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

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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.