Project Summary The principal goal of the Core B renewal is to provide in vitro and ex vivo human platforms to study mechanisms regulating excitation-contraction (EC) coupling in human airway smooth muscle (HASM). The objective of Core B is to establish, characterize and provide primary HASM cells, generate single HASM cell measurements of cytoskeletal (CSK) stiffness and force generation (magnetic twisting cytometry – MTC), and perform human precision cut lung slice (hPCLS) studies for all Projects and Cores. Our laboratory has over 25 years of experience in the establishment, cultivation, and characterization of primary HASM cells. Core B will also provide asthma- and non-asthma-derived HASM to characterize intrinsic differences engendered by disease-state and to capture heterogeneity of human responses. hPCLS will serve as the platform to measure physiologically relevant bronchoconstriction and dilation in intact human tissue. Additionally, we will use cytokine- and mast cell-exposure models to mimic a T2 inflammatory milieu. Aim 1 will provide novel cellular models of HASM cell hyperresponsiveness to explore the relationship among EC coupling, single cell shortening, and force generation to GPCR agonists and antagonists in asthma. Using these models, Project 1 will study mechanisms underlying TGF-β1 modulation of airway contractility; Projects 2 and 4 will elucidate mechanisms modulating TAS2R- and OGR1-mediated HASM relaxation; and Project 3 will develop targeted therapeutics to attenuate contractility and enhance relaxation of HASM. Aim 2 will provide an integrated tissue model of HASM contraction and airway hyperresponsiveness (AHR) to study GPCR signaling. Using hPCLS, Project 1 will examine mechanisms by which TGF-β1 modulates constriction and dilation of small airways; Projects 2 and 4 will elucidate mechanisms by which TAS2R and ORG1 mediate dilation of small airways; and Project 3 will discover novel ways to abrogate AHR and enhance bronchodilation with targeted therapeutics. Aim 3 will address the mechanisms by which inflammatory mediators alter GPCR function and bronchoconstriction in hPCLS. Using these platforms, Project 1 will address how cytokines modulate TGF-β1 effects on airway contractility; Projects 2 and 4 will characterize the roles of TAS2R- and OGR1-mediated bronchodilation in the context of a cytokine exposure or mast cell degranulation; and Project 3 will develop ligands that will attenuate AHR and restore bronchodilator hyporesponsiveness despite T2 inflammation. The contribution of Core B is significant as our models will elucidate molecular targets to abrogate AHR and enhance bronchodilation in the context of asthma. The innovation of Core B focuses on the state-of-the-art measurements of single cell force generation and small airway function of HASM using in vitro and ex vivo models. Further our models use only human cells and tissue mitigating species differences that confounds the study of EC coupling in ASM. A c...