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K+离子通道功能是如何在基因水平上受到细胞环境和发育过程的调控的。K+通道是兴奋性的重要调节者，在心脏中，它们负责终止心脏动作电位并抑制兴奋性。一个关键的K+通道，HERG，对心脏复极特别重要。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，这些转录本的不同表达会影响心脏细胞的电活动。因此，了解HERG通道转录的可塑性以及它是如何被细胞控制的是非常有意义的。背景资料。斑马鱼在发育生物学和分子遗传学研究中被用作比较模型。心脏发育的基因调控与人类相似，体外透明胚胎的快速发育与丰富的基因操作工具(如CRISPR)相结合，极大地促进了实验研究。在拟议的研究计划中，我们的目标是以斑马鱼作为模型系统，研究zkcnh(斑马鱼Zerg)转录表达在发育过程中的可塑性和细胞环境的变化，并将其与心脏功能的测量相关联。接近。我们提出了一种新的实验方法，将定量PCR和原位杂交的转录水平询问与新的CRISPR/Cas9技术相结合，以在胚胎中设计靶向基因编辑。我们打算追求以下几个目标：1)通过定位转录量和分布并将其与心功能(心率、心电图、心脏大小)和心脏电活动(从膜片钳上记录整个离体心的动作电位和电压敏感膜相关染料的光学定位)的测量相关联，来确定心脏zkcnh表达的发育规律；2)通过定位不同环境温度下鱼类对慢性冷热习服(6wk)的转录量和表达的适应性，来研究环境温度对心脏zkcnh表达的影响；3)利用CRISPR基因编辑技术操纵zkcnh基因的功能，进行靶向敲除目的基因，并研究其对心脏电活动的影响。影响：我们的发现将首次描述zkcnh转录本在斑马鱼中表达的发育和热可塑性，并旨在将环境应激源诱导的需求变化与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.