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

基本原理。本研究的目的是了解细胞环境和发育过程中K+离子通道的功能如何在基因水平上受到控制。K+通道是兴奋性的重要调节因子，在心脏中起着终止心脏动作电位和抑制兴奋性的作用。其中一个关键的K+通道hERG对心脏复极尤为重要。有趣的是，hERG基因产生不同的mRNA转录物，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通道转录物的可塑性及其如何受到细胞的控制具有重要的意义。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丰富的遗传操作工具（如CRISPR）相结合，极大地促进了实验研究。在我们提出的研究项目中，我们的目标是以斑马鱼为模型系统，研究zkcnh（斑马鱼zERG）转录物在发育和细胞环境变化过程中的可塑性，并将其与心脏功能的测量相关联。的方法。我们提出了一种新的实验方法，结合使用定量PCR和新型CRISPR/Cas9原位杂交技术对转录本水平的询问，在胚胎中进行靶向基因编辑。我们打算追求几个目标：1)通过绘制转录物的数量和分布，并将其与心功能（心率、心电图、心脏尺寸）和心电活动（膜片钳在整个离体心脏中的动作电位记录和电压敏感膜相关染料的光学作图）的测量相关联，确定心脏zkcnh表达的发育调节；2)研究环境温度对心脏ZKCNH表达的影响，绘制鱼在不同环境温度下对慢性冷暖驯化（6周）的转录量和表达的适应；3)利用CRISPR基因编辑技术操纵zkcnh基因功能，对感兴趣的基因进行靶向敲除，研究其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转录物表达的发育和热可塑性的第一个特征，并旨在将环境应激源引起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，并将推进CRISPR技术在斑马鱼和人类细胞中基因编辑的实用性和应用。

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.