UCSF

The sinoatrial (SA) node initiates the heart’s electrical impulse as the primary pacemaker. Since it leads the cardiac conduction system, its maturation at the tissue and cellular level are critical for proper cardiac function. Similar to cardiomyocytes (CMs), the maturing SA node is regulated by neuronal stimulation. Moreover, this tissue is more highly innervated than the working myocardium. Autonomic control is achieved through parasympathetic and sympathetic stimulation to slow down and speed up the heart rate, respectively. Previous studies have shown the importance of sympathetic neurons for the maturation of CMs by regulating cell size and cell cycle withdrawal. However, whether neuronal innervation similarly regulates the maturation of the SA node is not known. This dissertation seeks to bridge the gap between the shortage in knowledge of SA nodal development and innervation. Ultimately, we established a compartmentalized model system of the innervated SA node and myocardium.Chapter 1 details the background about the SA node and respective innervation of the heart. There is also a discussion about tissue engineering strategies that go into modeling different cardiac systems in vitro. Chapter 2 includes various studies that screen for different combinations of small molecules to selectively differentiate SA nodal cells (SANs) from hiPSCs. The culmination of studies supports previous reports that suggests Bmp4, RA, SB, and FGFi are the best cocktail for enriching SANs through transcriptional and functional assays. Due to the inefficiency of SAN differentiations compared to CMs, Chapter 3 describes our efforts to create a SHOX2:GFP reporter line for the identification and purification of SANs in heterogeneous differentiations. That chapter concludes with a study demonstrating the functionality of the reporter line through sorting and transcriptional analysis. Chapter 4 follows with functional analysis of SA nodal like pacemaker cells from the Protze Lab (SANLPCs). Different ratios of SANLPCs and CMs in aggregated microtissues demonstrate the importance of enriched SANLPC cultures for more applicable modeling of the SA node. Another study leverages the power of MEAs to assess the functional competency of SANLPCs to respond to neuronal signals prior to innervation. Chapter 5 conclusively describes the model system that was engineered to assess the differential innervation phenotypes and post-junctional changes between hiPSC-SANs (and SANLPCs) and hiPSC-CMs after co-culture with sympathetic neurons. Overall, this project suggests that SA nodal cells are preferentially targeted by autonomic neurons compared to cardiomyocytes, innervation induces cardiac maturation, and microfluidic systems can be a powerful tool to model and analyze complex heterotypic interactions.