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Purpose: This study compared the extended depth of focus (EDOF) intraocular lens (IOL) (ZXR00; Tecnis Symfony, Johnson & Johnson Vision, Santa Ana, CA, US) to a novel, higher-order aspheric monofocal IOL (ICB00; Tecnis Eyhance, Johnson & Johnson Vision, Santa Ana, CA, US) which uses the same platform and material. Methods: Medical records of patients undergoing cataract surgery with ZXR00 or ICB00 implantation between March 2020 and January 2021 and with the data available for the 3-month visit were reviewed. The uncorrected near, intermediate, and distance visual acuity (VA); corrected distance VA; and optical quality parameters were the main outcome measures. Results: Among the 174 enrolled patients, 72 and 102 received the ZXR00 and ICB00, respectively. The average patient ages were 59.6 ± 10.6 (range: 49 to 70) and 65.2 ± 8.2 (range: 45 to 82) years in the ZXR00 and ICB00 groups, respectively, with significantly older patients in the ICB00 group. The other baseline parameters were not different for the 2 groups. Compared to the ICB00 group, the ZXR00 group showed markedly superior near VA (P

Neddylation is a reversible post-translational modification that covalently attaches ubiquitin-like protein NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Through regulating the function of various cellular proteins, neddylation impacts diverse cellular processes and disease states. We have previously identified an indispensable role for neddylation in cardiac chamber maturation through sustaining YAP signaling in the developing heart. However, the importance of neddylation in post-mitotic hearts remains unclear. Here we report neddylation as a novel post-translation mechanism regulating mitochondrial homeostasis in adult heart. Inducible deletion of NAE1, a regulatory subunit of NEDD8 E1 enzyme, in adult mouse heart resulted in dilated cardiomyopathy, heart failure, and eventually premature lethality. The severe cardiac phenotype was preceded by accumulation of mitophagic vesicles and mitochondrial elongation, accompanied by pronounced upregulation of mitophagic markers and alterations in mitochondrial dynamics proteins. Moreover, we found that neddylation is required for mitochondrial ubiquitination and turnover. Consequently, pharmacological and genetic inhibition of neddylation accumulated damaged mitochondria, impaired mitochondrial membrane potential, inhibited mitochondrial respiration, and increased cardiomyocyte cell death in both neonatal and adult cardiomyocytes. Mechanistically, neddylation of Cullins is essential for the assembly of a functional multi-subunit Cullin-RING ubiquitin ligase, which appears to mediate mitochondrial ubiquitination following mitochondrial damage. Taken together, our findings suggest that neddylation serves as a novel mechanism surveilling mitochondrial quality and is essential for the function of normal adult heart.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane–associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca(2+)-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

Mitochondrial permeability transition (MPT) is a phenomenon that the inner mitochondrial membrane (IMM) loses its selective permeability, leading to mitochondrial dysfunction and cell injury. Electrophysiological evidence indicates the presence of a mega-channel commonly called permeability transition pore (PTP) whose opening is responsible for MPT. However, the molecular identity of the PTP is still under intensive investigations and debates, although cyclophilin D that is inhibited by cyclosporine A (CsA) is the established regulatory component of the PTP. PTP can also open transiently and functions as a rapid mitochondrial Ca

Background Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics. Methods We used azide-induced ATP depletion to incite cell stress in mouse embryonic fibroblasts and renal proximal tubular cells, and renal ischemia-reperfusion to induce stress in mice. We also used knockout cells and mice to determine the role of Bif-1, and used multiple techniques to analyze the molecular interaction between Bif-1 and prohibitin-2. Results Upon cell stress, Bif-1 translocated to mitochondria to bind prohibitin-2, resulting in the disruption of prohibitin complex and proteolytic inactivation of the inner membrane fusion protein OPA1. Bif-1-deficiency inhibited prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis. Domain deletion analysis indicated that Bif-1 interacted with prohibitin-2 via its C-terminus. Notably, mutation of Bif-1 at its C-terminal tryptophan-344 not only prevented Bif-1/prohibitin-2 interaction but also reduced prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis, supporting a pathogenic role of Bif-1/prohibitin-2 interaction. In mice, Bif-1 bound prohibitin-2 during renal ischemia/reperfusion injury, and Bif-1-deficiency protected against OPA1 proteolysis, mitochondrial fragmentation, apoptosis and kidney injury. Conclusions These findings suggest that during cell stress, Bif-1 regulates mitochondrial inner membrane by interacting with prohibitin-2 to disrupt prohibitin complexes and induce OPA1 proteolysis and inactivation.

Elevated levels of the excitatory amino acid homocysteine (Hcy) have been implicated in retinal diseases in humans including glaucoma and macular degeneration. It is not clear whether elevated Hcy levels are pathogenic. Models of hyperhomocysteinemia (Hhcy) have proven useful in addressing this including mice with deficiency in the enzyme cystathionine β-synthase (CBS). Cbs(+/−) mice have a ~two-fold increase in plasma and retinal Hcy levels. Previous studies of visual function and structure in Cbs(+/−) mice during the first 10 months of life revealed mild ganglion cell loss, but minimal electrophysiological alterations. It is not clear whether extended, chronic exposure to moderate Hhcy elevation will lead to visual function loss and retinal pathology. The present study addressed this by performing comprehensive analyses of retinal function/structure in 20 month Cbs(+/−) and Cbs(+/+) (WT) mice including IOP, SD-OCT, scotopic and photopic ERG, pattern ERG (pERG), and visual acuity. Eyes were harvested for histology and immunohistochemical analysis of Brn3a (ganglion cells), dihydroethidium (oxidative stress) and GFAP (gliosis). The analyses revealed no difference in IOP between groups for age/strain. Visual acuity measured ~0.36c/d for mice at 20 months in Cbs(+/−) and WT mice; contrast sensitivity did not differ between groups at either age. Similarly SD-OCT, scotopic/photopic ERG and pERG revealed no differences between 20 month Cbs(+/−) and WT mice. There was minimal disruption in retinal structure when eyes were examined histologically. Morphometric analysis revealed no significant differences in retinal layers. Immunohistochemistry revealed ~5 RGCs/100μm retinal length in both Cbs(+/−) and WT mice at 20 months. While there was greater oxidative stress and gliosis in older (20 month) mice versus young (4 month) mice, there was no difference in these parameters between the 20 month Cbs(+/−) and WT mice. We conclude that chronic, moderate Hhcy (at least due to deficiency of Cbs) is not accompanied by retinal structural/functional changes that differ significantly from age-matched WT littermates. Despite considerable evidence that severe Hhcy is toxic to retina, moderate Hhcy appears tolerated by retina suggesting compensatory cellular survival mechanisms.

Key points Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. Abstract Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.

SUMMARY Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/− mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging., In Brief Kim et al. demonstrate a molecular link between PDIA1 and Drp1 oxidoreduction, which protects against mitochondrial fragmentation and ROS elevation, limiting endothelial senescence. This study provides insights into restoring endothelial PDIA1 function or targeting Drp1 Cys oxidation as potential therapeutic strategies for treating diabetes-associated vascular and metabolic diseases., Graphical Abstract

The protein optic atrophy 1 (OPA1) is a dynamin-related protein associated with the inner mitochondrial membrane and functions in mitochondrial inner membrane fusion and cristae maintenance. Inner membrane-anchored long OPA1 (L-OPA1) undergoes proteolytic cleavage resulting in short OPA1 (S-OPA1). It is often thought that S-OPA1 is a functionally insignificant proteolytic product of L-OPA1 because the accumulation of S-OPA1 due to L-OPA1 cleavage is observed in mitochondrial fragmentation and dysfunction. However, cells contain a mixture of both L- and S-OPA1 in normal conditions, suggesting the functional significance of maintaining both OPA1 forms, but the differential roles of L- and S-OPA1 in mitochondrial fusion and energetics are ill-defined. Here, we examined mitochondrial fusion and energetic activities in cells possessing L-OPA1 alone, S-OPA1 alone, or both L- and S-OPA1. Using a mitochondrial fusion assay, we established that L-OPA1 confers fusion competence, whereas S-OPA1 does not. Remarkably, we found that S-OPA1 alone without L-OPA1 can maintain oxidative phosphorylation function as judged by growth in oxidative phosphorylation-requiring media, respiration measurements, and levels of the respiratory complexes. Most strikingly, S-OPA1 alone maintained normal mitochondrial cristae structure, which has been commonly assumed to be the function of OPA1 oligomers containing both L- and S-OPA1. Furthermore, we found that the GTPase activity of OPA1 is critical for maintaining cristae tightness and thus energetic competency. Our results demonstrate that, contrary to conventional notion, S-OPA1 is fully competent for maintaining mitochondrial energetics and cristae structure.

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Muller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Muller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Muller cells were exposed to L-Hcy-thiolactone [50μM–10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Muller cells. Unlike isolated RGCs, isolated Muller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, Muller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Muller cells harvested from Nrf2−/− mice. In contrast to WT Muller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2−/− Muller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Muller cells.

Aims Vascular smooth muscle cell (VSMC) migration in response to arterial wall injury is a critical process in the development of intimal hyperplasia. Cell migration is an energy-demanding process that is predicted to require mitochondrial function. Mitochondria are morphologically dynamic, undergoing continuous shape change through fission and fusion. However, the role of mitochondrial morphology in VSMC migration is not well understood. The aim of the study is to understand how mitochondrial fission contributes to VSMC migration and provides its in vivo relevance in the mouse model of intimal hyperplasia. Methods and results In primary mouse VSMCs, the chemoattractant PDGF induced mitochondrial shortening through the mitochondrial fission protein dynamin-like protein 1 (DLP1)/Drp1. Perturbation of mitochondrial fission by expressing the dominant-negative mutant DLP1-K38A or by DLP1 silencing greatly decreased PDGF-induced lamellipodia formation and VSMC migration, indicating that mitochondrial fission is an important process in VSMC migration. PDGF induced an augmentation of mitochondrial energetics as well as ROS production, both of which were found to be necessary for VSMC migration. Mechanistically, the inhibition of mitochondrial fission induced an increase of mitochondrial inner membrane proton leak in VSMCs, abrogating the PDGF-induced energetic enhancement and an ROS increase. In an in vivo model of intimal hyperplasia, transgenic mice expressing DLP1-K38A displayed markedly reduced ROS levels and neointima formation in response to femoral artery wire injury. Conclusions Mitochondrial fission is an integral process in cell migration, and controlling mitochondrial fission can limit VSMC migration and the pathological intimal hyperplasia by altering mitochondrial energetics and ROS levels.

The major function of mitochondria is production and supply of cellular energy. Mitochondria are highly dynamic organelles undergoing frequent shape changes via fission and fusion. Many studies have elucidated the molecular components mediating fission and fusion and their regulatory mechanisms, and mitochondrial shape change is now recognized as an essential cellular process that is closely associated with functional states of mitochondria. This review updates the recent advancements in fission and fusion mechanisms, and discusses the bi-directional relationship between mitochondrial morphology and energetic states in physio-pathological settings.

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.

Mitochondria produce the majority of cellular ATP through oxidative phosphorylation, and their capacity to do so is influenced by many factors. Mitochondrial morphology is recently suggested as an important contributor in controlling mitochondrial bioenergetics. Mitochondria divide and fuse continuously, which is affected by environmental factors, including metabolic alterations. Underscoring its bioenergetic influence, altered mitochondrial morphology is reported in tissues of patients and in animal models of metabolic dysfunction. In this study, we found that mitochondrial fission plays a vital role in the progression of nonalcoholic fatty liver disease (NAFLD). The development of hepatic steatosis, oxidative/nitrative stress, and hepatic tissue damage, induced by a high-fat diet, were alleviated in genetically manipulated mice suppressing mitochondrial fission. The alleviation of steatosis was recapitulated in primary hepatocytes with the inhibition of mitochondrial fission. Mechanistically, our study indicates that fission inhibition enhances proton leak under conditions of free fatty acid incubation, implicating bioenergetic change through manipulating mitochondrial fission. Taken together, our results suggest a mechanistic role for mitochondrial fission in the etiology of NAFLD. The efficacy of decreasing mitochondrial fission in the suppression of NAFLD suggests that mitochondrial fission represents a novel target for therapeutic treatment of NAFLD.

Dynamin-related membrane remodeling proteins regulate mitochondrial morphology by mediating fission and fusion. Although mitochondrial morphology is considered an important factor in maintaining mitochondrial function, a direct mechanistic link between mitochondrial morphology and function has not been defined. We report here a previously unrecognized cellular process of transient contraction of the mitochondrial matrix. Importantly, we found that this transient morphological contraction of mitochondria is accompanied by a reversible loss or decrease of inner membrane potential. Fission deficiency greatly amplified this phenomenon, which functionally exhibited an increase of inner membrane proton leak. We found that electron transport activity is necessary for the morphological contraction of mitochondria. Furthermore, we discovered that silencing the inner membrane-associated dynamin optic atrophy 1 (OPA1) in fission deficiency prevented mitochondrial depolarization and decreased proton leak without blocking mitochondrial contraction, indicating that OPA1 is a factor in coupling matrix contraction to mitochondrial depolarization. Our findings show that transient matrix contraction is a novel cellular mechanism regulating mitochondrial activity through the function of the inner membrane dynamin OPA1.

Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mitochondrial division inhibitor ( mdivi-1), a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated myotubes ( day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass, and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.

Mitochondrial fission and fusion have been recognized as critical processes in the health of mitochondria and cells. Two decades of studies have generated a great deal of information about mitochondrial fission and fusion; however, still much needs to be understood for the basic molecular mechanisms of these important cellular processes. The core protein factors for mitochondrial fission and fusion are dynamin proteins that possess membrane-remodeling properties. This short review covers a recent development and understanding of the mechanisms by which these mechanochemical enzymes mediate mitochondrial fission and fusion.

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.

In typical cultured animal cells, mitochondria exist in shapes ranging from long interconnected tubules, often clustered in the perinuclear region of the cell, to small spherical forms in the cell periphery. However, mitochondrial appearance at any given moment in time is only a snapshot of dynamic

Increased production of reactive oxygen species (ROS) from mitochondria is the main cause of hyperglycemic complications. We previously showed that hyperglycemic conditions induce mitochondrial fragmentation that is causal for ROS overproduction. This study was to identify signaling components that induce mitochondrial fragmentation in high-glucose stimulation. We found that exposing cells to the high-glucose concentration evokes increases in cytosolic Ca2+. Chelating Ca2+ in the high-glucose medium prevented not only the Ca2+ transient but also mitochondrial fragmentation and the ROS increase, indicating that the Ca2+ influx across the plasma membrane is an upstream event governing mitochondrial fission and the ROS generation in high-glucose stimulation. We found that the high-glucose-induced Ca2+ increase activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2). The Ca2+ chelation prevented the ERK1/2 activation, and inhibition of the ERK1/2 phosphorylation decreased mitochondrial fragmentation as well as ROS levels in high-glucose stimulation. In addition, the level of the mitochondrial fission protein dynamin-like protein 1 in mitochondria increased in high-glucose incubation in a Ca2+-dependent manner. In vitro kinase assays showed that ERK1/2 is capable of phosphorylating dynamin-like protein 1. These results demonstrate that high-glucose stimulation induces the activation of mitochondrial fission via signals mediated by intracellular Ca2+ and ERK1/2. Antioxid. Redox Signal. 14, 425–437.

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca2+) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca2+ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca2+ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca2+ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca2+ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca2+ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca2+-mediated ROS generation in cardiac myocytes. These results suggest that Ca2+, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six alpha-helices (alpha1-alpha6) out of which alpha2-alpha5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the alpha1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that alpha1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the alpha5 helix and the linker between alpha3 and alpha4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by alpha1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.

Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca2+/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart., β-adrenergic receptor signaling induces mitochondrial permeability transition pore (mPTP) opening. Here, Xu et al. show that this effect is mediated by phosphorylation of mitochondrial fission protein Drp1 by CamKII, which increases the frequency of transient mPTP opening.

We have previously demonstrated delta PKC (dPKC) involvement in cardiac myocyte energy deprivation via its interaction with dF1Fo and have developed a peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] of this interaction, which targets to the mitochondrial matrix / inner membrane. In the present study, a 10 min ligation of the left anterior descending coronary artery (LAD) increased dPKC-dF1Fo co-immunoprecipitation (co-IP) by 5-fold and elevated dPKC-phosphoserine 643 immunoreactivity (a dPKC autophosphorylation site 56 +/- 17% (n=7, p

Previously, we demonstrated protection against hypoxic injury in neonatal cardiac myocytes and reduced release of cardiac troponin I from perfused rat hearts by a novel peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-] of the delta protein kinase C (δPKC) interaction with the "d" subunit of mitochondrial F1Fo ATP synthase (dF1Fo). This peptide was developed in our laboratory and contains: an HIV-Tat protein transduction domain; a mitochondrial targeting motif; the δPKC-dF1Fo inhibitor sequence; and a FLAG epitope. In the present study the δPKC-dF1Fo inhibitor attenuated co-immunoprecipitation of δPKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size following simulated global ischemia/reperfusion (IR) exposures. Perfusion of hearts with this peptide prior to IR enhanced ATP levels 2.1-fold, improved ADP (state 3)- and FCCP (maximal)-stimulated respiration in mitochondrial oxygen consumption assays, and attenuated Ca(++)-induced mitochondrial swelling following ischemic injury. Mitochondrial membrane potential (assessed by JC-1) was also improved 1.6-fold by the inhibitor in hearts subsequently exposed to IR injury. Brief IR exposures did not cause mitochondrial loss of cytochrome c in the presence or absence of the inhibitor. Additionally, the inhibitor did not modify accumulation of the autophagy marker LC3II after brief IR injury. Our results support the potential for this first-in-class peptide as a translational agent for combating cardiac IR injury.

Significance: Cardiac function is energetically demanding, reliant on efficient well-coupled mitochondria to generate adenosine triphosphate and fulfill the cardiac demand. Predictably then, mitochondrial dysfunction is associated with cardiac pathologies, often related to metabolic disease, most commonly diabetes. Diabetic cardiomyopathy (DCM), characterized by decreased left ventricular function, arises independently of coronary artery disease and atherosclerosis. Dysregulation of Ca2+ handling, metabolic changes, and oxidative stress are observed in DCM, abnormalities reflected in alterations in mitochondrial energetics. Cardiac tissue from DCM patients also presents with altered mitochondrial morphology, suggesting a possible role of mitochondrial dynamics in its pathological progression. Recent Advances: Abnormal mitochondrial morphology is associated with pathologies across diverse tissues, suggesting that this highly regulated process is essential for proper cell maintenance and physiological homeostasis. Highly structured cardiac myofibers were hypothesized to limit alterations in mitochondrial morphology; however, recent work has identified morphological changes in cardiac tissue, specifically in DCM. Critical Issues: Mitochondrial dysfunction has been reported independently from observations of altered mitochondrial morphology in DCM. The temporal relationship and causative nature between functional and morphological changes of mitochondria in the establishment/progression of DCM is unclear. Future Directions: Altered mitochondrial energetics and morphology are not only causal for but also consequential to reactive oxygen species production, hence exacerbating oxidative damage through reciprocal amplification, which is integral to the progression of DCM. Therefore, targeting mitochondria for DCM will require better mechanistic characterization of morphological distortion and bioenergetic dysfunction. Antioxid. Redox Signal. 22, 1545–1562.

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

Mitochondrial fission is a highly regulated process mediated by a defined set of protein factors and is involved in the early stage of apoptosis. In mammals, at least two proteins, the dynamin-like protein DLP1/Drp1 and the mitochondrial outer membrane protein hFis1, participate in mitochondrial fission. The cytosolic domain of hFis1 contains six α-helices that form two tetratricopeptide repeat (TPR) motifs. Overexpression of hFis1 induces DLP1-mediated fragmentation of mitochondria, suggesting that hFis1 is a limiting factor in mitochondrial fission by recruiting cytosolic DLP1. In the present study, we identified two regions of hFis1 that are necessary for correct fission of mitochondria. We found that the TPR region of hFis1 participates in the interaction with DLP1 or DLP1-containing complex and that the first helix (α1) of hFis1 is required for mitochondrial fission presumably by regulating DLP1-hFis1 interaction. Misregulated interaction between DLP1 and hFis1 by α1 deletion induced mitochondrial swelling, in part by the mitochondrial permeability transition, but significantly delayed cell death. Our data suggest that hFis1 is a main regulator of mitochondrial fission, controlling the recruitment and assembly of DLP1 during both normal and apoptotic fission processes.

The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca2+. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca2+ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1. The complexity of protein–protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.

The mitochondrion is at the core of cellular energy metabolism, being the site of most ATP generation. Calcium is a key regulator of mitochondrial function and acts at several levels within the organelle to stimulate ATP synthesis. However, the dysregulation of mitochondrial Ca2+homeostasis is now recognized to play a key role in several pathologies. For example, mitochondrial matrix Ca2+overload can lead to enhanced generation of reactive oxygen species, triggering of the permeability transition pore, and cytochrome c release, leading to apoptosis. Despite progress regarding the independent roles of both Ca2+and mitochondrial dysfunction in disease, the molecular mechanisms by which Ca2+can elicit mitochondrial dysfunction remain elusive. This review highlights the delicate balance between the positive and negative effects of Ca2+and the signaling events that perturb this balance. Overall, a “two-hit” hypothesis is developed, in which Ca2+plus another pathological stimulus can bring about mitochondrial dysfunction.

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.

Intimal hyperplasia occurs in response to arterial injury and is also an important feature of atherosclerotic plaques. Vascular smooth muscle cell (VSMC) migration is one of the main processes contributing to intimal hyperplasia. Mitochondria are energy producing organelles that also generate reactive oxygen species as a byproduct of ATP synthesis. Mitochondria inside the cell redistribute frequently and undergo constant shape change mainly through fission and fusion, which is referred to as mitochondrial dynamics. Although the involvement of mitochondria in VSMC migration has been reported in the context of oxidative stress and mitochondrial dysfunction, the role of mitochondrial dynamics in VSMC migration has not been studied. In the present study, we found that platelet-derived growth factor (PDGF), a strong migratory stimulant, induced mitochondrial shortening in VSMCs. This phenomenon was accompanied by the translocation of the fission protein Drp1/DLP1 from the cytosol to mitochondria, suggesting th...

The large GTPase dynamin is a mechanoenzyme that mediates the liberation of nascent clathrin-coated pits from the plasma membrane during endocytosis. Recently, this enzyme has been demonstrated to comprise an extensive family of related proteins that have been implicated in a large variety of vesicle trafficking events during endocytosis, secretion and even maintenance of mitochondrial form. The potential contributions by the dynamin family to these diverse but related functions are discussed.

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

gamma-Tubulin is central to the nucleation of microtubule assembly in vivo. Although it is most obviously located at microtubule organizing centers, it is also found in soluble cytoplasmic complexes. Characterizing these complexes and identifying proteins that interact with gamma-tubulin in vivo will be necessary if gamma-tubulin function is to be understood fully. We have begun to investigate soluble complexes of gamma-tubulin in Aspergillus nidulans, the organism in which gamma-tubulin was discovered and in which a great deal of genetic and molecular genetic analysis of gamma-tubulin has been carried out. We find that approximately 32% of the gamma-tubulin in A. nidulans is soluble. Sucrose density gradients revealed that the soluble gamma-tubulin is in 8-20S complexes with little or no monomeric gamma-tubulin present. In the presence of 0.5 M KCl the average size of the complexes decreased and a peak was present between 4S and 11S. Cross-linking experiments with a zero-length cross-linker suggest that gamma-tubulin in isolated nuclei and in intact hyphae interacts physically with three proteins with molecular weights of approximately 105, 95, and 80 kDa.

Fission and fusion of mitochondrial tubules are the main processes determining mitochondrial shape and size in cells. As more evidence is found for the involvement of mitochondrial morphology in human pathology, it is important to elucidate the mechanisms of mitochondrial fission and fusion. Mitochondrial morphology is highly sensitive to changing environmental conditions, indicating the involvement of cellular signaling pathways. In addition, the well-established structural connection between the endoplasmic reticulum (ER) and mitochondria has recently been found to play a role in mitochondrial fission. This minireview describes the latest advancements in understanding the regulatory mechanisms controlling mitochondrial morphology, as well as the ER-mediated structural maintenance of mitochondria, with a specific emphasis on mitochondrial fission.

We have developed a procedure for purifying assembly-competent tubulin from Aspergillus nidulans. To our knowledge, this is the first report of the purification of assembly-competent tubulin from a filamentous fungus, and the procedure should be of great value in analyzing the large number of alpha- and beta-tubulin mutations that have been isolated and characterized in A. nidulans. Our procedure consists of overproduction of alpha- and beta-tubulin, partial purification by ion-exchange chromatography, and final purification by rounds of assembly and disassembly. We have found that taxol promotes the assembly of A. nidulans tubulin into microtubules, but a higher concentration of taxol is required for maximal assembly of A. nidulans tubulin than is required for brain tubulin. The critical concentration for assembly in the presence of taxol is also significantly higher for A. nidulans tubulin than for brain tubulin. In addition, A. nidulans microtubules that were assembled and maintained in the presence of taxol depolymerized in conditions in which taxol-stabilized mammalian microtubules remain intact. These results suggest that A. nidulans tubulin has a lower affinity for taxol than mammalian tubulin.