Investigating Mitogenic Mechanism and Therapeutic Efficacy of Adenosine Deaminase Knockout in Human and Murine Cardiomyocytes

研究人和小鼠心肌细胞中腺苷脱氨酶敲除的有丝分裂机制和治疗效果

基本信息

批准号：
10815529
负责人：
Sophia Bunnell DeLuca
金额：
$ 4.25万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2024-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10815529
关键词：
3-Dimensional Adenosine Adult Affect Biological Assay Birth Breeding Calcium Cardiac Cardiac Myocytes Cardiovascular Physiology Cell Cycle Chemicals Cicatrix Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Consumption Coronary artery Culture Media Custom Cyclic AMP Development Echocardiography Embryo Ethanol Metabolism Event Fibroblasts Flow Cytometry Foundations Future Gap Junctions Generations Genetic Genetic Screening Genus Hippocampus Glucose Glucosephosphate Dehydrogenase Heart Heart Injuries Human Hypertrophy Image Immunity In Vitro Injections Injury Inosine Ischemia Knock-out Letters Ligation MAP Kinase Gene MAPK3 gene Mediating Metabolic Metabolism Mitogens Modeling Molecular Mus Muscle function Myocardial Infarction Myocardium Natural regeneration Neonatal Nucleotides Pathway interactions Pentosephosphate Pathway Ploidies Pluripotent Stem Cells Polyploidy Production Proliferating Purine-Nucleoside Phosphorylase Purinergic P1 Receptors Rattus Receptor Signaling Recovery of Function Reperfusion Injury Reperfusion Therapy Reporting Research Resistance Respiration Ribose Rodent Role Sarcomeres Signal Transduction Stains Study models Tail Testing Therapeutic Therapeutic Effect Time Tissue Engineering Transgenic Mice Treatment Efficacy Veins Western Blotting Work Zebrafish adeno-associated viral vector adenosine deaminase aurora B kinase cardiac regeneration cardiac tissue engineering cardioprotection follow-up gene therapy genetic manipulation heart damage heart function heart metabolism improved in vitro Model in vitro testing in vivo induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes inducible Cre injured insight knockout gene lipid metabolism mouse model muscle form neovascularization novel nucleotide metabolism p38 Mitogen Activated Protein Kinase pharmacologic postmitotic purine metabolism ratiometric regeneration potential repaired stem cells sugar nucleotide therapeutic evaluation transcriptome sequencing

项目摘要

Abstract Mammalian cardiomyocytes (CMs) in embryonic and neonatal hearts are proliferative, which allows for heart regeneration to occur. This proliferative capacity of CMs is lost shortly after birth and in the adult hearts CMs are largely post-mitotic, renewing at very low rates of less than 1% per year. The inability of adult CMs to proliferate, along with the absence of resident stem cells capable of robust generation of new CMs, limit the ability of adult mammalian hearts to self-repair following injury such as myocardial infarction (MI). Preliminary results from my CRISPR knockout screen in cultured neonatal rat CMs show that knockout of the gene adenosine deaminase (ADA-KO) results in robust cell cycle activation in both primary rodent and pluripotent stem cell-derived human CMs in vitro. Follow-up RNA sequencing analysis suggests that ADA-KO alters CM metabolism. As metabolic alterations have been shown to underlie the pro-proliferative actions of other known cardiac mitogens, my proposed studies will determine the metabolic and molecular signaling mechanisms governing ADA-KO mediated proliferation in human CMs. Additionally, I will test the in vitro effects of ADA-KO on injured engineered cardiac tissues (ECTs) made using polyploid human CMs, which we have shown to be resistant to division similarly to polyploid CMs in vivo. Furthermore, in proof-of-concept in vivo studies, I will test therapeutic efficacy of ADA-KO in the setting of myocardial infarction by AAV delivery of ADA-KO sgRNAs to transgenic mice with CM-specific Cas9 expression, followed by studies of cardiac proliferation and function. When completed, these cross-species in vitro and in vivo studies will provide better understanding of the roles of ADA in cardiac metabolism and regeneration and will create a basis for the development of novel gene therapies for cardiac regeneration.

抽象的胚胎和新生儿心脏中的哺乳动物心肌细胞 (CM) 具有增殖能力，这使得心脏能够发生再生。 CM 的这种增殖能力在出生后不久就会丧失，在成人心脏 CM 中大部分是有丝分裂后的，更新率非常低，每年不到 1%。成人 CM 无法增殖，加上缺乏能够强劲生成新 CM 的常驻干细胞，限制了成年哺乳动物心脏在心肌梗塞（MI）等损伤后自我修复的能力。初步的我在培养的新生大鼠 CM 中进行 CRISPR 敲除筛选，结果表明该基因的敲除腺苷脱氨酶 (ADA-KO) 可导致原代啮齿动物和多能动物的细胞周期强劲激活体外干细胞衍生的人类 CM。后续 RNA 测序分析表明 ADA-KO 改变 CM 代谢。由于代谢改变已被证明是其他已知促增殖作用的基础心脏有丝分裂原，我提出的研究将确定代谢和分子信号传导机制控制 ADA-KO 介导的人类 CM 增殖。另外，我会测试ADA-KO的体外效果对使用多倍体人类 CM 制成的受损工程心脏组织 (ECT) 进行研究，我们已证明与体内多倍体 CM 类似，具有抗分裂能力。此外，在体内概念验证研究中，我将通过 AAV 递送 ADA-KO sgRNA 测试 ADA-KO 对心肌梗死的治疗效果具有 CM 特异性 Cas9 表达的转基因小鼠，随后进行心脏增殖和功能的研究。完成后，这些跨物种体外和体内研究将有助于更好地理解其作用 ADA 在心脏代谢和再生中的作用，将为新基因的开发奠定基础心脏再生疗法。