Abstract Body (Do not enter title and authors here): Introduction: Mitochondrial fission is mediated by dynamin related protein-1 (Drp1) activation; however, to complete fission, we have evidence that Drp1 requires assistance from the GTPase dynamin 2 (DNM2).
Hypothesis: DNM2 interacts with Drp1 to regulate mitotic fission and cell cycle progression, permitting the hyperproliferative phenotype of pulmonary artery smooth muscle cells (PASMC) in pulmonary arterial hypertension (PAH).
Methods: Colocalization of Drp1 and DNM2 was assessed using STED super resolution confocal microscopy. Interaction of Drp1 and DNM2 was confirmed by immunoprecipitation. The role of DNM2’s GTPase domain in mitochondrial targeting was assessed by heterologous expression of truncated constructs. Changes in transcriptomic signature from silencing DNM2 in PAH human PASMC (hPASMC) were measured by RNA-seq. DNM2 expression in control vs PAH was quantified in hPASMC (n=4-5/group) and lungs from human (n=5/group), monocrotaline (MCT) (n=4-5/group) and SU5416/hypoxia (Su/Hx) (n=4-5/group) induced rodents using immunoblots, immunohistochemistry and immunofluorescence. DNM2’s effects on cell proliferation, cell cycle and apoptosis were assessed by flow cytometry. siDNM2 was nebulized to rats with established MCT-PAH.
Results: DNM2 is significantly increased in human and rodent PAH and silencing DNM2 reduces mitochondrial fission and cell proliferation and induces apoptosis and cell cycle blockade in the G0/G1 phase in PAH PASMC. DNM2 and Drp1 interact at mitochondrial fission sites. DNM2’s translocation to the mitochondria is Drp-dependent and requires DNM2’s GTPase domain. Silencing DNM2 downregulates the Regulator of Cell Cycle, RGCC. Augmenting DNM2 in normal PASMC induces mitochondrial fission and cell proliferation. miR-124-3p, which negatively regulates DNM2, is decreased in PAH PASMC. Augmenting miR-124-3p decreases DNM2 expression, inhibits fission and proliferation and induces apoptosis in PAH PASMC. Silencing STAT3 (also negatively regulated by miR-124-3p) reduces DNM2 and inhibits fission. siDNM2 regresses PAH in MCT-PAH rats.
Conclusion: DNM2 binds to Drp1 and translocates to mitochondria, regulating fission. A pathological increase in DNM2 expression, driven by decreased miR-124a-3p and STAT3 activation, increases mitochondrial fission, proliferation and inhibits apoptosis in PAH PASMC. DNM2 regulates cell proliferation via RGCC. DNM2 is a fission mediator and its regulatory pathway offers potential PAH therapeutic targets.