PROJECT SUMMARY In the heart, pacemakers or sinoatrial node cells (SAN) initiate and maintain a rhythmic beating pattern that can respond to external stimuli, including neurotransmitters, paracrine, and endocrine signals. In addition to its clinical importance, resting heart rate is associated with lifespan across species, and has strong correlation with longevity within individuals in several large epidemiologic studies. Despite their key role in human health and physiology, the exact intracellular mechanism to maintain precise rhythmic oscillation is unknown, given the limited access to these cells in the heart. The principal investigator hypothesizes that gene expression analysis of the human sinoatrial node cell will identify cellular and physiologic features of human pacemaking function. Recent technologies have enabled generating functional human SAN from embryonic stem cells (hPSC-SAN) and performing molecular characterization at the single cell level. VSNL1, a calcium sensing protein, was identified as a marker specific to SAN cells. I hypothesize that VSNL1 is uniquely involved in heart rate regulation. Analyzing the genetic variants in VSNL1 gene and their effect on heart rate in large biobanks will enable validation of the functional role of this protein in heart rate physiology. The applicant will use large biobank cohorts and genetically engineered human embryonic stem cells to pursue the following aims: First, determine the association between genetic variants in VSNL1 with cardiovascular physiology in several large biobanks. Preliminary data in UK biobank (UKBB) cohort suggests significant association between genetic variants in VSNL1 gene and baseline heart rate. The applicant will further study the effect of VSNL1 variants on heart rate with exercise, atrial and ventricular function (on cardiac MRI), and cardiovascular morbidity and mortality outcomes in UKBB and other more diverse cohorts. Second, understand the effect of genetic variants associated with heart rate in UKBB on molecular and electrophysiological characteristics of hPSC-SAN cells in vitro. In preliminary data, the applicant has successfully confirmed the specific expression of VSNL1 in hPSC-SAN cells and their absence in the human embryonic stem cells (hESC) derived ventricular myocytes. Using CRISPR-Cas9 technology, the applicant has generated knock-out models of VSNL1 gene in hESC. The applicant will use hESC lines carrying VSNL1 null mutation to generate hPSC-SAN lacking functional VSNL1. Analyzing beating rate, calcium activity, and action potential using patch clamp will elucidate the cell type-specific role of VSNL1 in human SAN biology. Third, perform functional studies to test the role of VSNL1 in hPSC-SAN response to adrenergic and cholinergic signals. Recent studies support that VSNL1 is involved in neurotransmitter receptor trafficking. The applicant will generate loss of function (LoF) and gain of function (GoF) VSNL1 variants in hESC using the previous...