Project Background
Heart failure is often caused by the inability of the heart cells (cardiomyocytes) to clear damaged, mutant, or misfolded or proteins. Our cardiomyocytes have several specialized mechanisms to remove these harmful proteins. One such mechanism is called chaperone mediated autophagy (CMA), which can selectively clear a single diseased protein. CMA employs chaperones for the selective delivery of substrate protein containing KFERQ-like motif to lysosomes for degradation. Our goal is to combine this unique feature of CMA, together with adeno-associated viral (AAV) transgene delivery, to target specific mutant proteins for lysosomal degradation.
Project 1. We will focus on a targeting a missense mutation of αB-crystallin protein termed CryABR120G (R120G). which forms insoluble protein aggregates in the heart and causes cardiomyopathy and heart failure. Our goal is to test the ability of CMA-targeting AAV constructs to clear mutant R120G, in an in vitro model of human induced pluripotent stem cell differentiated cardiomyocytes, harboring the R120G mutation (hiPSC-CMR120G), vs. hiPSC-CM expressing the wild-type protein (hiPSC-CMWT).
Project 2: Apart from CMA, there are two additional protein degradation pathways: the ubiquitin proteasome system and the macroautophagy pathway. Both these processes play a beneficial role in maintaining proteostasis i.e., protein homeostasis however the coordinated interplay among the three pathways in response to physiological and pathophysiological interventions is not known. We will characterize the three degradation pathways in hiPSCs and mice hearts harboring R120G mutation.
In conclusion, our research aims to develop effective therapies to treat/prevent cardiac pathologies.
Student Role
The student will be actively involved in the daily research activities of the lab. He/she will interact closely with the mentor, post-docs, graduate students and other undergrads in the lab and in the process learn various biochemical and molecular biology techniques. The student will:
- Culture hiPSC and differentiate them into beating cardiomyocytes
- Test the dosing and time of CMA-targeting AAVs for efficient mutant protein clearance
- Assess CMA/proteasome/macroautophagy pathway in hiPSC-CMR120G and hiPSC-CMWT
- Determine protein expression levels of CryAB using immunoblotting and immunostaining
- Determine the levels of insoluble CryABR120G protein aggregates using confocal imaging
- Determine protein-protein interaction by colocalization and co-immunoprecipitation assays
- Determine CMA activity using a fluorogenic CMA activity assay
- Read and present/discuss research articles on the latest discovery in the field and
- Collect and analyze data and prepare figures for presentation (UCUR/NCUR/lab meetings) and manuscript submission.
Student Learning Outcomes and Benefits
Outcomes: At the end of their training, the students will learn various scientific techniques and methodologies pertaining to cardiac physiology, cell biology and molecular biology. They will learn about the daily schedule of a researcher and thereby improve their own time-management and organization skills. They will learn how to design experiments, evaluate data critically and think about the next logical step of an experiment. They will improve their written and oral communication skills through presentation in departmental, national and international meetings/seminars.
Benefits: Performing research in a lab can stir-up interest and enthusiasm among the students. The students will understand the broader impact of their and other related research, and think of ways to incorporate the results of the research into textbook learning. They will be able to think more critically about a subject and exhibit a higher level of reasoning. Their training will enable them to recognize what can and cannot be answered with the different types of analyses, about the strengths and weaknesses of those techniques, and how to troubleshoot assays when they don't work as planned. Most importantly, successful completion of the training will help them in laying a ground-work to pursue their career in the field of health and medicine and facilitate their transition into a successful medical/healthcare professional.
Rajeshwary Ghosh
Dr. Ghosh completed her PhD from the University of Calcutta, India, and post-doctoral training at the University of California, Davis, and University of South Dakota. She is currently a Research Assistant Professor in the Department of Nutrition and Integrative Physiology, College of Health, at the University of Utah, embedded in Dr. J. David Symon’s and Dr. Sihem Boudina’s laboratories. Dr. Ghosh’s research focuses on delineating the mechanisms of protein degradation pathways (macroautophagy, chaperone-mediated autophagy and proteasomal pathways) in the amelioration of cardiac pathologies.
Philosophy: Mentoring is an essential aspect of a student's growth at all levels. My goal is to teach complex concepts and ideas in a simple way and consequently improve the depth of a student's understanding on a subject. Additionally, I will try to provide constant help to the student to set and
achieve goals and to overcome challenging situations as they arise.
Mentoring Activities:
- I will constantly challenge the students to think more critically about scientific questions, giving them research papers to read, train them on how to ask more pointed, focused research questions, how to construct tighter, testable hypotheses, and how to plan out more intricate experimental designs. I enjoy carrying out my experiments and therefore available daily in the lab so the student can interact and ask questions whenever needed.
- We have weekly lab meetings where research in progress is presented and long term research strategies are discussed. The student will be encouraged to present his/her research findings in front of the lab members and other PIs. This will improve their oral communication skill as well as their critical thinking ability.
- The student will be encouraged to present their data at SPUR summer symposium, NCUR, UCUR and URS.
- The student will also be encouraged to attend national conferences (e.g. American Heart Association, BCVS meeting) to present their work to a more diverse audience and interact with other researchers and established scientists.