The work outlined in this proposal is intended to fund an intensive mentored training program that promotes the development of the applicant into a complete and multi-disciplinarily trained independent investigator. The emphasis and strengths of the application are aligned according to three areas: 1. applicant credentials, 2. training environment, and 3. Innovative research. 1. Applicant Credentials. Starting a new position in Dr. Dirksen laboratory """"""""aligned all stars in the sky"""""""" for Dr. Yarotskyy by creating ideal conditions for the proposed Mentored Research Scientist Development Award (K01) application: proven track-record of productivity, eligibility for program (obtaining permanent residence status), readiness to establish scientific independence, change in research direction, and exceptional technical and professional training opportunity. The applicant has outstanding skills and great expertise in electrophysiology and biophysics, but lacks essential in-depth training in other key components required for a successful independent career in academic research. The PI's short-term career goals include expanding scope of technical skills, begin to develop and establish an independent line of research, and to refine essential professional skills (including scientific writing, oral communication, instruction, trainee mentoring, and involvement in peer-review). Long-term goals include obtaining a tenure-track position, establishing an independent line of research, training future scientists, emerging as leader in chosen field of research field, and participation in professional and society leadership. This proposal is explicitly designed to: 1. Develop a new area of research for applicant, 2. provide needed additional technical training and expertise, 3. promote continued development of leadership, mentoring and professional skills, and 4. establish laboratory and project management skills. Completing the proposed training will achieve all short-term goals and provide expertise required to accomplish the applicant's long-term goals. 2. Training Environment. Dr. Dirksen is fully committed to the complete training of outstanding and bright young scientists. Dr. Dirksen's laboratory is an ideal place for the applicant's proposed training and first independent step to take place. The Dirksen laboratory employs a broad range of cutting-edge technologies routinely used by the research team with diverse expertise in imaging, photometry, molecular biology and biochemistry, adenoviral technology, mouse genetics and disease models. Dr. Dirksen's overall philosophy is to provide all trainees with an enriched and nurturing scientific environment that promotes trainee development into careful, critical-thinking problem-solvers and effective scientific communicators. Dr. Yarotskyy will benefit from intense training in new research techniques, mentoring, leadership, and professional skills. Being a proud member of the Department of Physiology and Pharmacology, Dr. Yarotskyy will also benefit from great support from other well-established senior investigators. Overall oversight of the training program will be closely supervised by an outstanding Mentoring Advisory Committee consisting of Drs. R. Dirksen, D. Yule and S.-S. Sheu. Toward the end of the K01 award, Dr. Yarotskyy will use results obtained during this project to justify and extend the scope of the project by formulating an independent New Investigator R01 application that will enable him to launch his independent research program and obtain a tenure track Assistant Professor position within the department. 3. Innovative Research. The central hypothesis to be tested here is that: """"""""Nuclear RyR1 (nRyR1) release channels are important regulators of nucleoplasmic Ca2+ signaling and gene regulation in skeletal muscle."""""""" The type I ryanodine receptor (RyR1) plays an essential role in skeletal muscle function. Mutations in RyR1 result in several debilitating and life-threatening myopathies, including malignant hyperthermia (MH) and central core disease (CCD). The molecular mechanisms underlying CCD core formation have yet to be fully clarified. The current paradigm is that mutant RyR1 channels in the sarcoplasmic reticulum (SR) alter cytoplasmic Ca2+ signaling that eventually leads to the MH and CCD phenotypes. We challenge this paradigm by proposing a role for mutant RyR1 channels in the nucleoplasmic reticulum (nRyR1) that result in aberrant nucleoplasmic Ca2+ signaling and altered transcription factor localization and activity that underlie the formation of cores and fiber type changes characteristic of CCD. In this project, we propose that: 1. nRyR1 regulates nucleoplasmic Ca2+ transients by controlling Ca2+ release from the perinuclear space during stimulation;2. nucleoplasmic Ca2+ impacts NFAT localization and activity which underlies fiber type specification;3. morphological changes in CCD result from altered gene expression driven by aberrant nRyR1 channel activity. This project will employ a multidisciplinary approach, including confocal imaging, biochemistry, molecular biology, and electrophysiology to test the central hypothesis. This project will test the validity of a novel pathophysiological mechanism by which altered RyR1 channel in impacts skeletal muscle disease.
Mutations in ryanodine receptors result in several debilitating and life-threatening skeletal muscle disorders (malignant hyperthermia and central core disease) and cardiomyopathy (arrhythmias and sudden death). Elucidating the molecular mechanisms of these channelopathies will have a huge economical impact. This project will provide novel insights into pathophysiological mechanisms that underlie malignant hyperthermia and central core disease.
