OBJECTIVES/SPECIFIC AIMS: The goals of this study are to develop a human-based screening assay for testing individual drug reactions and investigate the mechanism underlying susceptibility to develop diLQT. METHODS/STUDY POPULATION: We derived iPSC-CMs from 10 subjects with a high sensitivity to Sotalol (high-S group) and 10 subjects with no changes in QT interval after administration of the same drug (low-S group). Multielectrode array (MEA) was used to measure field potential duration, a surrogate to the QT interval in the electrocardiogram, in iPSC-CMs under basal conditions and in response to increasing concentrations of Sotalol. Transcriptomic profiling of iPSC-CMs from high-S Versus low-S groups was performed using RNA-sequencing. A parameter sensitivity analysis was performed on the Paci et al . iPSC-CM mathematical model to further support the lead hits identified via RNA-sequencing. RESULTS/ANTICIPATED RESULTS: Cardiac differentiation resulted in the generation of iPSC-CMs with appropriate cardiac channel expression and response to a hERG blocker E4031. MEA recordings showed a significantly higher response to Sotalol in iPSC-CMs from high-S compared with low-S subjects. Transcriptomic profiling identified upregulation or downregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization and calcium handling machinery as underlying high sensitivity to Sotalol. In silico parameter sensitivity analysis corroborated transcriptomic profiling of select genes; upregulated KCNE4 and downregulated CAMKV were predicted to positively and negatively correlate with iPSC-CM action potential duration when exposed to Sotalol, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest subject-specific iPSCs can be used to model functional abnormalities observed in diLQTS and offer novel insights into iPSC-based screening assays for toxic drug reactions. Success of this study may help identify key components underlying diLQT susceptibility to ultimately develop novel therapeutic agents.
Introduction: Emerging evidence suggests that post-transcriptional modifications of mRNAs are vital to their stability, nuclear export, cellular compartmentalization, translation to proteins, degradation and stem cell pluripotency. However, such mechanisms remain unexplored in mature post-mitotic tissues such as the mammalian myocardium, especially under its pathophysiological remodeling. Here, we investigated the role of N6-methyladenosine (m6A), the most prevalent chemical modification in RNA, in myocardial ischemia. Methods and Results: We studied post-mortem human tissues, murine and swine models of myocardial ischemia and demonstrated for the first time that m6A methylation of mRNA in the ischemic left ventricle (LV) is significantly increased when compared with non-ischemic LV. We found that increase in m6A methylation is conserved in human, pig and mice and have global effect in post-ischemic cardiac remodeling, affecting mRNA, miRNA and protein expressions, and myocyte function. We identified that the expression of m6A demethylase, fat mass and obesity-associated protein (FTO) is decreased in post-ischemic human and mouse myocardium. Using loss-of-function gain-of-function studies in isolated primary adult rat cardiomyocytes, we identified FTO as a direct regulator of m6A methylation, SERCA2a expression and cardiomyocyte function including Ca 2+ transient, decay and sarcomere shortening. Remarkably, in primary cardiomyocytes under hypoxia stress, FTO expression was down regulated resulting in increase in mRNA methylation, decrease in SERCA2a expression and compromised cardiomyocyte function, all of which was restored by adenovirus-mediated overexpression of FTO under hypoxia. Finally, using methylated RNA pull-down assay of RNA from human ischemic and control LV tissues, we have demonstrated that SERCA mRNA is hypermethylated under ischemia and that SERCA demethylation by FTO is essential for SERCA mRNA expression and protein synthesis. Conclusions: We have discovered a novel pathomechanism by which FTO, a RNA demethylase, plays a critical role in cardiac homeostasis and cardiomyocyte function under ischemia. Our discovery opens up a new paradigm for our understanding of post-ischemic cardiac remodeling.
Cardiac hypertrophy is an increase in the mass of the heart. It is a major risk factor for the development of myocardial infarction and congestive heart failure, diseases that afflict millions of patients worldwide. Hypertrophy can be caused by intrinsic defects of the proteins of the contractile apparatus of the heart, or by extrinsic stimuli such as hypertension. In this review, we will focus on the cytosolic signal transduction pathways that mediate the hypertrophic response to extrinsic stimuli. Although a large number of signaling molecules have been implicated in the hypertrophic response, we will review data that, we believe, suggest there may be only a few molecules that serve as signaling funnels through which many hypertrophic signals must pass on their way to the nucleus. These include the stress response protein kinases (the stress-activated protein kinases or SAPKs, and, possibly, the p38 kinases) and calcineurin. These molecules have as their primary targets transcription factors, many of which have been implicated in the complex yet stereotypic genetic response to hypertrophic stress. In most cases, it is not possible at present to complete the link from hypertrophic stimulus through a specific signaling molecule and a specific transcription factor to the induction of a specific gene that initiates a particular biologic response. We will attempt to identify some of the most important areas where major questions remain in the hopes of stimulating further research into this major cause of death and disability.
