Asish Das Gupta, Queen’s University

Supervisor: Stephen Archer, Queen’s University

This proposal focuses on advancing basic understanding of pulmonary arterial hypertension (PAH) and developing new therapies for this lethal vascular disease. In PAH vascular obstruction results in part from a proliferative, apoptosisresistant diathesis of pulmonary arterial smooth muscle cells (PASMC). This neoplastic-like phenotype results, in part, from a perturbation of mitochondrial dynamics, due in part to ~ 50% downregulation of the antiproliferative-fusion mediator, mitofusin-2 (Mfn2). Mfn2 is a GTPase in the outer mitochondrial membrane that promotes fusion. The mechanism by which Mfn2 is downregulated in PAH is unknown. Hypothesis: Protein kinase A (PKA) promotes degradation of Mfn2 by phosphorylating Mfn2 at serine 442, a predicted PKA phosphorylation site. Decreasing serine 442 phosphorylation may restore Mfn2 expression and augment its antiproliferative effects. Preliminary data: 1) Phosphorylation of Mfn2 at serine 442 increases its proteasomal degradation. This loss of Mfn2 is sufficient to increase mitochondrial fragmentation and cause hyperproliferation. 2) A phosphomimetic form of Mfn2 (S442D), behaves as if it were constitutively phosphorylated. This mutant Mfn2 increases proteasomal degradation of native Mfn2 protein and recapitulates many of the characteristics of PAH in normal PASMCs. 3) PKA agonists phosphorylate Mfn2 at serine 442 and impair mitochondrial fusion leading to mitochondrial fragmentation. Goals: We will determine whether posttranslational modification/mutation of serine 442 in Mfn2 occur in PAH. We will also assess whether this posttranslational mechanism of Mfn2 downregulation contributes to the proliferation/apoptosis imbalance in PAH. The therapeutic value of inhibiting PKA or inhibiting proteasome-mediated degradation to restore Mfn2 expression and improve mitochondrial fusion will be tested in human PAH cell lines and in preclinical rodent models of PAH. Finally, we will explore whether Mfn2 phospho-serine 442 can be exploited as a molecular-biomarker for early detection of PAH. Relevance: This proposal aims to improve the health of Canadians by furthering the field of vascular research.