Epigenetic Dysregulation of the Drp1 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

The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease

Background: Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. Methods: For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. Results: Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. Conclusion: Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress