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 unique technological innovations of single-molecule and single-cell micromechanical methods and integrative genetics and genomics approaches in clinically relevant human precision cut lung slices (hPCLS) and primary HASM cells derived from donor lungs of patients with and without severe asthma. When successful, the knowledge gained from these experimental and computational studies will: 1) shed new light on inflammation-dependent and -independent regulation of E-C coupling in HASM shortening; 2) uncover previously unidentified TAS2R paradigms to mitigate the physiological loss of β2AR function; and 3) establish new druggable targets and agents to treat β2-agonist-insenstivity in a large cohort of patients with difficult-to- control and severe asthma. This line of research is underappreciated in asthma and represents a clear shift in the asthma treatment paradigm.

PROJECT NARRATIVE: Human airway smooth muscle (HASM) represents the pivotal cell regulating bronchomotor tone. However, our incomplete understanding of type 2 (T2) inflammation-regulated excitation-contraction (E-C) coupling in HASM shortening has, for the past 60 years, hindered the development of new HASM bronchodilators in the treatment of obstructive lung disease. We assert that studying the molecular mechanisms of excitation-contraction (E-C) coupling that differentiate HASM shortening in asthma- and non-asthma-derived cells/tissues will uncover novel mechanical endotypes of airflow obstruction (inflammation-dependent and -independent) and identify improved bronchodilators that are less susceptible to tolerance and less affected by immune inflammatory responses in asthma.