Engineering a power switch to study the contribution of stem cell-derived cardiomyocytes on heart regeneration

项目总结 我是约翰·霍普金斯大学的一名医学讲师，我的目标是成为一名独立的 心脏再生领域的内科医生和科学家，专注于破译干细胞的机制 受损心肌的再生治疗。心脏再生疗法具有巨大的修复潜力 损害心脏并改善其功能。在过去的15年里，有几项试验测试了 不同类型的干细胞治疗在不同的心肌病模型中具有不同的结果。一位批评者 仍然没有答案的问题是干细胞和/或祖细胞是否可以分化为 能够与宿主心肌组织建立稳定的机电整合的心肌细胞 产生有意义的力量，从而显著改善收缩功能。或者，也有可能 有益的效果是由于旁分泌因子和外体分泌促进修复途径和 防止细胞死亡。在这项研究中，我提出了一种新的方法来直接测试这个基本问题，通过使用 一种有条件的电源开关，控制干细胞来源的心肌细胞的兴奋和收缩。 兴奋和收缩将通过有条件地表达名为Rem1的GTP酶来控制，该酶抑制 电压激活钙通道Cav1.2(ICa，L)，是心肌细胞兴奋所必需的通道。Rem1 在多西环素的控制下，将可诱导启动子导入干细胞，使其分化为 心肌细胞和在多西环素诱导时，可以打开或关闭。这项提案将引入以下内容 心肌细胞进入梗死大鼠心脏，并重复打开和关闭电源开关以评估其收缩功能 贡献。Rem1抑制Cav1.2对旁分泌因子和外切体分泌的影响 作为转录因子的表达和干细胞来源的心肌细胞的线粒体功能也将 学习。总体而言，这一建议将为心脏再生的机制提供重要的见解 这种疗法对于这种疗法的未来改进和广泛临床应用将是至关重要的。至 成功地执行了这一建议，我组建了一支优秀的团队。这包括心脏领域的领军人物 生理学，比如我的导师大卫·卡斯博士和我的顾问莱斯利·董博士，我的共同导师朱兰博士 我的顾问查尔斯·默里博士是心脏病领域的世界领先者。 再生和帕特里克·卡汉博士，生物信息学专家。他们将在以下方面提供出色的培训 我需要并将监督和支持我的科学进步和职业发展的每一种方法 独立调查员。我已经有了自己的实验室空间、实验室技术人员和资金，我将拥有完全访问权限 约翰霍普金斯大学核心设施和心脏病学部的最先进设备。

PROJECT SUMMARY I am an instructor of Medicine at Johns Hopkins University and my goal is to become an independent physician-scientist in the field of heart regeneration with a focus on deciphering the mechanisms of stem cell regeneration therapy in damaged myocardium. Cardiac regeneration therapy holds great potential to repair damaged hearts and improve their function. In the past 15 years, several trials have tested the efficacy of various types of stem cell-based therapies in different cardiomyopathy models with varying results. A critical question that remains unanswered is whether stem cells and/or progenitor cells can differentiate into cardiomyocytes capable of establishing stable electromechanical integration with the host cardiac tissue to generate meaningful force and thus significantly improve systolic function. Alternatively, it is also possible that the beneficial effects are due to paracrine factor and exosome secretions that boost reparative pathways and prevent cell death. In this study, I propose a novel approach to directly test this fundamental question by using a conditional power switch to control the excitation and contraction, of stem cell-derived cardiomyocytes. Excitation and contraction will be controlled by conditionally expressing a GTPase named Rem1, which inhibits the voltage-activated calcium channel Cav1.2 (ICa,L), a channel essential for cardiomyocyte excitation. Rem1 under control of a doxycycline-inducible promoter was introduced into stem cells that differentiate into cardiomyocytes and upon doxycycline induction can be turned on or off. This proposal will introduce these myocytes into infarcted rat hearts and repetitively turn the power switch on and off to assess their systolic contributions. The influence of Cav1.2 inhibition by Rem1 on paracrine factor and exosome secretions, as well as transcription factor expression and mitochondria function of stem cell-derived cardiomyocytes will also be studied. Overall, this proposal will provide important insights into the mechanisms of cardiac regeneration therapy that will be critical for future refinement and widespread clinical application of this treatment. To successfully carry out this proposal, I have assembled an outstanding team. This includes leaders in cardiac physiology such as my mentor Dr. David Kass and my consultant Dr. Leslie Tung, my co-mentor Dr. Chulan Kwon an expert in heart development, and my consultants Drs. Charles Murry a world leader in cardiac regeneration and Dr. Patrick Cahan an expert in bioinformatics. They will all provide outstanding training in every method I need and will oversee and support my scientific progress and career development as an independent investigator. I already have my own lab space, lab technician and funds and I will have full access to state-of-art equipment both at the Johns Hopkins University Core facilities and in the division of Cardiology.