PROJECT SUMMARY Heart failure (HF), a leading cause of morbidity and mortality, involves significant dysfunction of the sino-atrial node (SAN). SAN dysfunction (SND) can result from or worsen HF through a vicious cycle leading to HF progression and/or death. However, mechanisms of HF-induced SND due to alterations in expression and distribution of ion channels/receptors and their regulatory factors across the human SAN pacemaker complex have not been studied in human hearts but rather only in animal models that possess significantly different 3D SAN structure and molecular profile. To overcome these major obstacles, our multidisciplinary team has established a vigorous human heart research program to acquire viable explanted hearts with the long-term goal to develop a novel integrative framework to understand how structural and molecular remodeling contribute to SND and arrhythmias in HF and other diseases. We developed an integrated approach of intramural optical mapping, 3D structural imaging, and molecular mapping to examine the human SAN from molecular to organ levels. Our initial results with this approach suggest that the human SAN complex consists of multiple and redundant intranodal pacemakers and conduction pathways (SAN compartments), which allow for the robust regulation of heart rhythm. Our results also suggest that HF-induced impairments of SAN compartments and SND may be orchestrated in part by (1) fibrotic remodeling, (2) molecular remodeling in expression and distribution of adenosine A1 receptors and the G protein-coupled channel IK.Ado (GIRK1/GIRK4)() and/or (3) If pacemaker channels (HCN1/HCN4). Furthermore, our preliminary data suggest that HF-induced downregulation of If pacemaker channels is linked to the upregulation of several microRNAs (or miRs) in animal models, where SAN targeting treatment with antimiRs restored SAN function and alleviated HF. Finally, our preliminary data demonstrates that these miRs are selectively upregulated in human failing SAN and represent promising targets for SND treatment. Based on these recently published and preliminary data, our central hypothesis is that HF- induced heterogeneous structural and molecular remodeling of SAN compartments impairs the robustness of the human SAN complex and leads to SND. The overall objective is to define functional, structural, and molecular features of the human SAN complex altered by HF to reveal novel targets for SND treatment. This translational research will advance our understanding of human SAN function and expression profiles in normal and diseased hearts that is essential for the development of new therapies against SND.