PROJECT SUMMARY Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is the most common cause of PH worldwide, and at present, it has no proven effective therapies. Previously, we have found convincing evidence that PH-HFpEF-regulating sirtuin-3 (SIRT3) is predominantly decreased in the skeletal muscle. During the past grant period, we found a new endocrine signaling axis that links depressed skeletal muscle SIRT3 to production/secretion of signaling molecules that interact with pulmonary vasculature in PH-HFpEF. However, underlying mechanisms by which skeletal muscle SIRT3-mediated scretome regulates pathological changes in pulmonary vasculature are not well-understood at present. Based on a mass spectrometry-based plasma proteomics approach, our new data show that 2-microglobulin (B2M) is present at higher levels in patients with PH-HFpEF. Both circulating and skeletal muscle levels of B2M are markedly increased in mice with skeletal muscle SIRT3 deficiency. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients also show higher B2M levels with increased disease severity. Treatment with B2M recombinant protein reduces expression of WW domain-containing oxidoreductase (WWOX), which plays a role in repressing cell proliferation, in PASMCs. B2M treatment also increases expression of canopy fibroblast growth factor signaling regulator 2 (CNPY2), an unfolded protein response (UPR) initiator known to induce excessive angiogenesis and proliferation, in PAECs. While B2M treatment increases PAECs migration/proliferation and media conditioned by CNPY2-overexpressed PAECs reduces WWOX in PASMCs, the role of PAECs CNPY2 in PH-HFpEF remains elusive. Thus, we hypothesize that skeletal muscle-secreted B2M acts as an endocrine signaling molecule which induces remote pulmonary vascular remodeling via dysregulating PAECs CNPY2 and PASMCs WWOX. Specific Aim 1 will determine whether skeletal muscle B2M is an important signaling molecule affecting multiple pulmonary vascular pathophenotypes. Using wild-type and skeletal muscle-specific B2m KO mice, with or without high-fat feeding (HFD), which induces a PH-HFpEF phenotype, we will characterize the contribution of skeletal muscle B2M in PH-HFpEF. In Aim 2, we will determine the role of PAECs CNPY2 in pulmonary vascular remodeling and PH-HFpEF with EC-specific Cnpy2 KO mice. Specific Aim 3 will use plasma and muscle biopsies of patients with PH-HFpEF or HFpEF alone collected from biobanks and Phase II clinical trials to evaluate whether B2M can be a relevant biomarker of PH-HFpEF severity and prognosis. Inducible pluripotent stem cell (iPSC)-derived ECs of PH-HFpEF patients will also be used to evaluate clinical relevance of CNPY2. We expect that successful execution of the proposed research plan would provide new mechanistic understanding of PH-HFpEF pathogenesis and aid in providing important information to guide future research towards development of t...