Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells.

BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.

Metabolomic and transcriptomic signatures of chemogenetic heart failure.

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.

Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca²âº Leak Directed to Mitochondria in Pancreatic Islets and ß-Cells.

BACKGROUND/AIMS: In pancreatic ß-cells, the intracellular Ca²âº homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²âº store balances cellular Ca²âº. In this study basal ER Ca²âº homeostasis was evaluated in order to reveal potential ß-cell-specificity of ER Ca²âº handling and its consequences for mitochondrial Ca²âº, ATP and respiration. METHODS: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²âº and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3ß activity was measured with ELISA. RESULTS: An atypical ER Ca²âº leak was observed exclusively in pancreatic islets and ß-cells. This continuous ER Ca²âº efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3ß-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3ß. Expression of a presenlin-1 mutant that mimics GSK3ß-mediated phosphorylation established a ß-cell-like ER Ca²âº leak in HeLa and EA.hy926 cells. The ER Ca²âº loss in ß-cells was compensated at steady state by Ca²âº entry that is linked to the activity of TRPC3. CONCLUSION: Pancreatic ß-cells establish a cell-specific ER Ca²âº leak that is under the control of GSK3ß and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²âº handling for basal mitochondrial activity.

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and ß-Cells upon Elevated Glucose.

BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

Intracellular Ca2+ release decelerates mitochondrial cristae dynamics within the junctions to the endoplasmic reticulum.

Mitochondria are multifunctional organelles that essentially contribute to cell signaling by sophisticated mechanisms of communications. Live cell imaging studies showed that mitochondria are dynamic and complex structures that form ramified networks by directed movements, fission, and fusion events. There is emerging evidence that the morphology of mitochondria determines cellular functions and vice versa. Several intracellular signaling pathways and messengers including Ca2+ dynamically influence the architecture of mitochondria. Because electron microscopy cannot be utilized for an assessment of dynamics of mitochondrial morphology in intact cells, most studies were performed using wide-field or laser confocal fluorescence microscopies that, due to limitations of their spatial resolution, do not allow investigating sub-mitochondrial structures. Accordingly, our understanding of the dynamics of substructures of mitochondria is quite limited. Here, we present a robust super-resolution method to quantify the dynamics of mitochondrial cristae, the main substructures of the inner mitochondrial membrane, exploiting structured illumination microscopy (SIM). We observed that knockdown of the dynamin-like 120-kDa protein, which is encoded by the OPA1 gene, specifically reduces the dynamics of the mitochondrial cristae membranes (CM), while the inner boundary membrane (IBM) remained flexible. We further used dual color SIM to quantify the dynamics of CM in the junction between mitochondria and the endoplasmic reticulum (ER; mitochondrial associated membranes, MAMs). Intracellular Ca2+ release spatially reduced CM-dynamics in MAMs. Moreover, CM-dynamics was independent from matrix Ca2+ signal. Our data suggest that local Ca2+ signals specifically control CM-dynamics and structure to facilitate a well-balanced functional (Ca2+) interplay between mitochondria and the ER.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 µm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 µm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 µm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria.

BACKGROUND/AIMS: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. METHODS: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. RESULTS: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. CONCLUSIONS: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.

UCP2 modulates single-channel properties of a MCU-dependent Ca(2+) inward current in mitochondria.

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.

Generation of Red-Shifted Cameleons for Imaging Ca²âº Dynamics of the Endoplasmic Reticulum.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Molecularly distinct routes of mitochondrial Ca2+ uptake are activated depending on the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).

The transfer of Ca(2+) across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca(2+) uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca(2+) fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca(2+) uptake pathway or establish distinct routes for mitochondrial Ca(2+) sequestration. In this study, we show that a modulation of Ca(2+) release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca(2+) signals and consequently switches mitochondrial Ca(2+) uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca(2+) sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca(2+) across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca(2+) uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca(2+) rises.

Mitochondrial Ca(2+) uniporter (MCU)-dependent and MCU-independent Ca(2+) channels coexist in the inner mitochondrial membrane.

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic ß-cells.

In pancreatic ß-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic ß-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic ß-cells and their involvement in the positive feedback required for sustained insulin secretion.

Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Sensory ataxia and cardiac hypertrophy caused by neurovascular oxidative stress in chemogenetic transgenic mouse lines.

Oxidative stress is associated with cardiovascular and neurodegenerative diseases. Here we report studies of neurovascular oxidative stress in chemogenetic transgenic mouse lines expressing yeast D-amino acid oxidase (DAAO) in neurons and vascular endothelium. When these transgenic mice are fed D-amino acids, DAAO generates hydrogen peroxide in target tissues. DAAO-TGCdh5 transgenic mice express DAAO under control of the putatively endothelial-specific Cdh5 promoter. When we provide these mice with D-alanine, they rapidly develop sensory ataxia caused by oxidative stress and mitochondrial dysfunction in neurons within dorsal root ganglia and nodose ganglia innervating the heart. DAAO-TGCdh5 mice also develop cardiac hypertrophy after chronic chemogenetic oxidative stress. This combination of ataxia, mitochondrial dysfunction, and cardiac hypertrophy is similar to findings in patients with Friedreich's ataxia. Our observations indicate that neurovascular oxidative stress is sufficient to cause sensory ataxia and cardiac hypertrophy. Studies of DAAO-TGCdh5 mice could provide mechanistic insights into Friedreich's ataxia.

Leucine zipper EF hand-containing transmembrane protein 1 (Letm1) and uncoupling proteins 2 and 3 (UCP2/3) contribute to two distinct mitochondrial Ca2+ uptake pathways.

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.