Mitochondrial fission, regulated by dynamin‐related protein‐1 (Drp1), is a newly recognized determinant of mitochondrial function, but its contribution to left ventricular (LV) impairment following ischemia‐reperfusion (IR) injury is unknown. We report that Drp1 activation during IR results in LV dysfunction and that Drp1 inhibition is beneficial. In both isolated neonatal murine cardiomyocytes and adult rat hearts (Langendorff preparation) mitochondrial fragmentation and swelling occurred within 30 min of IR Drp1‐S637 (serine 637) dephosphorylation resulted in Drp1 mitochondrial translocation and increased mitochondrial fission. The Drp1 inhibitor Mdivi‐1 preserved mitochondrial morphology, reduced cytosolic calcium, and prevented cell death. Drp1 siRNA similarly preserved mitochondrial morphology. In Langendorff hearts, Mdivi‐1 reduced mitochondrial reactive oxygen species, improved LV developed pressure (92±5 vs. 28±10 mmHg, P <0.001), and lowered LV end diastolic pressure (10±1 vs. 86±13 mmHg, P <0.001) following IR Mdivi‐1 was protective if administered prior to or following ischemia. Because Drp1‐S637 dephosphorylation is calcineurin sensitive, we assessed the effects of a calcineurin inhibitor, FK506. FK506 treatment prior to IR prevented Drp1‐S637 dephosphorylation and preserved cardiac function. Likewise, therapeutic hypothermia (30°C) inhibited Drp1‐S637 dephosphorylation and preserved mitochondrial morphology and myocardial function. Drp1 inhibition is a novel strategy to improve myocardial function following IR.—Sharp, W. S., Fang, Y. H., Han, M., Zhang, H. J., Hong, Z., Banathy, A., Morrow, E., Ryan, J. J., Archer, S. L. Dynamin‐related protein 1 (Drp1)‐mediated diastolic dysfunction in myocardial ischemia‐reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission. FASEB J. 28, 316–326 (2014). www.fasebj.org

Rationale: Pulmonary arterial hypertension (PAH) is a lethal, female-predominant, vascular disease. Pathologic changes in PA smooth muscle cells (PASMC) include excessive proliferation, apoptosis-resistance, and mitochondrial fragmentation. Activation of dynamin-related protein increases mitotic fission and promotes this proliferation–apoptosis imbalance. The contribution of decreased fusion and reduced mitofusin-2 (MFN2) expression to PAH is unknown. Objectives: We hypothesize that decreased MFN2 expression promotes mitochondrial fragmentation, increases proliferation, and impairs apoptosis. The role of MFN2’s transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), was assessed. MFN2 therapy was tested in PAH PASMC and in models of PAH. Methods: Fusion and fission mediators were measured in lungs and PASMC from patients with PAH and female rats with monocrotaline or chronic hypoxia+Sugen-5416 (CH+SU) PAH. The effects of adenoviral mitofusin-2 (Ad-MFN2) overexpression were measured in vitro and in vivo. Measurements and Main Results: In normal PASMC, siMFN2 reduced expression of MFN2 and PGC1α; conversely, siPGC1α reduced PGC1α and MFN2 expression. Both interventions caused mitochondrial fragmentation. siMFN2 increased proliferation. In rodent and human PAH PASMC, MFN2 and PGC1α were decreased and mitochondria were fragmented. Ad-MFN2 increased fusion, reduced proliferation, and increased apoptosis in human PAH and CH+SU. In CH+SU, Ad-MFN2 improved walking distance (381 ± 35 vs. 245 ± 39 m; P < 0.05); decreased pulmonary vascular resistance (0.18 ± 0.02 vs. 0.38 ± 0.14 mm Hg/ml/min; P < 0.05); and decreased PA medial thickness (14.5 ± 0.8 vs. 19 ± 1.7%; P < 0.05). Lung vascularity was increased by MFN2. Conclusions: Decreased expression of MFN2 and PGC1α contribute to mitochondrial fragmentation and a proliferation–apoptosis imbalance in human and experimental PAH. Augmenting MFN2 has therapeutic benefit in human and experimental PAH.

Honokiol, an activator of Sirtuin-3 (SIRT3) preserves mitochondria and protects the heart from doxorubicin-induced cardiomyopathy in mice

Doxorubicin (Doxo) is a chemotherapeutic drug widely used to treat variety of cancers. One of the most serious side effects of Doxo is its dose-dependent and delayed toxicity to the heart. Doxo is known to induce cardiac mitochondrial damage. Recently, the mitochondrial sirtuin SIRT3 has been shown to protect mitochondria from oxidative stress. Here we show that overexpression of SIRT3 protects the heart from toxicity of Doxo by preventing the drug-induced mitochondrial DNA (mtDNA) damage. Doxo treatment caused depletion of Sirt3 levels both in primary cultures of cardiomyocytes and in mouse hearts, which led to massive acetylation of mitochondrial proteins. Doxo-induced toxicity to cardiomyocytes was associated with increased reactive oxygen species (ROS) production, mitochondrial fragmentation, and cell death. Overexpression of SIRT3 helped to attenuate Doxo-induced ROS levels and cardiomyocyte death. Sirt3 knockout (Sirt3.KO) mice could not endure the full dose of Doxo treatment, developed exacerbated cardiac hypertrophy, and died during the course of treatment, whereas Sirt3 transgenic (Sirt3.tg) mice were protected against Doxo-induced cardiotoxicity. Along with Sirt3, we also observed a concomitant decrease in levels of oxoguanine-DNA glycosylase-1 (OGG1), a major DNA glycosylase that hydrolyzes oxidized-guanine (8-oxo-dG) to guanine. Depletion of OGG1 levels was associated with increased mtDNA damage. Sirt3.KO mice and Doxo-treated mice showed increased 8-oxo-dG adducts in DNA and corresponding increase in mtDNA damage, whereas, 8-oxo-dG adducts and mtDNA damage were markedly reduced in Sirt3 overexpressing transgenic mice hearts. These results thus demonstrated that Sirt3 activation protects the heart from Doxo-induced cardiotoxicity by maintaining OGG1 levels and protecting mitochondria from DNA damage.

Rationale:: Closure of the ductus arteriosus (DA) is essential for the transition from fetal to neonatal patterns of circulation. Initial P O ₂ -dependent vasoconstriction causes functional DA closure within minutes. Within days a fibrogenic, proliferative mechanism causes anatomic closure. Though modulated by endothelial-derived vasodilators and constrictors, O ₂ sensing is intrinsic to ductal smooth muscle cells and oxygen-induced DA constriction persists in the absence of endothelium, endothelin, and cyclooxygenase mediators. O ₂ increases mitochondrial-derived H ₂ O ₂ , which constricts ductal smooth muscle cells by raising intracellular calcium and activating rho kinase. However, the mechanism by which oxygen changes mitochondrial function is unknown. Objective:: The purpose of this study was to determine whether mitochondrial fission is crucial for O ₂ -induced DA constriction and closure. Methods and Results:: Using DA harvested from 30 term infants during correction of congenital heart disease, as well as DA from term rabbits, we demonstrate that mitochondrial fission is crucial for O ₂ -induced constriction and closure. O ₂ rapidly (<5 minutes) causes mitochondrial fission by a cyclin-dependent kinase- mediated phosphorylation of dynamin-related protein 1 (Drp1) at serine 616. Fission triggers a metabolic shift in the ductal smooth muscle cells that activates pyruvate dehydrogenase and increases mitochondrial H ₂ O ₂ production. Subsequently, fission increases complex I activity. Mitochondrial-targeted catalase overexpression eliminates P O ₂ -induced increases in mitochondrial-derived H ₂ O ₂ and cytosolic calcium. The small molecule Drp1 inhibitor, Mdivi-1, and siDRP1 yield concordant results, inhibiting O ₂ -induced constriction (without altering the response to phenylephrine or KCl) and preventing O ₂ -induced increases in oxidative metabolism, cytosolic calcium, and ductal smooth muscle cells proliferation. Prolonged Drp1 inhibition reduces DA closure in a tissue culture model. Conclusions:: Mitochondrial fission is an obligatory, early step in mammalian O ₂ sensing and offers a promising target for modulating DA patency.