Date of Award

Spring 5-15-2023

Author's School

McKelvey School of Engineering

Author's Department

Biomedical Engineering

Degree Name

Doctor of Philosophy (PhD)

Degree Type



Mechanical loading plays critical roles in regulating heart and cardiomyocyte physiology, with volume overload playing an important role in healthy cardiac development from the fetus to adulthood. Inhibition of mechanical loading during fetal development leads to weak and dysfunctional heart chambers, hypertension, organ failure, and even fetal death in severe cases. Increased loading on the heart has also been clinically shown to increase arrhythmia risk in athletes, including those with no genetic predisposition.Clinical observations studying the effects of cardiac genetic disorders may reveal the mechanisms in which mechanical loading influences cardiomyocyte development. Genetic mutations affecting plakophilin-2 (PKP2), a protein in the desmosome complex that physically links adjacent cardiomyocytes, exacerbates the effects of high levels of mechanical loading and leads to increased arrhythmia formation. Physical activity coupled with a genetic mutation in a desmosome protein has been shown to increase the risk of sudden cardiac death by five-fold in young adults. While we understand that mechanical loading and cell-cell junctions play crucial roles in both normal and pathologic heart development, the mechanisms by which preloading induces electrophysiology changes remains unknown.To study cardiomyocyte development, engineered heart tissues have been created to more closely mimic adult cardiac tissue. Tissues provide necessary cues such as cell alignment and increased mechanical forces that have been shown to mature the cells. These systems have been to more accurately predict the effects of pro- and anti-arrhythmic drugs compared to monolayers. Despite the increased maturation achieved, the cardiomyocytes in these tissues remain smaller than those found in adults, and do not express ion channels in the same ratios as in an adult. It remains unknown how these systems induce cell maturation, with no systematic understanding of how to further improve them.In this dissertation I work to understand how engineered tissue formats induce changes in cardiomyocyte physiology, and the role desmosomes play in this adaptation. To enable this, I first created a method to enable the replication of high resolution stereolithography 3D printed features into elastomers. Materials used in high resolution SLA 3D printing inhibit the crosslinking of materials like PDMS, which are commonly used in micro-device fabrication. Utilizing hydrogels as an intermediary, this method was capable of replicating a wide range of shapes and features into PDMS while maintaining the resolution of the original 3D print. Additionally, this method limits the transfer of potentially toxic chemical leachates from the 3D print into the final PDMS. This new method will enable the quick design and implementation of new designs for engineered tissues previously not possible, and will facilitate the broader utilization of engineered tissues in lab groups for whom the barrier to entry was too high.Utilizing this new method, I created multiple different tissue geometries to determine how tissue prestress regulates cardiomyocyte electrophysiology. The results seen reveal how tissue prestress regulates cell morphology and ion channels that are important drivers of the cardiac action potential. Importantly, prestress alone is sufficient to drive a lower spontaneous beat-rate and form functional NaV1.5 current after only two weeks of culture. Prestress also appeared to regulate the expression of proteins, including Kir2.1 and Cx43, although to a lesser degree. Ultimately, the data shown here reveals that a prestress threshold is necessary for sodium channel function in cardiomyocytes, and that changes in are due to changes in specific ion channels and not global maturation.To understand the mechanisms by which tissue prestress regulates ion channel expression, PKP2-/- µHM of multiple geometries were created. Although the loss of PKP2 did not inhibit prestress-induced morphology changes on the cardiomyocytes, it starkly inhibited the electrophysiology changes seen in wild-type tissues due to tissue geometry and prestress. There was no functional Nav1.5 expression in plakophilin-2 deficient tissues. Cav1.2, hERG, Kir2.1, and Cx43 also appeared to be dependent as well, although to a lesser degree. Ultimately, the data shown here reveals that plakophilin-2 is necessary for cardiomyocytes to properly adjust their physiology according to tissue prestress.Ultimately, I found that tissue prestress is a major factor regulating cardiomyocyte physiology, and that a prestress threshold is necessary for proper expression of action potential-related ion channels. Importantly, cardiomyocytes were dependent on the presence of plakophilin-2 to sense and response to this stress. The work described here details the specificity with which prestress regulates certain aspects of cell electrophysiology, while others are not dependent on this type of mechanical loading. This understanding of cardiomyocyte development will allow for the creation of future in-vitro models that more accurately model adult human cardiac response, and can be used to study disease pathology, predict cardiac drug toxicity, and study heart development


English (en)


Nathaniel Huebsch

Committee Members

Sharon Cresci, Jianjun Guan, Stacey Rentschler, Jon Silva,

Available for download on Wednesday, May 15, 2024