snRNAseq for Modeling long QT syndrome using gene-edited pigs
To date, KCNH2 mutations identified in patients with long QT syndrome type 2 (LQT2) have been extensively studied using heterologous expression systems. While these systems allow for the evaluation of the pathogenicity of specific hERG mutations through the overexpression of mutant channels, they fail to recapitulate the full spectrum of electrophysiological alterations and ion channel remodeling that occur in cardiomyocytes under physiological conditions in LQT. Existing zebrafish and rodent models are also limited in their ability to faithfully model human QT interval abnormalities due to substantial differences in lifespan, size, anatomy, and physiology. For instance, rodents exhibit a much higher heart rate and significantly shorter action potential duration (APD) than humans. Moreover, IKr is the major repolarizing current in human ventricles, whereas its inhibition in rodents has minimal impact on ventricular repolarization, rendering genetic mouse models inadequate for the study of LQT2. Thus, there is a critical need to develop animal models that can more accurately mimic human inherited arrhythmias. To address this gap, we generated a miniature pig model of LQT2, capitalizing on the physiological similarities to humans, as well as advantages in breeding and genome editing compared to non-human primates. To investigate the mechanisms by which KCNH2 mutations lead to LQT, we performed single-nucleus RNA sequencing to compare the transcriptomes of ventricular tissues from KCNH2-mutant pigs and wild-type controls. Overall design: To investigate the underlying mechanisms, single-nucleus transcriptomic analysis was performed on ventricular myocardial tissue from WT and LQT(L5) pigs. Tissue samples were dissected from multiple layers—including the left and right ventricular endocardium, myocardium, epicardium, and the interventricular septum—then pooled for subsequent sequencing. After rigorous quality control, a total of 22,342 genes were analyzed across 8,103 cells from WT and 9,691 cells from the L5 group.