Advisory Committee Chair
Lufang Zhou
Advisory Committee Members
Jack Rogers
Jianyi Zhang
Min Xie
Xiaoguang Liu
Document Type
Dissertation
Date of Award
2020
Degree Name by School
Doctor of Philosophy (PhD) School of Engineering
Abstract
Mitochondria are an important organelle found in all human cells, they fulfill several crucial roles necessary for cellular function such as energy production, calcium handling, metabolic regulation, and inducing pre-programmed cell death. Mitochondrial dysfunction is associated with many different diseases such as heart failure, aging, and cancer, as well as multiple neurodegenerative diseases. While there are currently different tools and approaches for studying different aspects of both mitochondrial function and dysfunction, they tend to have major drawbacks that limit their practicality and conclusions that can be drawn using them. We hypothesized that by finding the correct genetic mitochondrial targeting sequence, expression of large or complex genes could be successfully targeted to mitochondria, allowing for more precise and accurate investigation into their many crucial functions.We first screened several mitochondrial targeting sequences until finally coming across one capable of targeting expression of channelrhodopsin 2 (ChR2) to the inner membrane of mitochondria. This allowed for highly precise light-induced mitochondrial depolarization. Upon further investigation, it was found that prolonged light stimulation of cells expressing this mitochondrial ChR2 resulted in significant apoptotic cell death. On the other hand, brief low-level light stimulation instead made cells more resilient to mitochondrial stressors. We also used this mitochondrial targeting sequence to transport a large genetic calcium indicator to mitochondria, allowing for simultaneous measurement of cytosolic and mitochondrial calcium in active cardiomyocytes. We found that while adult cardiomyocytes have a highly consistent synchronized beat-by-beat response, cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) showed a highly variable relationship between cytosolic and mitochondrial calcium dynamics. Finally, as part of an ongoing study, we adapted our mitochondrial optogenetic tool for use in vivo and used it to induce prolonged mitochondrial dysfunction in the hearts of mice in order to elucidate what, if any, causal relationship is there between mitochondrial dysfunction and heart failure. This treatment led to a marked decline in cardiac function. While these data are preliminary, they provide evidence that this tool can be effectively used in vivo, allowing for investigation into the role mitochondrial dysfunction plays in a number of tissue types and diseases.
Recommended Citation
Ernst, Patrick James, "Development of mitochondrial-targeted genetic tools to investigate how changes in mitochondrial function affect the heart" (2020). All ETDs from UAB. 782.
https://digitalcommons.library.uab.edu/etd-collection/782