SPUR 2021: Assessing the Role of Chaperone Mediated Autophagy in the Heart

Background

Heart failure is often caused by the inability of the heart cells (cardiomyocytes) to clear damaged, mutant or misfolded 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. Although CMA is known to be active in the heart, its role in this organ has been rarely studied. We will define the function of CMA in the heart under physiological and pathological conditions.

Project 1: Our preliminary data show that genetically upregulating CMA is beneficial against hypoxic- and proteotoxicity-induced stress in cardiac cells. One significant question we will address is whether CMA-activating drugs can eliminate the pathological proteins in the heart, as well. We are in the process of developing a novel CMA reporter mouse model to access CMA’s function in the heart in response to various stresses. Using various biochemical and molecular biology approaches, we will examine the effect of CMA-activating drugs in the amelioration of cardiac pathologies in the CMA reporter mice as well as in cultured cardiomyocytes, subjected to different kinds of cardiac stresses.

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 myocardial samples obtained from heart failure patients and normal donor controls

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:

  1. Assess the CMA pathway in animal and human heart failure samples
  2. Isolate primary cardiomyocytes from rat pups
  3. Determine protein expression levels using immunoblotting and immunostaining in cultured cells and in tissue sections
  4. Assess cardiac function by echocardiography
  5. Determine protein-protein interaction by colocalization and co-immunoprecipitation assays
  6. Confocal imaging
  7. Isolate lysosomes from cultured cells and tissues to assess CMA function
  8. Assess ubiquitin proteasome pathway and macroautophagy pathways
  9. Read and present/discuss research articles on the latest discovery in the field and
  10. Collect and analyze data and prepare figures for presentation and manuscript submission.

Student Learning Outcomes & 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.

Remote Contingency Plan

I will meet with the student via video conferencing 3 to 4 times a week to discuss the basic theoretical concepts of molecular biology and cell biology techniques relevant to the “Project Background”. During the process, they will learn how to lay out the experimental design, analyze data, prepare the corresponding graphs and images, and perform statistics. Briefly the student will learn to apply various state-of-the-art software to:

  1. Quantify protein level changes in cardiac cells and whole heart lysates (using western-blot data analysis software Image Studio Lite)
  2. Quantify the fluorescence intensity levels of immunostained cells and tissues (using microscopy analysis software like ImageJ and MetaMorph).
  3. Learn how to quantify luciferase activity in the cells transfected with the CMA reporter or in mice heart tissues expressing the CMA reporter gene
  4. Learn how to use GraphPad Prism to compute the statistical significance between different experimental groups.

The student will be given the opportunity to present their analyzed data during weekly lab and/or departmental meeting. They will be encouraged to read original and review articles related to the field of cardiac protein quality control. They can present the up-to-date findings during the meetings. Further, the student will be energized to write and contribute a section to the manuscript aimed for publication. At the end of the training, the student will gain experience in various scientific analysis methods, scientific literature review and writing, all of which would help them in pursuing their career in the field of health and medicine.

Rajeshwary Ghosh
Research Assistant Professor

Nutrition & Integrative Physiology
College of Health

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 arose.

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 improve 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. Experimental Biology) to present their work to a more diverse audience and interact with other researchers and established scientists.