Light Alcohol Consumption Promotes Early Neurogenesis Following Ischemic Stroke in Adult C57BL/6J Mice.

Ischemic stroke is one of the leading causes of death and disability worldwide. Neurogenesis plays a crucial role in postischemic functional recovery. Alcohol dose-dependently affects the prognosis of ischemic stroke. We investigated the impact of light alcohol consumption (LAC) on neurogenesis under physiological conditions and following ischemic stroke. C57BL/6J mice (three months old) were fed with 0.7 g/kg/day ethanol (designed as LAC) or volume-matched water (designed as control) daily for eight weeks. To evaluate neurogenesis, the numbers of 5-bromo-2-deoxyuridine (BrdU)+/doublecortin (DCX)+ and BrdU+/NeuN+ neurons were assessed in the subventricular zone (SVZ), dentate gyrus (DG), ischemic cortex, and ischemic striatum. The locomotor activity was determined by the accelerating rotarod and open field tests. LAC significantly increased BrdU+/DCX+ and BrdU+/NeuN+ cells in the SVZ under physiological conditions. Ischemic stroke dramatically increased BrdU+/DCX+ and BrdU+/NeuN+ cells in the DG, SVZ, ischemic cortex, and ischemic striatum. The increase in BrdU+/DCX+ cells was significantly greater in LAC mice compared to the control mice. In addition, LAC significantly increased BrdU+/NeuN+ cells by about three folds in the DG, SVZ, and ischemic cortex. Furthermore, LAC reduced ischemic brain damage and improved locomotor activity. Therefore, LAC may protect the brain against ischemic stroke by promoting neurogenesis.

Mn(III) Porphyrin, MnTnBuOE-2-PyP5+, Commonly Known as a Mimic of Superoxide Dismutase Enzyme, Protects Cardiomyocytes from Hypoxia/Reoxygenation Induced Injury via Reducing Oxidative Stress.

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Endothelial Specific Deletion of Autotaxin Improves Stroke Outcomes.

Autotaxin (ATX) is an extracellular secretory enzyme (lysophospholipase D) that catalyzes the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA). The ATX-LPA axis is a well-known pathological mediator of liver fibrosis, metastasis in cancer, pulmonary fibrosis, atherosclerosis, and neurodegenerative diseases. Additionally, it is believed that LPA may cause vascular permeability. In ischemic stroke, vascular permeability leading to hemorrhagic transformation is a major limitation for therapies and an obstacle to stroke management. Therefore, in this study, we generated an endothelial-specific ATX deletion in mice (ERT2 ATX-/-) to observe stroke outcomes in a mouse stroke model to analyze the role of endothelial ATX. The AR2 probe and Evans Blue staining were used to perform the ATX activity and vascular permeability assays, respectively. Laser speckle imaging was used to observe the cerebral blood flow following stroke. In this study, we observed that stroke outcomes were alleviated with the endothelial deletion of ATX. Permeability and infarct volume were reduced in ERT2 ATX-/- mice compared to ischemia-reperfusion (I/R)-only mice. In addition, the cerebral blood flow was retained in ERT2 ATX-/- compared to I/R mice. The outcomes in the stroke model are alleviated due to the limited LPA concentration, reduced ATX concentration, and ATX activity in ERT2 ATX-/- mice. This study suggests that endothelial-specific ATX leads to increased LPA in the brain vasculature following ischemic-reperfusion and ultimately disrupts vascular permeability, resulting in adverse stroke outcomes.

Pathological Sequelae Associated with Skeletal Muscle Atrophy and Histopathology in G93A*SOD1 Mice.

Amyotrophic lateral sclerosis (ALS) is a complex systemic disease that primarily involves motor neuron dysfunction and skeletal muscle atrophy. One commonly used mouse model to study ALS was generated by transgenic expression of a mutant form of human superoxide dismutase 1 (SOD1) gene harboring a single amino acid substitution of glycine to alanine at codon 93 (G93A*SOD1). Although mutant-SOD1 is ubiquitously expressed in G93A*SOD1 mice, a detailed analysis of the skeletal muscle expression pattern of the mutant protein and the resultant muscle pathology were never performed. Using different skeletal muscles isolated from G93A*SOD1 mice, we extensively characterized the pathological sequelae of histological, molecular, ultrastructural, and biochemical alterations. Muscle atrophy in G93A*SOD1 mice was associated with increased and differential expression of mutant-SOD1 across myofibers and increased MuRF1 protein level. In addition, high collagen deposition and myopathic changes sections accompanied the reduced muscle strength in the G93A*SOD1 mice. Furthermore, all the muscles in G93A*SOD1 mice showed altered protein levels associated with different signaling pathways, including inflammation, mitochondrial membrane transport, mitochondrial lipid uptake, and antioxidant enzymes. In addition, the mutant-SOD1 protein was found in the mitochondrial fraction in the muscles from G93A*SOD1 mice, which was accompanied by vacuolized and abnormal mitochondria, altered OXPHOS and PDH complex protein levels, and defects in mitochondrial respiration. Overall, we reported the pathological sequelae observed in the skeletal muscles of G93A*SOD1 mice resulting from the whole-body mutant-SOD1 protein expression.

Alteration of Cellular Energy Metabolism through LPAR2-Axin2 Axis in Gastric Cancer.

Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and the malignant behavior of cancer cells through G-protein-coupled receptors. However, the role of LPA in ß-catenin-mediated gastric cancer is unknown. Here, we have noted the high expression of LPAR2 in human gastric cancer tissues, and that LPA treatment significantly increased the proliferation, migration, and invasion of human gastric cancer cells. Results from our biochemical experiments showed that an LPA exposure increased the expression of ß-catenin and its nuclear localization, increased the phosphorylation of glycogen synthase kinase 3ß (GSK-3ß), decreased the expression of Axin2, and increased the expression of the target genes of the ß-catenin signaling pathway. The LPA2 receptor (LPAR2) antagonist significantly reduced the LPA-induced nuclear localization of ß-catenin, the primary signaling event. The knockdown of LPAR2 in the gastric cancer cell lines robustly reduced the LPA-induced ß-catenin activity. An LPA exposure increased the ATP production by both oxidative phosphorylation and glycolysis, and this effect was abrogated with the addition of an LPAR2 antagonist and XAV393, which stabilizes the Axin and inhibits the ß-catenin signaling pathway. Based on our findings, the possibility that LPA contributes to gastric cancer initiation and progression through the ß-catenin signaling pathway as well as by the dysregulation of the energy metabolism via the LPAR2 receptor and Axin2, respectively, provides a novel insight into the mechanism of and possible therapeutic targets of gastric cancer.

Electrophilic Aldehyde 4-Hydroxy-2-Nonenal Mediated Signaling and Mitochondrial Dysfunction.

Reactive oxygen species (ROS), a by-product of aerobic life, are highly reactive molecules with unpaired electrons. The excess of ROS leads to oxidative stress, instigating the peroxidation of polyunsaturated fatty acids (PUFA) in the lipid membrane through a free radical chain reaction and the formation of the most bioactive aldehyde, known as 4-hydroxynonenal (4-HNE). 4-HNE functions as a signaling molecule and toxic product and acts mainly by forming covalent adducts with nucleophilic functional groups in proteins, nucleic acids, and lipids. The mitochondria have been implicated as a site for 4-HNE generation and adduction. Several studies clarified how 4-HNE affects the mitochondria's functions, including bioenergetics, calcium homeostasis, and mitochondrial dynamics. Our research group has shown that 4-HNE activates mitochondria apoptosis-inducing factor (AIFM2) translocation and facilitates apoptosis in mice and human heart tissue during anti-cancer treatment. Recently, we demonstrated that a deficiency of SOD2 in the conditional-specific cardiac knockout mouse increases ROS, and subsequent production of 4-HNE inside mitochondria leads to the adduction of several mitochondrial respiratory chain complex proteins. Moreover, we highlighted the physiological functions of HNE and discussed their relevance in human pathophysiology and current discoveries concerning 4-HNE effects on mitochondria.

Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy.

Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.

Rotenone-Induced 4-HNE Aggresome Formation and Degradation in HL-1 Cardiomyocytes: Role of Autophagy Flux.

Reactive oxygen species (ROS) cause oxidative stress by generating reactive aldehydes known as 4-hydroxynonenal (4-HNE). 4-HNE modifies protein via covalent adduction; however, little is known about the degradation mechanism of 4-HNE-adducted proteins. Autophagy is a dynamic process that maintains cellular homeostasis by removing damaged organelles and proteins. In this study, we determined the role of a superoxide dismutase (SOD) mimetic MnTnBuOE-2-PyP5+ (MnP, BMX-001) on rotenone-induced 4-HNE aggresome degradation in HL-1 cardiomyocytes. A rotenone treatment (500 nM) given for 24 h demonstrated both increased ROS and 4-HNE aggresome accumulation in HL-1 cardiomyocytes. In addition, cardiomyocytes treated with rotenone displayed an increase in the autophagy marker LC3-II, as shown by immunoblotting and immunofluorescence. A pre-treatment with MnP (20 µM) for 24 h attenuated rotenone-induced ROS formation. An MnP pre-treatment showed decreased 4-HNE aggresomes and LC3-II formation. A rotenone-induced increase in autophagosomes was attenuated by a pre-treatment with MnP, as shown by fluorescent-tagged LC3 (tfLC3). Rotenone increased tubulin hyperacetylation through the ROS-mediated pathway, which was attenuated by MnP. The disruption of autophagy caused HL-1 cell death because a 3-methyladenine inhibitor of autophagosomes caused reduced cell death. Yet, rapamycin, an inducer of autophagy, increased cell death. These results indicated that a pre-treatment with MnP decreased rotenone-induced 4-HNE aggresomes by enhancing the degradation process.

The ATX-LPA Axis Regulates Vascular Permeability during Cerebral Ischemic-Reperfusion.

Endothelial permeability is a major complication that must be addressed during stroke treatment. Study of the mechanisms underlying blood−brain barrier (BBB) disruption and management of the hypoxic stress-induced permeability of the endothelium following reperfusion are both urgently needed for stroke management. Lysophosphatidic acid (LPA), a bioactive lipid essential for basic cellular functions, causes unfavorable outcomes during stroke progression. LPA-producing enzyme autotaxin (ATX) is regulated in ischemic stroke. We used an electrical cell-substrate impedance sensor (ECIS) to measure endothelial permeability. Mitochondrial bioenergetics were obtained using a Seahorse analyzer. AR-2 probe fluorescence assay was used to measure ATX activity. LPA increased endothelial permeability and reduced junctional protein expression in mouse brain microvascular endothelial cells (MBMEC). LPA receptor inhibitors Ki16425 and AM095 attenuated the LPA-induced changes in the endothelial permeability and junctional proteins. LPA significantly diminished mitochondrial function in MBMEC. ATX was upregulated (p < 0.05) in brain microvascular endothelial cells under hypoxic reperfusion. ATX activity and permeability were attenuated with the use of an ATX inhibitor in a mouse stroke model. The upregulation of ATX with hypoxic reperfusion leads to LPA production in brain endothelial cells favoring permeability. Inhibition of the ATX−LPA−LPAR axis could be therapeutically targeted in stroke to achieve better outcomes.

The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes.

Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1's direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1's subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1's localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1's mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1's structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1's C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.

Chronic Low-Dose Alcohol Consumption Promotes Cerebral Angiogenesis in Mice.

Chronic alcohol consumption dose-dependently affects the incidence and prognosis of ischemic stroke. We determined the influence of chronic alcohol consumption on cerebral angiogenesis under physiological conditions and following ischemic stroke. In in vitro studies, acute exposure to low-concentration ethanol significantly increased angiogenic capability and upregulated vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) in C57BL/6J mouse brain microvascular endothelial cells (MBMVECs). The increased angiogenic capability was abolished in the presence of a VEGFR2 inhibitor. In addition, the increased angiogenic capability and upregulated VEGF-A and VEGFR2 remained in chronically low-concentration ethanol-exposed MBMVECs. In in vivo studies, 8-week gavage feeding with low-dose ethanol significantly increased vessel density and vessel branches and upregulated VEGF-A and VEGFR2 in the cerebral cortex under physiological conditions. Furthermore, vessel density, vessel branches, and expression of VEGF-A and VEGFR2 in the peri-infarct cortex were significantly greater in low-dose ethanol-fed mice at 72 h of reperfusion. Although low-dose ethanol did not alter cerebral vasoreactivity and regional cerebral blood flow (rCBF) either before or during ischemia, it significantly augmented post-ischemic hyperemia during reperfusion. In contrast, exposure to high-concentration ethanol and 8-week gavage feeding with high-dose ethanol only had a mild inhibitory effect on angiogenic capability and cerebral angiogenesis, respectively. We conclude that heavy alcohol consumption may not dramatically alter cerebral angiogenesis, whereas light alcohol consumption significantly promotes cerebral angiogenesis.

Disrupted Blood-Brain Barrier and Mitochondrial Impairment by Autotaxin-Lysophosphatidic Acid Axis in Postischemic Stroke.

Background The loss of endothelial integrity increases the risk of intracerebral hemorrhage during ischemic stroke. Adjunct therapeutic targets for reperfusion in ischemic stroke are in need to prevent blood-brain barrier disruption. Recently, we have shown that endothelial permeability is mediated by lysophosphatidic acid (LPA), but the role of autotaxin, which produces LPA, remains unclear in stroke. We investigate whether autotaxin/LPA axis regulates blood-brain barrier integrity after cerebral ischemia. Methods and Results Ischemic stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by 24-hour reperfusion. The therapeutic efficacy of autotaxin/LPA receptor blockade was evaluated using triphenyl tetrazolium chloride staining, Evans blue permeability, infrared imaging, mass spectrometry, and XF24 analyzer to evaluate blood-brain barrier integrity, autotaxin activity, and mitochondrial bioenergetics. In our mouse model of ischemic stroke, the mRNA levels of autotaxin were elevated 1.7-fold following the cerebral ischemia and reperfusion (I/R) group compared with the sham. The enzymatic activity of autotaxin was augmented by 4-fold in the I/R group compared with the sham. Plasma and brain tissues in I/R group showed elevated LPA levels. The I/R group also demonstrated mitochondrial dysfunction, as evidenced by decreased (P<0.01) basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity. Treatment with autotaxin inhibitors (HA130 or PF8380) or autotaxin/LPA receptor inhibitor (BrP-LPA) rescued endothelial permeability and mitochondrial dysfunction in I/R group. Conclusions Autotaxin-LPA signaling blockade attenuates blood-brain barrier disruption and mitochondrial function following I/R, suggesting targeting this axis could be a new therapeutic approach toward treating ischemic stroke.

Chronic Low-Dose Alcohol Consumption Attenuates Post-Ischemic Inflammation via PPARγ in Mice.

Ischemic stroke is one of the leading causes of death and permanent disability in adults. Recently, we found that light alcohol consumption (LAC) suppresses post-ischemic inflammatory response, which plays an important role in ischemic brain damage. Our goal was to determine the role of peroxisome proliferator-activated receptor-gamma (PPARγ) in the anti-inflammatory effect of LAC against transient focal cerebral ischemia. In in vivo study, male C57BL/6J wild type (WT) and endothelial-specific conditional PPARγ knockout mice were gavage fed with 0.7 g/kg/day ethanol or volume-matched water daily for 8 weeks. From the 7th week, 3 mg/kg/day GW9662 (a selective PPARγ antagonist) was intraperitoneally given for two weeks. Cerebral ischemia/reperfusion (I/R) injury and expression of manganese superoxide dismutase (MnSOD) and adhesion molecules, neutrophil infiltration, and microglial activation in the cerebral cortex before and following a 90 min unilateral middle cerebral artery occlusion (MCAO)/24 h reperfusion were evaluated. In in vitro study, the impact of chronic alcohol exposure on expression of PPARγ and MnSOD in C57BL/6J mouse brain microvascular endothelial cells (MBMVECs) was measured. PPARγ and MnSOD were significantly upregulated in the cerebral cortex of ethanol-fed WT mice and low-concentration ethanol-exposed C57BL/6J MBMVECs. GW9662 significantly inhibited alcohol-induced upregulation of MnSOD. Eight-week ethanol feeding significantly reduced cerebral I/R injury and alleviated the post-ischemic inflammatory response (upregulation of intercellular adhesion molecule-1 (ICAM-1) and E-selectin, microglial activation, and neutrophil infiltration). Treatment with GW9662 and endothelial-specific conditional knockout of PPARγ did not alter cerebral I/R injury and the inflammatory response in the control mice but abolish the neuroprotective effect in ethanol-fed mice. In addition, GW9662 and endothelial-specific conditional knockout of PPARγ diminished the inhibitory effect of LAC on the post-ischemic expression of adhesion molecules and neutrophil infiltration. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing the post-ischemic inflammation via activation of PPARγ.

Protective Effect of Low-Dose Alcohol Consumption against Post-Ischemic Neuronal Apoptosis: Role of L-PGDS.

Ischemic stroke is one of the leading causes of permanent disability and death in adults worldwide. Apoptosis is a major element contributing to post-ischemic neuronal death. We previously found that low-dose alcohol consumption (LAC) protects against neuronal apoptosis in the peri-infarct cortex following transient focal cerebral ischemia. Lipocalin-type prostaglandin D2 synthase (L-PGDS), which is mainly localized in the central nervous system (CNS), was previously shown to inhibit neuronal apoptosis. Therefore, we determined whether L-PGDS is involved in the protective effect of LAC against post-ischemic neuronal apoptosis. Wild-type (WT), CaMKIIαCreERT2/+/L-PGDS+/+, and CaMKIIαCreERT2/+/L-PGDSflox/flox mice on a C57BL/6J background were gavage fed with ethanol or volume-matched water once a day for 8 weeks. Tamoxifen (2 mg/day) was given intraperitoneally to CaMKIIαCreERT2/+/L-PGDS+/+ and CaMKIIαCreERT2/+/L-PGDSflox/flox mice for 5 days during the fourth week. AT-56 (30 mg/kg/day), a selective inhibitor of L-PGDS, was given orally to AT-56-treated WT mice from the fifth week for four weeks. Cerebral ischemia/reperfusion (I/R) injury, TUNEL-positive neurons, and cleaved caspase-3-positive neurons were measured at 24 h of reperfusion after a 90 min unilateral middle cerebral artery occlusion (MCAO). We found that 0.7 g/kg/day but not 2.8 g/kg/day ethanol significantly upregulated L-PGDS in the cerebral cortex. In addition, 0.7 g/kg/day ethanol diminished cerebral ischemia/reperfusion (I/R) injury and TUNEL-positive and cleaved caspase-3-positive neurons in the peri-infarct cortex in WT and CaMKIIαCreERT2/+/L-PGDS+/+ mice. Furthermore, the neuroprotective effect of 0.7 g/kg/day ethanol was alleviated in AT-56-treated WT and CaMKIIαCreERT2/+/L-PGDSflox/flox mice. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing post-ischemic neuronal apoptosis via an upregulated L-PGDS.

Dysfunctional Mitochondrial Dynamic and Oxidative Phosphorylation Precedes Cardiac Dysfunction in R120G-αB-Crystallin-Induced Desmin-Related Cardiomyopathy.

Background The mutated α-B-Crystallin (CryABR120G) mouse model of desmin-related myopathy (DRM) shows an age-dependent onset of pathologic cardiac remodeling and progression of heart failure. CryABR120G expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryABR120G mice show an accumulation of electron-dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryABR120G mice. In the present study, we report that the CryABR120G mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin-related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryABR120G hearts. Mitochondria isolated from CryABR120G transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM-associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryABR120G hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.

Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function.

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of "binge and crash" methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of "binge and crash" methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to "binge and crash" methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.

SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy.

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy.

Pleiotropic effects of mdivi-1 in altering mitochondrial dynamics, respiration, and autophagy in cardiomyocytes.

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion events to adapt to changes in the cellular environment. Aberrant mitochondrial fission has been associated with several types of cardiovascular dysfunction; inhibition of pathologically aberrant mitochondrial fission has been shown to be cardioprotective. Pathological fission is mediated by the excessive activation of GTPase dynamin-related protein 1 (Drp1), making it an attractive therapeutic target in numerous cardiovascular diseases. Mitochondrial division inhibitor (mdivi-1) is widely used small molecule reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate injury. The purpose of our study was to understand the pleiotropic effects of mdivi-1 on mitochondrial dynamics, mitochondrial respiration, electron transport activities, and macro-autophagy. In this study, we found that mdivi-1 treatment decreased Drp1 expression, proteolytically cleaved L-OPA1, and altered the expression of OXPHOS complex proteins, resulting in increased superoxide production. The altered expression of OXPHOS complex proteins may be directly associated with decreased Drp1 expression, as Drp1 siRNA knockdown in cardiomyocytes showed similar effects. Results from an autophagy flux assay showed that mdivi-1 induced impaired autophagy flux that could be restored by Atg7 overexpression, suggesting that mdivi-1 mediated inhibition of macro-autophagy in cardiomyocytes. Treatment with mdivi-1 resulted in increased expression of p62, which is required for Atg7 overexpression-induced rescue of mdivi-1-mediated impaired autophagy flux. In addition, mdivi-1-dependent proteolytic processing of L-OPA1 was associated with increased mitochondrial superoxide production and altered expression of mitochondrial serine/proteases. Overall, the novel pleiotropic effect of mdivi-1 in cardiomyocytes included proteolytically cleaved L-OPA1, altered expression of OXPHOS complex proteins, and increased superoxide production, which together resulted in defects in mitochondrial respiration and inhibition of macro-autophagy.

Doxorubicin-induced cardiomyopathy associated with inhibition of autophagic degradation process and defects in mitochondrial respiration.

Doxorubicin (Dox) is a highly effective anticancer drug but cause acute ventricular dysfunction, and also induce late-onset cardiomyopathy and heart failure. Despite extensive studies, the pathogenic sequelae leading to the progression of Dox-associated cardiomyopathy remains unknown. We assessed temporal changes in autophagy, mitochondrial dynamics, and bioenergetics in mouse models of acute and chronic Dox-cardiomyopathy. Time course study of acute Dox-treatment showed accumulation of LC3B II in heart lysates. Autophagy flux assays confirmed that the Dox-induced accumulation of autophagosomes occurs due to blockage of the lysosomal degradation process. Dox-induced autophagosomes and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. Mitochondria isolated from acute Dox-treated hearts showed significant suppression of oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity also exhibited time-dependent accumulation of LC3B II levels and increased accumulation of green puncta in GFP-LC3 Tg hearts. Mitochondria isolated from chronic Dox-treated hearts also showed significant suppression of mitochondrial OCR. The in vivo impairment of autophagic degradation process and mitochondrial dysfunction data were confirmed in vitro using cultured neonatal cardiomyocytes. Both acute and chronic Dox-associated cardiomyopathy involves a multifocal disease process resulting from autophagosomes and autolysosomes accumulation, altered expression of mitochondrial dynamics and oxidative phosphorylation regulatory proteins, and mitochondrial respiratory dysfunction.