PROJECT SUMMARY/ABSTRACT Myocardial Infarction (MI) claims nearly one million lives in the US per year, a number expected to increase by 30% by 2060. Survivors are at increased risk for stroke, heart failure, and recurrent MI. Neutrophils play a critical, yet dual role in regulating cardiac recovery following MI. Depending on their inflammatory state, these innate immune cells can both initiate a healing response or drive further tissue damage. A current gap in understanding how neutrophils integrate chemical and mechanical signals to regulate this inflammatory state has resulted in failure of existing neutrophil-targeted therapies to treat MI. Recent innovations from the Beebe and Huttenlocher labs enable interrogating the role of mechanical and chemical cues on regulating neutrophil inflammatory state. The Beebe lab has introduced a system of liquid- walled microfluidic channels that model the dynamic mechanical environment over the course of MI. The Huttenlocher lab’s introduction of genetically tractable human iPSC-derived neutrophils enables dissection of the signaling mechanisms that regulate response to varying mechanical cues. This work has identified a key signal, myeloid-derived growth factor (MYDGF), a leukocyte-secreted factor that mediates cardiac repair following MI in mice, to also regulate neutrophil response to mechanical cues. This fellowship proposal will further utilize these technologies to investigate the mechanisms behind MYDGF- mediated cardiac healing. We hypothesize that MYDGF paracrine signaling directly counteracts mechanical activation through competition in HIF-1α signaling and regulates cardiomyocyte regeneration within a zebrafish cardiac wound model. Aim 1 will use our organotypic system to first characterize the role of mechanical stress on activation state of primary neutrophils. Within this system we will then use primary and human iPSC-derived neutrophils to test they hypothesis that MYDGF paracrine signaling counteracts mechanical activation through competition in HIF-1α pathway signaling. Within Aim 2, we will employ a zebrafish cardiac injury and regeneration model to investigate the role of MYDGF in regulating cardiac repair through modulation of neutrophil response. The goal of this work is to identify key signals that regulate neutrophil activation state for future therapeutic targeting to treat human MI. The proposal will provide specific training in cell signaling and in vivo imaging. The project will synthesize the engineering expertise of Dr. Beebe and cell biology expertise of Dr. Huttenlocher, with the vast medical, scientific, and translational resources available at the University of Wisconsin – Madison. This pre-doctoral fellowship will drive further development of research, clinical, mentorship, innovation, and communication skills necessary for my career as an independent physician scientist.