks and non-rigid motion with a twisting motion or tangential component. The MRI tissue-tagging technique gives researchers the first glimpse into how the heart actually beats. This research uses the tagged MRI images of the heart to create a three dimensional model of a beating heart indicating the stress of a region. Tagged MRI techniques are still developing and vary vastly, meaning that there needs to be a methodology that can adapt to these changes rapidly and effectively, to meet the needs of the evolving technology. The focus of this research is to develop and test such a methodology by the means of a Strain Estimation Pipeline along with an effective way of validating any changes made to the individual processes that it comprises of

Physics-Based Probabilistic Motion Compensation of Elastically Deformable Objects

A predictive tracking approach and a novel method for visual motion compensation are introduced, which accurately reconstruct and compensate the deformation of the elastic object, even in the case of complete measurement information loss. The core of the methods involves a probabilistic physical model of the object, from which all other mathematical models are systematically derived. Due to flexible adaptation of the models, the balance between their complexity and their accuracy is achieved

DEVELOPMENT AND IMPLEMENTATION OF NOVEL STRATEGIES TO EXPLOIT 3D ULTRASOUND IMAGING IN CARDIOVASCULAR COMPUTATIONAL BIOMECHANICS

Introduction In the past two decades, major advances have been made in cardiovascular diseases assessment and treatment owing to the advent of sophisticated and more accurate imaging techniques, allowing for better understanding the complexity of 3D anatomical cardiovascular structures1. Volumetric acquisition enables the visualization of cardiac districts from virtually any perspective, better appreciating patient-specific anatomical complexity, as well as an accurate quantitative functional evaluation of chamber volumes and mass avoiding geometric assumptions2. Additionally, this scenario also allowed the evolution from generic to patient-specific 3D cardiac models that, based on in vivo imaging, faithfully represent the anatomy and different cardiac features of a given alive subject, being pivotal either in diagnosis and in planning guidance3. Precise morphological and functional knowledge about either the heart valves\u2019 apparatus and the surrounding structures is crucial when dealing with diagnosis as well as preprocedural planning4. To date, computed tomography (CT) and real-time 3D echocardiography (rt3DE) are typically exploited in this scenario since they allow for encoding comprehensive structural and dynamic information even in the fourth dimension (i.e., time)5,6. However, owing to its cost-effectiveness and very low invasiveness, 3D echocardiography has become the method of choice in most situations for performing the evaluation of cardiac function, developing geometrical models which can provide quantitative anatomical assessment7. Complementing this scenario, computational models have been introduced as numerical engineering tools aiming at adding qualitative and quantitative information on the biomechanical behavior in terms of stress-strain response and other multifactorial parameters8. In particular, over the two last decades, their applications have been ranging from elucidating the heart biomechanics underlying different patho-physiological conditions9 to predicting the effects of either surgical or percutaneous procedures, even comparing several implantation techniques and devices10. At the early stage, most of the studies focused on FE modeling in cardiac environment were based on paradigmatic models11\u201315, being mainly exploited to explore and investigate biomechanical alterations following a specific pathological scenario or again to better understand whether a surgical treatment is better or worse than another one. Differently, nowadays the current generation of computational models heavily exploits the detailed anatomical information yielded by medical imaging to provide patient-specific analyses, paving the way toward the development of virtual surgical-planning tools16\u201319. In this direction, cardiac magnetic resonance (CMR) and CT/micro-CT are the mostly accomplished imaging modality, since they can provide well-defined images thanks to their spatial and temporal resolutions20\u201325. Nonetheless, they cannot be applied routinely in clinical practice, as it can be differently done with rt3DE, progressively became the modality of choice26 since it has no harmful effects on the patient and no radiopaque contrast agent is needed. Despite these advantages, 3D volumetric ultrasound imaging shows intrinsic limitations beyond its limited resolution: i) the deficiency of morphological detail owing to either not so easy achievable detection (e.g., tricuspid valve) or not proper acoustic window, ii) the challenge of tailoring computational models to the patient-specific scenario mimicking the morphology as well as the functionality of the investigated cardiac district (e.g., tethering effect exerted by chordal apparatus in mitral valve insufficiency associated to left ventricular dilation), and iii) the needing to systematically analyse devices performances when dealing with real-life cases where ultrasound imaging is the only performable technique but lacking of standardized acquisition protocol. Main findings In the just described scenario, the main aim of this work was focused on the implementation, development and testing of numerical strategies in order to overcome issues when dealing with 3D ultrasound imaging exploitation towards predictive patient-specific modelling approaches focused on both morphological and biomechanical analyses. Specifically, the first specific objective was the development of a novel approach integrating in vitro imaging and finite element (FE) modeling to evaluate tricuspid valve (TV) biomechanics, facing with the lack of information on anatomical features owing to the clinically evident demanding detection of this anatomical district through in vivo imaging. \u2022 An innovative and semi-automated framework was implemented to generate 3D model of TV, to quantitively describe its 3D morphology and to assess its biomechanical behaviour. At this aim, an image-based in vitro experimental approach was integrated with numerical models based on FE strategy. Experimental measurements directly performed on the benchmark (mock circulation loop) were compared with geometrical features computed on the 3D reconstructed model, pinpointing a global good consistency. Furthermore, obtained realistic reconstructions were used as the input of the FE models, even accounting for proper description of TV leaflets\u2019 anisotropic mechanical response. As done experimentally, simulations reproduced both \u201cincompetent\u201d (FTR) and \u201ccompetent-induced\u201d (PMA), proving the efficiency of such a treatment and suggesting translational potential to the clinic. The second specific aim was the implementation of a computational framework able to reproduce a functionally equivalent model of the mitral valve (MV) sub-valvular apparatus through chordae tendineae topology optimization, aiming at chordae rest length arrangement to be able to include their pre-stress state associated to specific ventricular conformation. \u2022 We sought to establish a framework to build geometrically tractable, functionally equivalent models of the MV chordae tendineae, addressing one of the main topics of the computational scientific literature towards the development of faithful patient-specific models from in vivo imaging. Exploiting the mass spring model (MSM) approach, an iterative tool was proposed aiming to the topology optimization of a paradigmatic chordal apparatus of MVs affected by functional regurgitation, in order to be able to equivalently account for tethering effect exerted by the chordae themselves. The results have shown that the algorithm actually lowered the error between the simulated valve and ground truth data, although the intensity of this improvement is strongly valve-dependent.Finally, the last specific aim was the creation of a numerical strategy able to allow for patient-specific geometrical reconstruction both pre- and post- LVAD implantation, in a specific high-risk clinical scenario being rt3DE the only available imaging technique to be used but without any acquisition protocol. \u2022 We proposed a numerical approach which allowed for a systematic and selective analysis of the mechanism associated to intraventricular thrombus formation and thrombogenic complications in a LVAD-treated dilated left ventricle (LV). Ad-hoc geometry reconstruction workflow was implemented to overcome limitations associated to imaging acquisition in this specific scenario, thus being able to generate computational model of the LV assisted with LVAD. In details, results suggested that blood stasis is influenced either by LVAD flow rate and, to a greater extent, by LV residual contractility, being the positioning of the inflow cannula insertion mandatory to be considered when dealing with LVAD thrombogenic potential assessment