PROJECT SUMMARY: Asthma is characterized by chronic inflammation and bronchial obstruction due to human airway smooth muscle (HASM) shortening. However, the underlying basis for an enhanced shortening or the hyper-contractile state of HASM in asthma is not known. Further, our incomplete understanding of type 2 (T2) inflammation- regulated excitation-contraction (E-C) coupling in HASM shortening has hindered the development of new HASM bronchodilators with a novel mechanism of action for over 60 years. This application seeks to gain a foundational knowledge on the mechanical endotypes of HASM shortening in asthma (inflammation-dependent and -independent) and identify improved bronchodilators that are less susceptible to tolerance and less affected by immune inflammatory responses in asthma, focusing on previously unrecognized mechanisms evoked by bitter taste receptors (TAS2Rs) expressed on HASM. Our preliminary data, in pre-clinical models, support a premise that the immunologic and/or pathogenic mechanisms associated with a sustained mechanical reinforcement of HASM shortening, and the loss of β2-adrenoceptor (β2AR)-mediated bronchodilation, involve a transcriptional repressor function of the polycomb group (PcG) protein EZH2 (enhancer of zeste homolog 2). Further, our preliminary studies find a mechanistic role for microRNA-214 (miR-214) in TAS2R-evoked translational inhibition of EZH2. Based on these results, we hypothesize that TAS2Rs on HASM inhibit T2 cytokine-regulated E-C coupling in HASM shortening and the physiological loss of β2AR function in EZH2- and miR-214-dependent manners. Our goals are, first, to characterize T2- and non-T2- mediated molecular kinetics and mechanics of E-C coupling in HASM shortening and, second, determine miR- epigenetic nexus (i.e., non-genetic mechanisms) by which TAS2R activation promotes the functional efficacy of β2ARs and inhibits the mechanical endotypes of HASM shortening in asthma. Toward this end, we will leverage our un