Clara Borie
Research Mentor: Adam Helms
Mentor Department: Internal Medicine / Cardiovascular Medicine, Medicine
Author(s): Clara Borie, Esraa Ismail, Adam Helms
Session: Session 1 (9:00 AM – 9:50 AM)
Presentation Type: Poster 66
Abstract
NPPA and NPPB are genes that encode atrial/brain natriuretic peptides (ANP/BNP). These peptides regulate blood pressure through signaling pathways including promoting sodium and water excretion by the kidneys (natriuresis) and inducing vascular smooth muscle relaxation (vasodilation). During heart failure, NPPA/B are upregulated in response to mechanical stress. Clinically, BNP is widely used as a biomarker to diagnose heart failure. Although the mechanosensitivity of these genes is well studied, the cis-regulatory elements (CREs) underlying this response remain poorly defined. Multiplex CRISPR Interference screens facilitate the simultaneous testing of many CREs to determine their functional contributions to gene expression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the upregulation of both genes following enhanced contractile activity in induced pluripotent stem cell-derived cardiomyocytes (iPSC-derived CMs). A comprehensive mapping of CREs will be performed using a gRNA library to inhibit individual elements and assess their impact on gene expression. The RNA of NPPA and NPPB will be quantified using Flow Fluorescence In Situ Hybridization (FISH), followed by fluorescence-activated cell sorting (FACS) after multiplex CRISPR perturbation. To advance this workflow, FISH probes targeting genes of interest had to be validated. RT-qPCR confirmed that enhanced contractile activity upregulated NPPA (p=0.020) and NPPB (p=0.008) expression, while non-contractile cells showed reduced expression, particularly for NPPB (p=0.005). Optimal FlowFISH/FACS conditions were established and multiplex RNA staining was validated using Alexa Fluor 568 probes for NPPB and Alexa Fluor 488 probes for RPL13 with a multifold increase in fluorescence compared to unstained controls. This work establishes a CRISPRi-FlowFISH framework to study cardiac stress regulation using NPPA and NPPB as key heart failure markers. The approach is broadly applicable to other genes of interest for high-throughput CRE mapping to identify functional regulatory elements that can serve as candidate therapeutic targets.
