Poster T19 - In Vitro Modelling of Iron Overload Cardiomyopathy

Introduction: The systemic iron levels in the body are tightly regulated through complex feedback loops between organ systems, and too much or too little iron can lead to iron overload or anemia, respectively. Iron overload can be caused by mutations in genes associated with iron handling or by secondary causes such as multiple blood transfusions required in patients with sickle cell disease. While iron overload affects multiple organs, one serious complication is a form of heart failure known as iron overload cardiomyopathy (IOC). Hearts from patients with IOC show systolic and diastolic dysfunction, fibrosis, and arrhythmias. Much of our understanding of IOC comes from excellent studies in small animal models; however, small animals do not always recapitulate the human disease phenotype due to physiological differences between species. Moreover, it has been challenging to study human IOC in isolation due to the complex feedback loops that govern systemic iron homeostasis. Our goals were 1) to develop completely defined in vitro models that could recapitulate key aspects of human IOC, 2) apply these models to understand the mechanisms underlying human IOC, and 3) to demonstrate how these tools can be harnessed for in vitro testing of potential therapeutics.

Materials and

Methods: We used stem cell technologies, tissue engineering, and in vitro reconstitution assays to model key aspects of IOC. Importantly, our completely defined assays enable us to selectively test the contributions of different factors in the disease pathogenesis. While there are multiple cell types in the heart, we focused on two key cell types involved in heart failure, cardiomyocytes (CMs) and cardiac fibroblasts (CFs); however, the approaches laid out here could easily be extended to include additional cell types. CMs and CFs were derived from human pluripotent stem cells using small molecule modulation of the Wnt pathway. We then examined these cells in isolation using fluorescence-based assays to determine the cell-specific effects of iron on cell viability, iron import, and the production of reactive oxygen species. We also examined the combined effects of these cells by incorporating them into 3D human engineered heart tissues (EHTs). Using EHTs, we examined the electrophysiological, calcium handling, and contractile properties of tissues treated with iron using both fluorescence assays and mechanical measurements. To examine the molecular mechanisms underlying functional changes seen in EHTs, we used purified proteins in in vitro reconstitution assays. Finally, we applied these systems to examine the effects of small molecules on IOC pathology.

Results, Conclusions, and Discussions: We observed that human CMs have higher rates of iron import (p < 0.0001), and higher levels of reactive oxygen species (p < 0.0001) compared to CFs, however, this did not affect cell viability (p = 0.66 for CMs and p = 0.64 for CFs). Moreover, we saw that in the context of human EHTs, iron overload causes changes in both the action potential and calcium transient which could contribute to the arrhythmias observed in patients. In EHTs, we observed that the calcium transient is increased and prolonged (Ƭ30 (p = 0.0004) and Ƭ50 (p = 0.001) decay values) while the force of tissue beating is reduced (p < 0.0001). This is consistent with the reduced systolic function seen in patients. Moreover, this result suggests that iron overload reduces the coupling between calcium and contraction. To better understand this reduced coupling, we measured molecular contractility using in vitro reconstitution assays consisting of purified proteins. We observed that iron causes oxidative damage to the contractile proteins in muscle and it directly inhibits myosin’s contractile function. Finally, we tested whether our simplified in vitro systems could be applied to understand potential therapeutics for IOC. We observed that it was possible to improve cellular and molecular function using both iron chelators and calcium channel blockers that are currently being investigated in clinical trials. Taken together, our results clearly demonstrate how simplified in vitro systems can be applied to model key aspects of human iron overload cardiomyopathy and test potential therapeutic mechanisms.

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