Epigenetic Dysregulation of the Dynamin-Related Protein 1 Binding Partners MiD49 and MiD51 Increases Mitotic Mitochondrial Fission and Promotes Pulmonary Arterial Hypertension: Mechanistic and Therapeutic Implications

Background -Mitotic fission is increased in pulmonary arterial hypertension (PAH), a hyperproliferative, apoptosis-resistant disease. The fission mediator, dynamin related protein 1 (Drp1) must complex with adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here we examine the role of two recently discovered, poorly understood, Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51) in normal vascular cells and explore their dysregulation in PAH. Methods -Immunoblots of pulmonary artery smooth muscle cells (PASMC, control, n=6; PAH, n=8) and immunohistochemistry of lung sections (control, n=6; PAH, n=6) were used to assess the expression of MiD49 and MiD51. The effects of manipulating MiDs on cell proliferation, cell cycle, and apoptosis were assessed in human and rodent PAH PASMC using flow cytometry. Mitochondrial fission was studied by confocal imaging. A microRNA (miR) involved in the regulation of MiD expression was identified using microarray techniques andin silicoanalyses. The expression of circulatory miR was assessed using qRT-PCR in healthy volunteers (HV) vs PAH patients from Sheffield, UK (plasma, HV, n=29, PAH, n=27; whole blood, HV, n=11, PAH, n=14), and then confirmed in a cohort from Beijing, China (plasma, HV, n=19, PAH, n=36; whole blood, HV, n=20, PAH, n=39). This work was replicated in monocrotaline and SU5416-hypoxia, preclinical PAH models. siRNA targeting MiDs or a miR mimic were nebulized to rats with monocrotaline-induced PAH (n=4-10). Results -MiD expression is increased in PAH PASMC, which accelerates Drp1-mediated mitotic fission, increases cell proliferation and decreases apoptosis. Silencing MiDs (but not other Drp1 binding partners, Fis1 or MFF) promotes mitochondrial fusion and causes G1-phase cell cycle arrest, through ERK1/2 and CDK4-dependent mechanism. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased miR-34a-3p expression. Circulatory miR-34a-3p expression is decreased in both PAH patients and in preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. Conclusions -In health, MiDs regulate Drp1-mediated fission whilst in disease, epigenetic upregulation of MiDs increases mitotic fission, which drives pathologic proliferation and apoptosis resistance. The miR-34a-3p-MiD pathway offers new therapeutic targets for PAH.