PROJECT SUMMARY No effective pharmacological or medical device interventions are available to treat heart failure with preserved ejection fraction (HFpEF). We have pioneered the concept of cell therapy for HFpEF: Cardiosphere-derived cells (CDCs) dramatically improve diastolic function and reduce arrhythmias, while attenuating fibrosis and inflammation. Most, if not all, of these beneficial effects are mediated by exosomes secreted by CDCs (CDCEXO). Here we seek to establish detailed molecular signatures of HFpEF; to use those molecular signatures as roadmaps to identify key, potentially causal pathways by dissecting the responses to CDCEXO; and to discover novel defined molecular entities, based on CDCEXO cargo, with disease-modifying bioactivity in HFpEF. Our hypotheses, backed by strong preliminary data, are: • Underlying HFpEF are bewilderingly extensive changes in myocardial transcriptomics and proteomics. Sorting causal from associative changes presents a major challenge, but it is doable. • A subset of these HFpEF-related proteome changes are reversed by CDCs or CDCEXO and some correlated to the reversal of the key functional abnormalities of HFpEF. We posit that focusing on CDCEXO-responsive pathways will facilitate the search for causal abnormalities, enabling targeted hypothesis testing. • By mining the RNA and protein contents of CDCEXO, we have the potential to pinpoint defined factors which have disease-modifying bioactivity in HFpEF. Such defined factors may themselves be viable therapeutic candidates, or can inspire the creation of new chemical entities as therapeutic candidates. The overall goal of this proposal is to understand better the pathogenesis of HFpEF, and to develop novel cell- free approaches to treat this disease. Three aims are proposed. In Aim 1 we will perform and analyze tissue and single cell transcriptomics and proteomics (including numerous protein post-translational modifications) of HFpEF, in three different models (pig, mouse and rat) that represent different comorbidities and compare their disease signature to those in human HFpEF heart tissue. Commonalities in the OMICS responses among species will help distinguish causal versus associative pathways in HFpEF pathogenesis. Aim 2 will analyze molecular signatures of therapeutic efficacy by comparing transcriptomics and proteomics with and without exposure to CDCEXO. Because CDCs and their exosomes strikingly reverse the HFpEF phenotype, identification of CDCEXO-induced molecular changes will further refine pathway prioritization in terms of causal versus associative. Here we will study ventricular tissue and single cells from rat, mouse and pig models of HFpEF (and controls), with and without in vivo exposure to CDCEXO. In Aim 3 we will mine CDCEXO cargo to identify critical factors underlying disease-modifying bioactivity in a rat model of HFpEF. This will allow us to define and/or create specific molecular entities (either RNA species or proteins) tha...