Maturing Engineered Heart Tissues for Titin-Based Disease Modeling

Abstract

Engineered heart tissues (EHTs) are three-dimensional, fibrin-based heart muscle constructs developed as an advanced disease modeling tool for therapeutic development and personalized medicine, but at a fraction of the time and cost of traditional animal models. EHT maturity can be evaluated by identifying the isoforms of titin, a giant filamentous protein that regulates sarcomere organization and provides passive stiffness to cardiomyocytes, and has been implicated in cardiomyopathies. For example, dilated cardiomyopathy (DCM) patients have been shown to undergo eccentric remodeling with a switch from a stiffer N2B isoform to a more compliant N2BA isoform. Current neonatal rat EHTs express immature titin isoform expression patterns and require additional maturation to improve clinical relevancy. This dissertation examines different approaches to maturing titin isoform expression in EHTs using angiotensin II and triiodothyronine supplementation, chronic electrical stimulation, and extracellular matrix (ECM) modifications. Angiotensin II supplementation at 20 µM significantly increased mature N2B titin isoform expression while triiodothyronine supplementation had no significant effect. Chronic electrical stimulation at 0.5 Hz significantly enhanced the force production, fractional shortening, and contraction velocity in EHTs. However, paced EHTs exhibited a negative force-frequency relationship indicating other factors like calcium handling should also be considered for maturation. Surprisingly only N2BA titin isoform expression was significantly increased in paced EHTs. Sarcomere width and ECM, myofiber, mitochondria, and void fractional area were also significantly increase in paced EHTs while significant reduction was observed in Z-disk width, sarcomere length, collagen fibril width, and cytoplasm, cardiomyocyte, and empty fractional area. Decreasing the fibrinogen concentration lowered the Young’s Modulus of the initial fibrin gel used for generating EHTs. This change in extracellular matrix stiffness enhanced contraction kinetics during development and significantly increased the mature N2B titin isoform expression. Finally, EHTs generated from human induced pluripotent stem cells differentiated into cardiomyocytes were examined for maturity level. Contraction kinetics reveal significantly lower values in comparison to neonatal rat EHTs and only fetal cardiac titin isoform was detected in titin analysis. Overall, these findings have only scratched the surface of the intricate overlap of biological, mechanical, and electrical cues necessary to fully mature EHTs.