Nuclear ryanodine receptor type 1: Role in skeletal muscle physiology and disease

描述(由申请人提供)：本提案中概述的工作旨在资助一项强化的导师培训计划，以促进申请人发展成为一名完整的、受过多学科培训的独立调查人员。申请的重点和优势根据三个方面进行调整：1.申请者资质；2.培训环境；3.创新研究。1.申请人资格证书。在Dirksen博士的实验室开始一个新的职位，为Yarotsky博士“调整天空中的所有星星”，为拟议的导师研究科学家发展奖(K01)申请创造理想的条件：经过证明的生产力记录，符合计划的资格(获得永久居留身份)，准备建立科学独立，改变研究方向，以及特殊的技术和专业培训机会。申请人在电生理学和生物物理学方面有突出的技能和丰富的专业知识，但缺乏在其他关键组成部分方面的必要深入培训，这是在学术研究中取得成功的独立职业生涯所必需的。PI的短期职业目标包括扩大技术技能的范围，开始发展和建立独立的研究领域，并完善基本的专业技能(包括科学写作、口头交流、指导、实习生指导和参与同行审查)。长期目标包括获得终身教职，建立独立的研究领域，培训未来的科学家，在选定的研究领域脱颖而出成为领导者，以及参与专业和社会领导。该建议明确旨在：1.为申请者开发一个新的研究领域，2.提供所需的额外技术培训和专业知识，3.促进领导力、指导和专业技能的持续发展，以及4.建立实验室和项目管理技能。完成拟议的培训将实现所有短期目标，并提供实现申请者长期目标所需的专业知识。2.培训环境。德克森博士全心全意致力于培养优秀、聪明的青年科学家。Dirksen博士的实验室是申请者建议进行培训的理想场所，也是进行独立培训的第一步。Dirksen实验室采用了研究团队经常使用的各种尖端技术，这些团队在成像、光度学、分子生物学和生物化学、腺病毒技术、小鼠遗传学和疾病模型方面拥有不同的专业知识。德克森博士的总体理念是为所有受训者提供一个丰富和有益的科学环境，促进受训者发展为细心、批判性思维的问题解决者和有效的科学沟通者。雅洛茨基博士将从新的研究技术、指导、领导力和专业技能方面的强化培训中受益。作为生理学和药理学系的一名自豪成员，亚罗茨基博士还将受益于其他知名高级研究人员的大力支持。培训计划的全面监督将由一个由R.Dirksen博士、D.Yule博士和S.-S.Sheu博士组成的杰出的指导咨询委员会密切监督。在K01奖项接近尾声时，亚罗特斯基博士将利用在这个项目中获得的结果，通过制定一个独立的New Investigator R01申请来证明和扩大项目的范围，这将使他能够启动他的独立研究计划，并在系内获得终身教职跟踪助理教授的职位。3.创新研究。这里要检验的中心假说是：“核RyR1(NRyR1)释放通道是骨骼肌中核质钙信号和基因调控的重要调节器。”I型Ryanodine受体(RyR1)在骨骼肌功能中起着重要作用。RyR1基因突变会导致几种衰弱和危及生命的肌病，包括恶性高热(MH)和中枢性核心病(CCD)。电荷耦合器件核心形成的分子机制尚未完全阐明。目前的研究范式是肌浆网(SR)中突变的RyR1通道改变细胞质中的钙信号，最终导致MH和Ccd表型。我们通过提出突变的RyR1通道在核质网(NRyR1)中的作用来挑战这一范式，这些突变的RyR1通道导致核质钙信号的异常和转录因子的定位和活性改变，这是形成核心和纤维类型变化的基础。在本项目中，我们认为：1.nRyR1通过控制刺激过程中核周间隙的钙释放来调节核质内的钙瞬变；2.核质内的钙影响NFAT的定位和活性，这是纤维类型规范的基础；3.Ccd的形态变化是由nRyR1通道活性异常引起的基因表达变化所致。该项目将采用多学科方法，包括共聚焦成像、生物化学、分子生物学和电生理学来检验中心假说。这个项目将测试一种新的病理生理学机制的有效性，通过这种机制改变RyR1通道来影响骨骼肌疾病。

DESCRIPTION (provided by applicant): 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.