The clinical use of doxorubicin (DOX, and anticancer drug) is limited due to its cardiotoxic side effects. The mechanism of DOX-induced cardiotoxicity (DIC) is not totally understood. We recently showed that DOX causes mitochondrial (as opposed to total cellular) iron accumulation, and that a reduction in mitochondrial iron leads to protection against DIC. However, these studies were predominantly performed in vitro and in animal models, and it is not clear whether higher mitochondrial iron contributes to DIC susceptibility in patients. To extend these findings beyond animal models, we have established several lines of human induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CM) from patients who either experienced DIC (DIC-CM) or did not have cardiotoxicity (noDIC-CM) after exposure to DOX, and showed that they recapitulate the patients? phenotype in vitro, and that they are susceptible to a special form of iron-mediated cell death called ?ferroptosis?. Using RNA- seq, we have also identified a number of mitochondrial iron genes to be altered in DIC samples after DOX treatment. Finally, we have demonstrated that hiPSC-CM from DIC patients display elevated mitochondrial iron after DOX exposure. Our central hypothesis is that mitochondrial iron plays a key role in the susceptibility to DIC in humans, and that a reduction in mitochondrial iron (either by iron chelators or by modulation of genes involved in mitochondrial iron homeostasis) alter cardiomyocyte susceptibility to DOX. We also hypothesize that DOX clearance from subcellular compartments is regulated by mitochondrial iron through mitophagy, and that this process is impaired in DIC.
In Aim 1, we will determine whether DIC-CM are more susceptible to DOX toxicity due to mitochondrial iron accumulation, and that a reduction in mitochondrial iron improves cell viability in these cells. We will modulate mitochondrial iron in DIC-CM and noDIC-CM using mitochondrial permeable iron chelators (including new chelators identified in our lab), followed by exposure to DOX and assessment of ferroptosis and cell viability.
In Aim 2, we will determine whether modulation of mitochondrial iron-related genes identified in our RNA-seq analysis alters susceptibility to DOX. Our studies demonstrated that mRNA levels of ABCB7 and ABCB8 (proteins that mediate mitochondrial iron export) and MFRN2 (a mitochondrial iron importer) are altered with DOX treatment. We will delete ABCB7 or ABCB8 in hiPSC from noDIC (which would result in an increase in mitochondrial iron) and MFRN2 in hiPSC from DIC (resulting in a reduction in mitochondrial iron) using CRISPR-Cas9, followed by generation of hiPSC-CM, exposure to DOX, and assessment of cell viability. Finally, in Aim 3, we will determine whether mitochondrial iron regulates DOX clearance and that inherent defects in DOX clearance promotes susceptibility to DIC. We will assess DOX clearance, mitochondrial dynamics and mitophagy in noDIC-CM and DIC-CM after DOX and in the presence and absence of iron chelators, and with modulation of mitochondrial iron genes. We will also assess DOX clearance and mitophagy in the hearts of Abcb8 knockout and transgenic mouse models after DOX.

Public Health Relevance

Doxorubicin is an anti-cancer drug with the side effect of causing damage to the heart and ?cardiomyopathy?. We have previously shown that the mechanism for doxorubicin-induced cardiomyopathy is due to mitochondrial iron accumulation in tissue culture and in mouse models. In this proposal, we will extend our studies to human cells and will assess the role of mitochondrial iron in this disorder using human induced pluripotent stem cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL140927-01A1
Application #
9916208
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Schwartz, Lisa
Project Start
2020-03-15
Project End
2024-02-29
Budget Start
2020-03-15
Budget End
2021-02-28
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611