Advancing 2D and 3D in vitro human skeletal muscle model designs that are simple and reproducible would provide a valuable method to extract relevant structural and functional data for use in disease modeling and drug therapy development. Such models could offer a method to investigate the initiation mechanisms behind Facioscapulohumeral Muscular Dystrophy (FSHD), an incurable genetic disorder that results in total loss of functional motility. This thesis describes the approach taken to create an in vitro model in 2D and 3D. The role of the substrate and extra cellular matrix are considered key in the 2D patterning of skeletal myoblasts, while the development of the hydrogel matrix for growing elongated myobundles is perfected in 3D. Attempts to optimize these models for the study of human skeletal muscles in vitro so they are consistently replicable have been made.

Heart disease remains the number one cause of death in the world leading many researchers to pursue fundamental research in understanding heart structure, cardiac disease, and healing. But even as more research is performed, patient treatment options are still limited and do not fully restore heart function. To provide better treatment options to patients, researchers have focused on understanding the structure of the heart and its relationship to cardiac output. As a pump providing nutrients to the body, the heart’s structure is highly organized across multiple length scales ranging from the 3D organ, down to the force producing units inside cardiomyocytes, sarcomeres. Due to interactions between multiple, complex feedback mechanisms, understanding the structure-function relationship is not trivial. In this dissertation, we aimed to understand how perturbations such as structural changes and inflammatory cytokines affect force generated by cardiac tissue. By employing tools such as microcontact printing and muscular thin films, we were able to mimic 2D cardiac tissue structure and measure the force these tissues produced. Via the creation of novel tissues organized at different length scales, we began to determine whether the size of cellular organization affects the force these tissues can produce. We were also able to design experiments to determine the effect that inflammatory cytokines have on force production. Even with these advances, many mysteries remain, leaving decades of work to be done as heart disease becomes more prevalent due to an aging population. As a result, more scientists and engineers will be needed to take on research roles to provide better treatment options to patients. Therefore, to encourage a new generation of students, a high school tissue engineering summer program, CardioStart, was created in the hopes of inspiring students to pursue degrees in STEM. An in-person summer program was modified to create an online course with the aims to increase student access and engagement. While the in-person program included hands-on activities to supplement presentations, students learned the same material through the online platform overall knowledge at the end of the program was comparable. Furthermore, the online platform provided more accessibility to students with five-fold the enrollment rate over the in-person program. In the future, this program can be expanded by working with other researchers in different fields for students to further their knowledge. With this increased knowledge, more students would be interested in pursuing STEM and taking over research roles to further fundamental cardiac research. This would include further studies in understanding cardiac remodeling and incorporating more complexity back into the simplified systems used in this dissertation.

The myocardium consists of complex tissue structures that are intricately organized to maintain proper organ function. Though many methods exist for studying cardiac tissue architecture, current in vitro studies do not use consistent approaches for characterizing changes in myocardium structure, which is exist in heart disease. This lack of consistency makes it challenging to compare organization between multiple constructs and tissue types. This work will demonstrate in vitro tools that can quantify the orientational organization of multiple cardiac tissue constructs for comparison as well as quantify the correlation between pairs of constructs. To show the benefits of this method, this study explores the results of the orientational order parameter of multiple spatial and temporal scales and the co-orientational order parameter on engineered cardiac tissues. The results of this study quantitatively show characteristic spatial and temporal scales of organization as well as the level of correlation between pairs of structures. The information gained from this method provides insight into many architectural properties that can be directly compared to data from a variety of other intracellular constructs in normal, diseased, and stem cell-derived cardiac tissues.

The reduction of blood flow, or ischemia, compromises the contractile function of the heart by damaging cardiomyocytes. Macrophages from the immune system play a key role during cardiac repair to preserve the heart physical integrity (i.e preventing leaks) as well as potentially acting to preserve some of the function. Therefore, studying the interaction between cardiomyocytes and macrophages would help to better understand the underlying mechanisms of cardiac repair. However, co-culture of cardiomyocytes and macrophages has not been extensively studied due to technical challenges. The first part of this thesis work aimed to explore whether macrophage culture media components affect cardiomyocyte health during in vitro culture. As part of this, the effects of media components on cellular health were investigated by observing cell size and morphology changes. It was observed that different media compositions altered cell size and morphology, suggesting that macrophages and/or macrophage-induced factors had a negative impact on healthy cardiomyocytes. An ischemia model was tested in the second part of this study. The effectiveness of an inexpensive and easy-to-implement model was tested to introduce hypoxia on cardiac fibroblasts and cardiomyocytes. The results showed that there was no significant reduction in both primary cardiac fibroblasts and neonatal cardiomyocytes population, suggesting that the model failed to establish hypoxia during the time period studied. Although the model was unsuccessful, this research provided preliminary data for creating simple ischemic devices.

Data that can be plotted across multiple dimensions of attributes are most often managed using spreadsheet programs, which is both error prone and time inefficient. In comparison, databases are highly scalable and effective solutions, but the perspective and skill sets necessary to implement systems for custom needs are often lacking in biomedical engineering laboratories. In this work, we illustrate the usage of common software in Matlab and Microsoft Access to create a data pipeline to convert raw data into structured formats then aggregate the data in a variety of ways using queries in a database. Methods and visuals for our specific context as well as generalized recommendations are included to provide others with a framework to construct their own custom data pipeline and databases. We also discovered a novel relationship within our own data set using the elucidated methods.

Through the creation of in vitro and in silico models of discontinuous electrical propagation, data can be collected about the stress generation of sarcomeres in abnormal cardiac tissues. Diseased and malformed cardiac muscle causes improper spread of action potentials, thus inhibiting normal cardiac contractile function. Biomedical solutions to common cardiovascular problems would benefit from a reproducible model. Here, we create a model to collect contractile force data in discontinuous engineered cardiac tissues in vitro and confirm theoretical predictions about the effect of discontinuity on stress generation. The formulation of a computational model about the probability of sarcomere firing events is also discussed, together with the methods of how this model will be validated by the in vitro model. While further development and experimental testing is needed, these designs are an efficient way to measure structure-function relationships in irregular cardiac muscle.

A consistently reproducible 2D and 3D model of human skeletal muscle would provide the necessary tools to investigate the relevant structural and functional properties observed in muscle tissue. For dystrophies, including Fascioscapulohumeral muscular dystrophy (FSHD), a model would act as a tool to investigate the symptoms of such an affliction. The importance arises in investigating the mechanisms involved with muscle degradation and reduction in motility. Using microcontact printing to pattern myocytes allows us to investigate the possibility of adapting a muscular thin film protocol to better understand the observable mechanisms in 2D. Alternatively, adapting a more biomimetic 3D model through a hydrogel-produced myobundle would allow for a comprehensive investigation of the structure at various layers, as well as contractility studies with stronger twitches than that of a 2D model. A platform to study human skeletal muscles ​in vitro ​has been optimized to improve reproducibility and consistency.

Hypoxia in the heart, marked by insufficient oxygenation of cardiac tissue, disrupts cellular energetics, compromises ATP availability, and initiates a cascade of detrimental effects that undermine cardiac contractility and often maladaptive structural remodeling. Within the sarcomere, the fundamental contractile unit of muscle, ATP is critical for cross-bridge cycling between actin and myosin filaments, driving the force generation required for contraction. When ATP levels decline, the sarcomere’s efficiency in generating and sustaining force is compromised, exacerbating contractile dysfunction. While ATP's chemical energy is required for mechanical work, the binding of ATP to cross-bridges actually drives muscle relaxation as well, thus, reduced ATP levels also drive impaired relaxation kinetics observed in conditions such as rigor. Beyond metabolic stress, the immune response plays a critical role in cardiac adaptation to hypoxic injury, where resident and infiltrating macrophages dynamically modulate inflammatory and reparative processes. Although immune cells initially facilitate clearance of damaged cells and extracellular debris, their prolonged activation can contribute to maladaptive remodeling, further disrupting cardiac function and even leading to increased susceptibility to future hypoxic events. Despite extensive research, limitations persist in both sarcomere models, which struggle to fully capture metabolite-dependent dynamics, and in vitro and in vivo systems to study hypoxic cardiac injury, which often lack the complexity necessary to study cardiomyocyte-macrophage interactions in hypoxic contexts.

This dissertation addresses these gaps by advancing a novel spatially explicit, stochastic-mechanical sarcomere model that appropriately incorporates ATP, ADP, and inorganic phosphate concentrations within the kinetic framework of a cross-bridge cycle, enabling more accurate simulations of sarcomeric force production under varied metabolic conditions. Additionally, we present an innovative immuno-heart on a chip platform that recreates the hypoxic cardiac microenvironment in vitro, facilitating controlled studies of cardiomyocyte-macrophage reciprocal interactions and their impact on each cell type's function. The platform’s modular design allows precise modulation of macrophage phenotype and density, providing a robust system for probing the impacts of immune modulation on cardiac structure and contractility. Findings from this work reveal key insights into the energetic dependencies of sarcomere function and uncover the dual protective and detrimental roles macrophages play in cardiac contractility under hypoxia. Together, these contributions deepen our understanding of metabolic and immune influences on cardiac performance, offering a foundation for continued research and targeted therapies to mitigate hypoxia-induced cardiac dysfunction and improve recovery outcomes in ischemic heart disease.

Although it is widely acknowledged that heart disease is the number one killer of Americans, what may not be so commonly known is that genetic mutations can cause heart diseases. According to numerous studies, many mutated genes cause heart diseases such as cardiomyopathies, and arrhythmias, yet the practical diagnosis and treatment for them are scarce. Lamin A/C gene (LMNA) is one of the genes that can cause dilated cardiomyopathy, arrhythmia, and heart failure. This gene codes for proteins, which create a mesh-like layer under the nucleus envelope known as the nuclear lamina. Even though nuclear lamina exists in almost every nucleated cell in the body, there are individuals with LMNA splice site mutation (c.357-2A>G) who mainly have heart problems. The mechanisms by which LMNA mutations cause heart dysfunctions remain a mystery. In this project, human-induced Pluripotent Stem Cells (hiPSCs) derived cardiomyocytes have been used to develop an in vitro model of the consequences of the mutation on heart function. We demonstrated that it is possible to recapitulate the pathological phenotype in vitro by using patient-specific derived cells and tissue engineering techniques, even though the patients do not exhibit symptoms until later in life. Moreover, this in vitro model of cardiomyocytes tissues can be utilized to correlate gene profiles, structure, and function of the hiPSC-derived cardiomyocytes; thus, elucidating the disease-causing mechanisms.

Through a variety of mechanisms, a healthy heart is able to regulate its structure and dynamics across multiple length scales. Disruption of these mechanisms can have a cascading effect, resulting in severe structural and/or functional changes that permeate across different length scales. Due to this hierarchical structure, there is interest in understanding how the components at the various scales coordinate and influence each other. While there has been much progress at the molecular scale, there is a growing need for theoretical models to address interactions at the cellular and subcellular scales. In particular, understanding the mechanisms guiding the formation and organization of the cytoskeleton in individual cardiomyocytes can aid tissue engineers in developing functional cardiac tissue.

In this dissertation, we developed computational models which integrate interactions at both the cellular and subcelluar scale to enhance our understanding of how cardiomyocytes self-assemble at different length scales. Experimental data, which consisted of single cell cardiomyocytes having fixed area but variable aspect ratio, was used to test and validate our models. Cells were analyzed for structural consistency and contractility using estab- lished metrics. The metrics where then applied to our model simulations for comparison. We demonstrated that our model simulations are capable of reproducing the stochasticity observed in experimental cells at different length scales while also mimicking structural consistency. In addition to recreating known patterns present in the experimental cells, our models have provided insight towards possible mechanisms that can be explored by experi- mentalists.