PROJECT SUMMARY/ABSTRACT Heart cells must precisely control the flow of calcium ions (Ca) within the cell to maintain a healthy heartbeat. Contraction is initiated when L-type Ca channels (LTCCs) on the cell surface open and induce sarcoplasmic reticulum (SR) Ca channels (RyR2) to release more Ca. This process is known as Ca-induced Ca release (CICR). People with Ca handling dysfunctions develop arrhythmia and are at risk for sudden cardiac death and heart failure. Yet, a detailed molecular and structural basis for CICR regulation in health and its dysregulation in disease remains a mystery. The goal of this project is to use cutting-edge developments in cryo-electron tomography (Cryo-ET), correlative light and electron microscopy, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and murine disease models, and CRISPR genome editing to determine the structures and organization of CICR proteins in multiple clinically relevant states. In the mentored phase of this award, the project will determine the localization of CICR proteins, the complexes they form, and their conformational state, both at rest and during β-adrenergic receptor stimulation. In the independent phase of the award, the project will use time-resolved imaging to capture short-lived but functionally important assemblies to dissect CICR refractoriness. At both stages of the project, healthy cells will be compared to disease models. This work will provide unprecedented insight into the molecular mechanisms that regulate CICR and how mutations in CICR proteins lead to arrhythmia. By connecting structural and cardiovascular biology, this project will provide a proof-of-concept for a new approach to study diverse cardiovascular processes and aid the development of precise therapeutics. It will also give Dr. Woldeyes the training and expertise necessary to start an academic career with a focus on using Cryo-ET for cardiovascular imaging. With the training support of this award and guidance from her mentors, her advisory committee members, and collaborators, Dr. Woldeyes will be well positioned to establish her independent research career. Dr. Woldeyes’ long-term goal is to dissect the mechanisms of cardiovascular disease at high spatial and temporal resolution. She is jointly mentored by Dr. Wah Chiu, a leader in the field of cryo-electron microscopy/tomography and Dr. Joseph Wu, a leader in the use of patient-derived iPSC-CMs to study cardiovascular diseases. Both have excellent track records in mentoring and transitioning trainees to independent academic careers. Their labs are an ideal environment for conducting the proposed experiments. With the resources and faculty available at the Cardiovascular Institute, SLAC National Laboratory, Stanford, and the MOSAIC UE5 program, she will have the training, support and intellectual input needed to ensure the success of this research project, enhance her career development, and prepare her for the transition to a successfu...