Using zebrafish to study developmental and environmental plasticity of cardiac K+ channel transcript expression

利用斑马鱼研究心脏 K 通道转录表达的发育和环境可塑性

• 批准号: RGPIN-2020-04429
• 负责人: Claydon, Thomas
• 金额: $ 3.06万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739957
• 关键词: Using; zebrafish; study; developmental; environmental

Rationale. The goal of this research is to understand how K+ ion channel function is controlled at the gene level by the cellular environment and during development. K+ channels are important regulators of excitability and in the heart they are responsible for termination of the cardiac action potential and dampening excitability. One key K+ channel, hERG, is particularly important for cardiac repolarization. Interestingly, the hERG gene produces different mRNA transcripts that code for three different forms of the channel protein, and variable expression of these influences electrical activity in heart cells. Understanding hERG channel transcript plasticity and how it is controlled by the cell is therefore of significant interest. Background. Zebrafish are used as a comparative model in developmental biology and molecular genetics studies. The genetic regulation of cardiac development is similar to that in humans and the rapid development of transparent embryos outside of the body combined with a wealth of tools for genetic manipulation (e.g. CRISPR) greatly facilitate experimental study. In the proposed program of research, we aim to use zebrafish as a model system to study plasticity of zkcnh (zebrafish zERG) transcript expression during development and cellular environmental change and to correlate this with measures of cardiac function. Approach. We propose a novel experimental approach that combines interrogation of transcript levels using quantitative PCR and in situ hybridization with novel CRISPR/Cas9 technology to engineer targeted gene-edits in embryos. We intend to pursue several objectives: 1) to determine the developmental regulation of cardiac zkcnh expression by mapping transcript amount and distribution and correlating this with measures of cardiac function (heart rate, ECG, heart dimensions) and cardiac electrical activity (action potential recordings in whole isolated hearts from patch clamp and optical mapping of voltage-sensitive membrane-associated dyes); 2) to investigate the effects of environmental temperature on cardiac zkcnh expression by mapping adaptations of transcript amount and expression in response to chronic warm or cold acclimation (6 wk) of fish at a range of environmental temperatures; 3) to manipulate zkcnh gene function using CRISPR gene-editing technology to perform targeted knock-out of genes of interest and investigate the consequence on cardiac electrical activity. Impact: Our findings will provide the first characterization of developmental and thermal plasticity of zkcnh transcript expression in zebrafish and aim to correlate the influence on cardiac functional adaptations to the changes in demand induced by environmental stressors. These findings will have relevance to the understanding of transcript regulation in fish and humans and will advance the utility and application of CRISPR technologies for gene-editing in zebrafish and human cells.

理由。本研究的目标是了解细胞环境和发育过程中K+离子通道功能如何在基因水平上控制。K+通道是兴奋性的重要调节剂，在心脏中，它们负责终止心脏动作电位和抑制兴奋性。一个关键的K+通道，hERG，对心脏复极化特别重要。有趣的是，hERG基因产生不同的mRNA转录本，编码三种不同形式的通道蛋白，这些蛋白的可变表达影响心脏细胞的电活动。因此，了解hERG通道转录可塑性及其如何受细胞控制具有重要意义。 背景斑马鱼被用作发育生物学和分子遗传学研究的比较模型。心脏发育的遗传调控与人类相似，体外透明胚胎的快速发育与大量遗传操作工具（如CRISPR）相结合，极大地促进了实验研究。在拟议的研究计划中，我们的目标是使用斑马鱼作为模型系统来研究发育和细胞环境变化过程中zkcnh（斑马鱼zERG）转录表达的可塑性，并将其与心脏功能的测量相关联。 Approach.我们提出了一种新的实验方法，将使用定量PCR和原位杂交的转录水平询问与新型CRISPR/Cas9技术相结合，以在胚胎中进行靶向基因编辑。本研究的主要目的是：1）通过绘制zkcnh基因转录本的数量和分布图，并将其与心功能指标相关联，来确定zkcnh基因表达的发育调控（心率、ECG、心脏尺寸）和心脏电活动（来自膜片钳和电压敏感膜相关染料的光学映射的整个分离心脏中的动作电位记录）; 2）通过绘制不同温度下鱼类心脏zkcnh基因转录量和表达量对慢性温、冷适应（6 wk）的适应性图谱，研究环境温度对zkcnh基因表达的影响; 3）使用CRISPR基因编辑技术操纵zkcnh基因功能，以进行目标基因的靶向敲除，并研究对心脏电活动的影响。影响：我们的研究结果将提供斑马鱼zkcnh转录本表达的发育和热可塑性的第一个特征，并旨在将环境应激诱导的需求变化对心脏功能适应的影响联系起来。这些发现将有助于理解鱼类和人类的转录调控，并将推动CRISPR技术在斑马鱼和人类细胞中进行基因编辑的实用性和应用。