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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：
10536370
负责人：
Sophia Bunnell DeLuca
金额：
$ 4.15万
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/10536370
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 Injections Injury Inosine Ischemia Knock-out Letters Life Ligation MAP Kinase Gene MAPK3 gene Mediating Metabolic Metabolism Mitogens Mitotic Modeling Molecular Mus Muscle function Myocardial Infarction Myocardium Neonatal Nucleotides Pathway interactions Pentosephosphate Pathway Pharmacology Ploidies Pluripotent Stem Cells Polyploidy Production Proliferating Purine-Nucleoside Phosphorylase Purinergic P1 Receptors Purines 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 human model improved in vivo induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes injured insight knockout gene lipid metabolism mouse model muscle form neovascularization novel nucleotide metabolism p38 Mitogen Activated Protein Kinase purine metabolism ratiometric regeneration potential repaired stem 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.

摘要