Defining the molecular basis of thrombospondin-1-induced fibro-fatty diaphragm remodeling--a driver of obesity-associated respiratory dysfunction

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ABSTRACT Breathing disorders affect most individuals with obesity—and while respiratory muscle dysfunction occurs with elevated BMI, its underlying pathophysiology and potential therapeutic targeting in obesity-associated pulmonary compromise are largely uninvestigated. Mice subjected to diet-induced obesity (DIO), develop diaphragm weakness. In these animals, intra-diaphragmatic adiposity, and extracellular matrix (ECM) content quantitatively correlate with reductions in muscle contractile force. Resident mesenchymal cells called fibro-adipogenic progenitors (FAPs) are the source of all adipocytes and many ECM-depositing cells in the obese diaphragm. With DIO, diaphragm FAPs assume a proliferative, fibrogenic phenotype, and differentiate toward a Thy1- expressing sub-population previously linked to muscle degeneration. Thrombospondin-1 (THBS-1) is a matricellular protein produced by cell types including macrophages, adipocytes and FAPs. Physiologically induced by injury and ischemia, THBS1 levels chronically increase in obesity. A FAP mitogen and activator of TGF signaling, THBS-1 has been linked to fibrotic and degenerative changes in genetic myopathies. Global Thbs1 knockout subjected to DIO gain equivalent weight to WT controls, but do not exhibit FAP pool expansion or shift toward the Thy-1 expressing sub-type. Moreover, Thbs1-null diaphragms are protected from obesity-induced increases in adiposity and ECM deposition. Strikingly, Thbs1 knockout mice maintain normal diaphragm contractile force and motion with DIO. We hypothesize mesenchymal THBS1 promotes TGF-mediated, FAP-driven stromal expansion that can be targeted to mitigate diaphragm weakness and respiratory dysfunction in obesity. We will test the hypothesis through cell and tissue-based systems, in genetically engineered mouse models, and in human subjects: (1) First, we will employ primary cell culture to interrogate TGF and other putative THBS1 effectors as drivers of FAP proliferation, fibrogenesis and adipogenesis. We will then leverage a FAP-myobundle organoid system to determine whether the negative impact of THBS1 on muscle contractile function depends on FAPs. (2) Next, we will apply mouse models of inducible cell type-specific Thbs1 ablation (macrophage, adipocyte, FAP) to establish the cellular source of pathogenic THBS1 in obesity. We will also treat DIO mice with agents targeting THBS1/TGF-driven fibrosis to test whether this maneuver is sufficient to forestall or reverse pathological diaphragm remodeling and respiratory dysfunction. (3) Finally, we will analyze a translational weight loss cohort to define the relationship among circulating THBS1 levels, diaphragm motion (ultrasound measurement of diaphragm excursion amplitude), and diaphragm fibro-adipogenic remodeling (ultrasound measurement of muscle echo intensity). Together, the proposed experiments will define targetable cellular and molecular mechanisms underlying obesity-induced respiratory dysfunction.