Dynamin-related Protein 1 Inhibitor Research Articles

The Critical Role of Dynamin-Related Protein 1 in Hypoxia-Induced Pulmonary Vascular Angiogenesis.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by pulmonary vascular obstruction due in part to excessive pulmonary artery endothelial cells (PAECs) migration and proliferation. The mitochondrial fission protein dynamin-related protein-1 (DRP1) has important influence on pulmonary vascular remodeling. However, whether DRP1 participates in the development and progression of pulmonary vascular angiogenesis has not been reported previously. To test the hypothesis that DRP1 promotes the angiogenesis via promoting the proliferation, stimulating migration, and inhibiting the apoptosis of PAECs in mitochondrial Ca(2+)-dependent manner, we performed following studies. Using hemodynamic analysis and morphometric assay, we found that DRP1 mediated the elevation of right ventricular systemic pressure (RVSP), right heart hypertrophy, and increase of pulmonary microvessels induced by hypoxia. DRP1 inhibition reversed tube network formation in vitro stimulated by hypoxia. The mitochondrial Ca(2+) inhibited by hypoxia was recovered by DRP1 silencing. Moreover, pulmonary vascular angiogenesis promoted by DRP1 was reversed by the specific mitochondrial Ca(2+) uniporter inhibitor Ru360. In addition, DRP1 promoted the proliferation and migration of PAECs in mitochondrial Ca(2+)-dependent manner. Besides, DRP1 decreased mitochondrial membrane potential, reduced the DNA fragmentation, and inhibited the caspase-3 activation, which were all aggravated by Ru360. Therefore, these results indicate that the mitochondrial fission machinery promotes migration, facilitates proliferation, and prevents from apoptosis via mitochondrial Ca(2+)-dependent pathway in endothelial cells leading to pulmonary angiogenesis.

Abstract 391: Endogenous Drp1 Modulates Cardiac Respiration Through mPTP and Independent of Fission

Background: The cardiac mitochondria exhibit a stable morphology with a rather low level of dynamic changes. However, fission and fusion proteins, such as dynamin-related protein 1 (DRP1) are abundant in the heart. Whether these proteins bear other functions in the heart than mitochondrial dynamics regulation are largely unknown. We hypothesize that endogenous DRP1 in the heart regulates mitochondrial respiration independent of fission. Methods: Mitochondrial respiration was determined by measuring the OCR with Seahorse assay or Clark type electrode in adult rat cardiomyocytes or mitochondria isolated from adult mouse heart. Confocal imaging was used to quantify mitochondrial morphology in adult cardiomyocytes and H9C2 myoblasts. To evaluate the role of mitochondrial permeability transition pore (mPTP), we monitored superoxide flashes (SOF) and laser-induced mPTP openings, and used cyclophilin D knockout mice (CypD KO). Mitochondrial ROS and Ca2+ were also monitored. Results: Inhibiting the DRP1 GTPase activity by Mdivi-1 or overexpression of the dominant-negative mutant (DRP1-K38A) induced mild mitochondrial morphological changes in adult cardiomyocytes, and inhibited mitochondrial respiration. Modulation of fission/fusion by overexpressing DRP1 or treating cells with S3, a compound facilitates fusion, exhibited significant morphological changes, but failed to influence respiration. Therefore, endogenous DRP1 activity may regulate respiration in the heart and this effect is dissociated with morphological changes. Further, inhibiting DRP1 activity attenuated the frequency of SOF, indicating decreased transient mPTP openings, delayed laser-induced permanent mPTP opening, and increased mitochondrial Ca2+. Inhibiting DRP1 activity decreased mitochondrial ROS levels. The role of DRP1 inhibition on respiration absents in CypD KO myocytes, suggesting the involvement of mPTP in the modulation of respiration by endogenous DRP1. Conclusion: These results suggest that endogenous DRP1 positively regulates respiration in the heart. This effect is likely independent of its role in mitochondrial fission. DRP1 regulation of respiration may involve transient opening of mPTP and contribute to mitochondrial Ca2+ and ROS signaling.

Effects of Dynamin‐Related Protein‐1 (DRP‐1) Inhibition with MDIVI‐1 on Myocardial Injury, Mitochondrial Respiration and Stress‐Signalling in the Murine Heart

Mitochondrial fission protein, dynamin‐related protein‐1 (DRP‐1), may be a key determinant of cardiac resistance to ischemia‐reperfusion (I‐R) and other stresses. We examined the impact of I‐R on myocardial expression of DRP‐1 (together with Akt and Erk1/2), mitochondrial respiration, and injury in I‐R hearts in 4mo male C57Bl/6 mice subject to DRP‐1 inhibition. Functional recoveries of Langendorff perfused hearts subjected to 25 min ischemia/45 min reperfusion were significantly improved by pre‐treatment with 1 µM MDIVI‐1. Cell death (LDH efflux) was only modified by a higher 5 µM concentration. Protective effects were not replicated by an alternate inhibitor dynasore hydrate (1 µM). Ischemic insult repressed mitochondrial O2 consumption during early reperfusion, with a ~50% fall in complex I activity that was effectively reversed by MDIVI‐1. Functional benefits were associated with MDIVI‐1 dependent reductions in mitochondrial DRP‐1 expression and cytosolic Erk1/2 phosphorylation during I‐R, while phosphorylation of cardiac Akt was significantly augmented by MDIVI‐1. A similar pattern was evident in parallel studies of H2O2 (400 µM) challenged H9c2 myoblasts, with MDIVI‐1 negating oxidative‐stress Erk1/2 phosphorylation whereas phosphoactivation of Akt was preserved. These data support a key role for DRP‐1 in repressing I‐R tolerance, mitochondrial function, and protective Akt phosphorylation (whilst also augmenting Erk1/2 activation). This role of DRP‐1 in control of stress‐resistance, mitochondrial function and survival‐kinase signalling supports the potential value of targeting the protein to protect ischemic myocardium.

Mitochondrial dynamics in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an idiopathic cardiopulmonary disease characterized by obstruction of small pulmonary arteries. Vascular obstruction is the consequence of excessive proliferation and apoptosis resistance of vascular cells, as well as inflammation, thrombosis, and vasoconstriction. Vascular obstruction increases the afterload faced by the right ventricle (RV), leading to RV failure. The proliferative, obstructive vasculopathy of PAH shares several mitochondrial abnormalities with cancer, notably a shift to aerobic glycolysis and mitochondrial fragmentation. Mitochondria in the pulmonary artery smooth muscle cell (PASMC) normally serve as oxygen sensors. In PAH, acquired mitochondrial abnormalities, including epigenetic silencing of superoxide dismutase (SOD2), disrupt oxygen sensing creating a pseudo-hypoxic environment characterized by normoxic activation of hypoxia-inducible factor-1α (HIF-1α). The resulting metabolic shift to aerobic glycolysis (the Warburg phenomenon) reflects inhibition of pyruvate dehydrogenase by pyruvate dehydrogenase kinases. In addition, altered mitochondrial dynamics result in mitochondrial fragmentation. The molecular basis of this structural change includes upregulation and activation of fission mediators, notably dynamin-related protein 1 (DRP-1), and downregulation of fusion mediators, especially mitofusin-2 (MFN2). These pathogenic mitochondrial abnormalities offer new therapeutic targets. Inhibition of mitotic fission or enhancement of fusion in PAH PASMC slows cell proliferation, causes cell cycle arrest, and induces apoptosis. DRP-1 inhibition or MFN2 gene therapy can regress PAH in experimental models of PAH. This review focuses on the etiology of mitochondrial fragmentation in PAH and explores the therapeutic implications of mitochondrial dynamics in the pulmonary vasculature and RV.

Abstract 16983: Inhibition of Dynamin-Related Protein 1 Accelerates Vascular Calcification in apoE-Deficient Mice through AKT Activation

Background: Mitochondrial dysfunction has been implicated in the pathogenesis of cardiovascular disease. Dynamin-related protein 1 (DRP1) regulates mitochondrial function by driving membrane fission and allowing mitochondria to divide. How DRP1 acts in atherosclerosis, including the development of vascular calcification however remains unknown. Methods and Results: Aortic smooth muscle mitochondria from apoE-deficient mice (apoE-/-) exhibited a 47% increase in size compared to mitochondria from wild-type mice fed the same atherogenic diet for 18 weeks (N=103-129 mitochondria from 3 mice/group; p<0.05). In agreement with the observed changes in mitochondrial morphology, inhibitory phosphorylation of DRP1 on Ser637 was enriched in apoE-/- mouse aortas. DRP1 immunoreactivity increased around calcified areas of apoE-/- mouse aortic sections. Additionally, DRP1 mRNA was increased 1.8-fold in calcified over non-calcified areas (N=8; p<0.05). To determine the role of DRP1 inhibition on aortic lesion calcification, apoE-/- mice maintained on an atherogenic diet for 13 weeks (a point prior to calcified lesion development) were subsequently given the DRP1 inhibitor, MDIVI-1 (50mg/kg, i.p., once a week) for 5 more weeks. MDIVI-1 administration led to a 65% increase in calcified lesion area without altering lesion size (N=26 mice/group; p<0.05). A single injection of MDIVI-1 resulted in a 3-fold increase in the ratio of phospho-AKT to total pan-AKT along with a strong increase in Runx2 protein in the apoE-/- mouse aorta 6 hours post-injection. MDIVI-1 administration did not alter bone remolding as detected by micro-CT in wild type or apoE-/- mice (N=5 mice/group). Conclusion: We provide novel roles of the mitochondrial fission protein DRP1 in the vascular calcification process, and demonstrate that inhibition of DRP1 through administration of the DRP1 inhibitor MDIVI-1 in apoE-/- mice accelerates vascular calcification through the AKT/Runx2 pathway.

Quantitative Phosphoproteomic Study of Pressure-Overloaded Mouse Heart Reveals Dynamin-Related Protein 1 as a Modulator of Cardiac Hypertrophy

Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

Abstract 72: Inhibition of Dynamin Related Protein 1 Mediated Mitochondrial Fission During Cardiac Arrest Is Cardioprotective and Associated with Therapeutic Hypothermia

Introduction: Mitochondrial fission increases cellular reactive oxygen species (ROS) and apoptosis but its role in cardiac arrest (CA) is unknown. Dynamin related protein 1 (DRP1) enhances fission by translocating to the mitochondria and is regulated by its phosphorylation at Serine 616 (activating) and Serine 637 (inhibiting). We hypothesized that inhibiting fission and DRP1 during CA would be cardioprotective. We also tested whether therapeutic hypothermia (TH) inhibited fission and DRP1. Methods: Cardiomyocytes isolated from 1-2 day old C57BL6/J mice were infected with a modified baculovirus containing mitochondrial targeted green fluorescent protein and then exposed to simulated ischemia/reperfusion (IR) at 37C or 30C (TH) to assess mitochondrial morphology. Hearts from Sprague Dawley rats were exposed to 30 min of global IR at 37C ± 5 uM mitochondrial inhibitor of division 1 (Mdivi-1) or 30C (TH) in a Langendorff perfusion system. ROS was measured using MitoSOX tissue staining. Left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), DRP1 gene/protein expression, and mitochondrial morphology (electron microscopy) were assessed. Heart samples were also collected from C57BL/6 mice after an 8-min cardiac arrest induced by KCl. Results: IR increased cell death, mitochondrial fragmentation, ROS, and LVEDP (62±1 mm Hg vs 9±1 mmHg, n=5, p<0.05) while decreasing LVDP (26±11 vs 77±8 mm Hg, p<0.05) compared to perfusion only controls. In IR, DRP1 Serine 637 decreased two-fold (n=4, p<0.05) while total DRP1 in mitochondrial protein fractions increased two fold (n=4, p<.0001). TH and Mdivi-1 maintained mitochondrial morphology, decreased ROS, inhibited DRP1 Serine 637 dephosphorylation and mitochondrial translocation. TH and Mdivi-1 also maintained LVEDP (12±1 and 14±2 mm Hg, n=5) and LVDP (84±6 and 84±2 mm Hg, n=5) at levels similar to perfusion-only controls. Conclusions: IR is associated with increased mitochondrial fission, DRP1 activation, ROS generation, and contractile dysfunction in cellular and organ models of IR and in vivo CA. Inhibition of fission and DRP1 with Mdivi-1 and TH are cardioprotective. DRP1 may be a therapeutic target for patients suffering CA or undergoing cardiopulmonary bypass.

Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation. We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1α activation by CoCl(2) or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH. DRP-1-mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

A hyperfused mitochondrial state achieved at G 1 –S regulates cyclin E buildup and entry into S phase

Mitochondria undergo fission-fusion events that render these organelles highly dynamic in cells. We report a relationship between mitochondrial form and cell cycle control at the G(1)-S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at G(1)-S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early G(1) to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for G(1)-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at G(1)-S is linked to cyclin E buildup for regulation of G(1)-to-S progression.