Heart Mitochondria Research Articles

Comorbidity affects Вcl-2 levels in mitochondria in C57BL/6 mice with transplantable B16/F10 melanoma.

e21581 Background: Overexpression of the Bcl-2 protein inhibits apoptosis and promotes carcinogenesis. Stress causes signaling leading to cell buffering with Bcl-2 protein above acceptable levels. The purpose of the study was to analyze the influence of comorbidity – chronic neurogenic pain (CNP) – on the Bcl-2 levels in mitochondria of cells of melanoma, the heart, skin and brain in female mice with growing tumors. Methods: Female С57ВL/6 mice were divided into groups: intact group (n = 21); control group with a CNP model – bilateral sciatic nerve ligation (n = 21); group M – B16/F10 melanoma (n = 63); CNP+M group – B16/F10 melanoma was transplanted 3 weeks after the CNP model creation (n = 63). The concentration of Bcl-2 (ng/mg of protein) was determined in mitochondrial samples by ELISA (Thermo Fisher Scientific, Austria). Statictical analysis of results: Statistica 10.0. Results: CNP decreased the Bcl-2 level in heart mitochondria by 1.3 times (p < 0.05), but increased it in skin and brain mitochondria by 5.8 and 1.3 times, respectively. Similar changes were observed in melanoma growth 1 week after its transplantation: Bcl-2 levels decreased in heart mitochondria by 1.3 times, and increased in the skin and brain by 8.9 and 1.3 times, respectively. After 2 weeks of the tumor growth, Bcl-2 in brain mitochondria decreased by 1.7 times, and it started declining in the skin by the 3rd week – by 4 times, compared to intact females. Bcl-2 in tumor mitochondria exceeded the values in the skin by more than 4 times throughout the experiment. Tumor growth in presence of CNP caused a decrease in Bcl-2 in brain mitochondria by 2.4 times after 3 weeks, and in the heart and skin – by 2 and 1.7 times, respectively, after 2 weeks. Bcl-2 in tumor mitochondria in presence of CNP was lower than in the intact skin on average by 1.8 times throughout the experiment. Conclusions: CNP as a comorbidity caused a modulating effect on the mechanisms of survival and apoptosis of cells both in the tumor and in the main organs providing the vital functions of the body - the brain and heart, and also affects the target organ of melanoma - the skin. The results demonstrated the ability of comorbidity to change levels of Bcl-2 in mitochondria depending on the stage of tumor development.

Aging is Associated with a Decline in Atg9b‐mediated Autophagosome Formation and Appearance of Enlarged Mitochondria in the Heart

Advancing age is a major risk factor for developing heart disease and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age-associated defects in autophagy remain poorly characterized. Here, using young (4 months) and aged (24 months) male mice, we have investigated how the autophagic process is altered in aged mouse hearts. We found that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. The Atg9 proteins are critical in delivering membrane to the growing autophagosome and we confirmed that siRNA knockdown of Atg9b in cells leads to a reduction in autophagosome formation. We also found that both Atg9a and Atg9b are expressed in the myocardium and that both are rapidly increased in response to myocardial infarction in the young heart. Atg9b-positive vesicles are localized to vesicles throughout the cytosol whereas Atg9a-positive vesicles are more closely associated with mitochondria, indicating that these proteins have distinct functions in autophagy. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We found increased levels of Parkin-positive, ubiquitinated mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. Moreover, we found that the decreased mitochondrial clearance coincided with formation of megamitochondria in the aged heart. Overall, our findings suggest that aging leads to reduced formation of autophagosomes with subsequent accumulation of mitochondria that have been labeled for mitophagy. The decreased clearance of Parkin-positive, ubiquitinated mitochondria in cells leads to enhanced compensatory mitochondrial fusion to dilute damaged components.

Increasing Glycolysis Protects Cardiac Function Against High Fat Diet‐Induced Cardiomyopathy

The healthy heart has a dynamic capacity to respond and adapt to changes in both nutrient availability and energy demands. This metabolic flexibility is critical for the maintenance of proper tissue function and its disruption has deleterious effects on the heart. For example, a signature characteristic of diabetic cardiomyopathy is metabolic inflexibility whereby the heart increases reliance on fatty acids to meet energetic demands. We hypothesize that increased rates of glycolysis can restore metabolic flexibility and mitigate cardiac dysfunction in the diabetic heart. Our objective was to examine how the heart coordinates changes in metabolic flexibility under normal and pro-glycolytic conditions. Wild type (WT) and transgenic mice with cardiac-specific increased glucose utilization (GlycoHi) were fed a low fat (LFD) or a high fat diet (HFD) for 16 weeks to induce the metabolic inflexibility seen in type 2 diabetes. Metabolic profiles were determined by measuring mitochondrial respiration and targeted quantitative proteomic and metabolomic analysis. Echocardiography was used to assess cardiac function. Heart mitochondria from GlycoHi mice on LFD exhibited increased rates of glucose-substrate supported respiration compared to WT/LFD. Mitochondria of both groups on HFD exhibited increased fatty acid-supported respiration. However, GlycoHi mitochondria showed a decrease in fatty acid reliance compared to WT. Quantitative proteomics data further supported that GlycoHi hearts have resistance to HFD induced metabolic inflexibility. Increasing glycolysis also had beneficial effects on cardiac function. Echocardiography analysis revealed a decrease of diastolic function in WT/HFD that was not seen in GlycoHi/HFD hearts. In this study, mitochondrial function analysis showed a resistance in GlycoHi mitochondria to the HFD induced metabolic changes seen in WT/HFD. Echocardiography analysis suggests cardio-protection against a HFD in GlycoHi hearts. Together these results provide strong evidence that increasing glucose usage in diabetic hearts can be protective against cardiomyopathy.

The Relationship between Mitochondrial Reactive Oxygen Species Production and Mitochondrial Energetics in Rat Tissues with Different Contents of Reduced Coenzyme Q.

We investigated the relationship between mitochondrial production of reactive oxygen species (ROS) and mitochondrial energetics in various rat tissues with different contents of the reduced coenzyme Q (Q) pool (Q9 + Q10). Our results indicate that similar to the tissue level, mitochondrial H2O2 release under nonphosphorylating conditions was strongly dependent on the amount of the reduced Q pool. Namely, in brain and lung mitochondria, less H2O2 release corresponded to a less reduced Q pool, while in liver and heart mitochondria, higher H2O2 release corresponded to a more reduced Q pool. We can conclude that the differences observed in rat tissues in the size of the reduced Q pool reflect different levels of ROS production and hence may reflect different demands for reduced Q as an antioxidant. Moreover, differences in mitochondrial H2O2 release were observed in different types of rat mitochondria during the oxidation of succinate (complex II substrate), malate plus glutamate (complex I substrate), and their mixture under phosphorylating and nonphosphorylating conditions. Our results indicate the existence of a tissue-specific maximum respiratory chain capacity in ROS production, possibly related to the membrane potential-mediated control of oxidative phosphorylation. We propose the use of a new parameter for the study of isolated mitochondria, RCRROS, the ratio between the formation of mitochondrial ROS under nonphosphorylating and phosphorylating conditions, which represents the maximum factorial increase in mitochondrial ROS formation that can be achieved after all ADP is phosphorylated.

Changes in the respiratory function of the heart and brain mitochondria of animals after chronic alcohol intoxication affected by a new GABA derivative

Introduction: Chronic ethanol consumption leads to significant functional and structural changes in the mitochondria of the heart and brain, increasing generation of reactive oxygen species. Therefore, the search for substances, which improve the functional state of the mitochondria and, meantime, reduce the oxidative stress, is relevant.
 Materials and methods: 10-months-old Wistar female rats were used in the experiments. Chronic alcohol intoxication (CAI) was modelled by replacing drinking water with a 10% ethanol solution containing sucrose (50 g/L) for 24 weeks. Four groups were formed: 1 – intact animals; 2 – animals after chronic alcohol consumption; 3 – rats after CAI which were administered RSPU-260 (25 mg/kg); 4 – rats after CAI which were administered the reference drug Mildronate (50 mg/kg). The intensity of lipid peroxidation (LPO) and the rate of oxygen consumption in various metabolic states were determined.
 Results and discussion: Administration of the compound RSPU-260 to the animals exposed to alcohol over a long period of time resulted in an increase in both the rate of oxygen consumption (state 3) and the respiratory control ratio (RCR) of the mitochondria of heart and brain cells. The use of a GABA derivative promoted a decrease in malonic dialdehyde in the mitochondria of the heart and brain. Total SOD activity in the mitochondria of heart cells was significantly increased in the groups of rats treated with RSPU-260. In terms of efficiency, the compound RSPU-260 was comparable to the reference drug Mildronate.
 Conclusions: The compound RSPU-260, and the reference drug Mildronate improve mitochondrial oxidative phosphorylation in heart and brain cells, the functioning of antioxidant enzymes in animals after CAI, and can be used to correct alcoholic damage to these organs.

Effects of Apocynin on Heart Muscle Oxidative Stress of Rats with Experimental Diabetes: Implications for Mitochondria.

Diabetes mellitus (DM) constitutes one of the public health problems today. It is characterized by hyperglycemia through a defect in the β-cells function and/or decreased insulin sensitivity. Apocynin has been tasted acting directly as an NADPH oxidase inhibitor and reactive oxygen species (ROS) scavenger, exhibiting beneficial effects against diabetic complications. Hence, the present study’s goal was to dissect the possible mechanisms by which apocynin could mediate its cardioprotective effect against DM-induced oxidative stress. Male Wistar rats were assigned into 4 groups: Control (C), control + apocynin (C+A), diabetes (D), diabetes + apocynin (D+A). DM was induced with streptozotocin. Apocynin treatment (3 mg/kg/day) was applied for 5 weeks. Treatment significantly decreased blood glucose levels and insulin resistance in diabetic rats. In cardiac tissue, ROS levels were higher, and catalase enzyme activity was reduced in the D group compared to the C group; the apocynin treatment significantly attenuated these responses. In heart mitochondria, Complexes I and II of the electron transport chain (ETC) were significantly enhanced in the D+A group. Total glutathione, the level of reduced glutathione (GSH) and the GSH/ oxidized glutathione (GSSG) ratio were increased in the D+A group. Superoxide dismutase (SOD) and the glutathione peroxidase (GSH-Px) activities were without change. Apocynin enhances glucose uptake and insulin sensitivity, preserving the antioxidant defense and mitochondrial function.

Distinct properties of Ca2+ efflux from brain, heart and liver mitochondria: The effects of Na+, Li+ and the mitochondrial Na+/Ca2+ exchange inhibitor CGP37157

Mitochondrial Ca2+ transport is essential for regulating cell bioenergetics, Ca2+ signaling and cell death. Mitochondria accumulate Ca2+ via the mitochondrial Ca2+ uniporter (MCU), whereas Ca2+ is extruded by the mitochondrial Na+/Ca2+ (mtNCX) and H+/Ca2+ exchangers. The balance between these processes is essential for preventing toxic mitochondrial Ca2+ overload. Recent work demonstrated that MCU activity varies significantly among tissues, likely reflecting tissue-specific Ca2+ signaling and energy needs. It is less clear whether this diversity in MCU activity is matched by tissue-specific diversity in mitochondrial Ca2+ extrusion. Here we compared properties of mitochondrial Ca2+ extrusion in three tissues with prominent mitochondria function: brain, heart and liver. At the transcript level, expression of the Na+/Ca2+/Li+ exchanger (NCLX), which has been proposed to mediate mtNCX transport, was significantly greater in liver than in brain or heart. At the functional level, Na+ robustly activated Ca2+ efflux from brain and heart mitochondria, but not from liver mitochondria. The mtNCX inhibitor CGP37157 blocked Ca2+ efflux from brain and heart mitochondria but had no effect in liver mitochondria. Replacement of Na+ with Li+ to test the involvement of NCLX, resulted in a slowing of mitochondrial Ca2+ efflux by ∼70 %. Collectively, our findings suggest that mtNCX is responsible for Ca2+ extrusion from the mitochondria of the brain and heart, but plays only a small, if any, role in mitochondria of the liver. They also reveal that Li+ is significantly less effective than Na+ in driving mitochondrial Ca2+ efflux.

Skeletal muscle specific mitochondrial dysfunction and altered energy metabolism in a murine model (oim/oim) of severe osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder with patients exhibiting bone fragility and muscle weakness. The synergistic biochemical and biomechanical relationship between bone and muscle is a critical potential therapeutic target, such that muscle weakness should not be ignored. Previous studies demonstrated mitochondrial dysfunction in the skeletal muscle of oim/oim mice, which model a severe human type III OI. Here, we further characterize this mitochondrial dysfunction and evaluate several parameters of whole body and skeletal muscle metabolism. We demonstrate reduced mitochondrial respiration in female gastrocnemius muscle, but not in liver or heart mitochondria, suggesting that mitochondrial dysfunction is not global in the oim/oim mouse. Myosin heavy chain fiber type distributions were altered in the oim/oim soleus muscle with a decrease (−33 to 50%) in type I myofibers and an increase (+31%) in type IIa myofibers relative to their wildtype (WT) littermates. Additionally, altered body composition and increased energy expenditure were observed oim/oim mice relative to WT littermates. These results suggest that skeletal muscle mitochondrial dysfunction is linked to whole body metabolic alterations and to skeletal muscle weakness in the oim/oim mouse.

Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT-FoxO1 signalling pathway.

Previous studies have shown that the expression of inwardly rectifying potassium channel 6.1 (Kir6.1) in heart mitochondria is significantly reduced in type 1 diabetes. However, whether its expression and function are changed and what role it plays in type 2 diabetic cardiomyopathy (DCM) have not been reported. This study investigated the role and mechanism of Kir6.1 in DCM. We found that the cardiac function and the Kir6.1 expression in DCM mice were decreased. We generated mice overexpressing or lacking Kir6.1 gene specifically in the heart. Kir6.1 overexpression improved cardiac dysfunction in DCM. Cardiac‐specific Kir6.1 knockout aggravated cardiac dysfunction. Kir6.1 regulated the phosphorylation of AKT and Foxo1 in DCM. We further found that Kir6.1 overexpression also improved cardiomyocyte dysfunction and up‐regulated the phosphorylation of AKT and FoxO1 in neonatal rat ventricular cardiomyocytes with insulin resistance. Furthermore, FoxO1 activation down‐regulated the expression of Kir6.1 and decreased the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. FoxO1 inactivation up‐regulated the expression of Kir6.1 and increased the ΔΨm in cardiomyocytes. Chromatin immunoprecipitation assay demonstrated that the Kir6.1 promoter region contains a functional FoxO1‐binding site. In conclusion, Kir6.1 improves cardiac dysfunction in DCM, probably through the AKT‐FoxO1 signalling pathway.

A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Protectors of the Mitochondrial Permeability Transition Pore Activated by Iron and Doxorubicin.

The study is aimed at examining of action of iron, DOX, and their complex on the Mitochondrial Permeability Transition Pore (MPTP) opening and detecting of possible protectors of MPTP in the conditions close to mitochondria-dependent ferroptosis. The Toxicity of Doxorubicin (DOX) is mainly associated with free iron accumulation and mitochondrial dysfunction. DOX can provoke ferroptosis, iron-dependent cell death driven by membrane damage. The Mitochondrial Permeability Transition Pore (MPTP) is considered as a common pathway leading to the development of apoptosis, necrosis, and, possibly, ferroptosis. The influence of DOX on the Ca2+ -induced MPTP opening in the presence of iron has not yet been studied. The study was conducted on isolated liver and heart mitochondria. MPTP and succinate- ubiquinone oxidoreductase were studied as targets of DOX in mitochondria-dependent ferroptosis. The iron chelator deferoxamine (DFO), the lipid radical scavenger butyl-hydroxytoluene (BHT), and rutenium red (Rr), as a possible inhibitor of ferrous ions uptake in mitochondria, were tested as MPTP protectors. The role of medium alkalization was also examined. Changes of threshold calcium concentrations required for MPTP opening were measured by a Ca2+ selective electrode, mitochondrial membrane potential was registered by tetraphenylphosphonium (TPP+)-selective electrode, and mitochondrial swelling was recorded as a decrease in absorbance at 540 nm. The activity of Succinate Dehydrogenase (SDH) was determined by the reduction of the electron acceptor DCPIP. MPTP and the respiratory complex II are identified as the main targets of the iron-dependent action of DOX on the isolated mitochondria. All MPTP protectors tested abolished or weakened the effect of iron and a complex of iron with DOX on Ca2+ -induced MPTP opening, acting in different stages of MPTP activation. These data open new approaches to the modulation of the toxic influence of DOX on mitochondria with the aim to reduce their dysfunction.

Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice.

Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.

Temporal evolution of cardiac mitochondrial dysfunction in a type 1 diabetes model. Mitochondrial complex I impairment, and H2O2 and NO productions as early subcellular events

The aim of this work was to study the early events that occur in heart mitochondria and to analyse the temporal evolution of cardiac mitochondrial dysfunction in a type 1 diabetes model. Male Wistar rats were injected with Streptozotocin (STZ, single dose, 60 mg × kg−1, i.p.) and hyperglycemic state was confirmed 72 h later. The animals were sacrificed 10 or 14 days after STZ-injection. Heart mitochondrial state 3 O2 consumption sustained by malate-glutamate (21%) or by succinate (16%), and complexes I-III (27%), II-III (24%) and IV (22%) activities were lower in STZ group, when animals were sacrificed at day 14, i.e. ~11 days of hyperglycemia. In contrast, after 10 days of STZ-injection (~7 days of hyperglycemia), only the state 3 O2 consumption sustained by malate-glutamate (23%) and its corresponding respiratory control (30%) were lower in diabetic rats, in accordance with complex I-III activity reduction (17%). Therefore, this time (~7 days of hyperglycemia) has been considered as an “early stage” of cardiac mitochondrial dysfunction. At this point, mitochondrial production rates of H2O2 (117%), NO (30%) and ONOO− (~225%), and mtNOS expression (29%) were higher; and mitochondrial SOD activity (15%) and [GSH + GSSG] (28%) were lower in diabetic rats. Linear correlations between the modified mitochondrial parameters and glycemias were observed. PGC-1α expression was similar between groups, suggesting that mitochondrial biogenesis was not triggered in this initial phase of mitochondrial dysfunction. Consequently, complex I, H2O2 and NO could be considered early subcellular signals of cardiac mitochondrial dysfunction, with NO and H2O2 being located upstream de novo synthesis of mitochondria.

Abstract 16602: Mitolamban is a Novel Cardiac-Enriched Mitochondrial Microprotein That Regulates Respiratory Complex III

Introduction: Emerging evidence suggests that many RNA molecules currently annotated as noncoding contain short open reading frames that code for functional small proteins called microproteins. Microproteins play critical roles in a diverse range of essential biological processes. To identify novel cardiac-expressed microproteins, we used a comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid transmembrane protein that is abundantly expressed in the heart. Methods and Results: Subcellular localization studies showed that Mtlbn localized specifically to the inner mitochondrial membrane. Immunoprecipitations and mass spectrometry analysis indicated that Mtlbn interacted with subunits of complex III of the electron transport chain (ETC) and we observed the presence of Mtlbn in complex III respiratory supercomplexes (SCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). Cardiac-specific Mtlbn overexpressing transgenic (TG) mice and Mtlbn gene-deleted (knockout, KO) mice were generated to dissect the molecular function of this protein in the heart. Mtlbn TG mice developed cardiomyopathy and died prematurely with histological, biochemical and ultrastructural pathologic features. Metabolomic analysis indicated that hearts from TG mice had signs of increased oxidative stress and mitochondrial dysfunction. While Mtlbn KO mice had normal cardiac function and histological appearance, BN-PAGE analysis of purified mitochondria from KO hearts showed altered complex III composition. Functional assessment of purified heart mitochondria from Mtlbn KO mice revealed a reduction in complex III activity and metabolomic analysis of KO heart tissue indicated an altered metabolite profile consistent with deficiencies in complex III activity. Conclusions: Mtlbn is a novel heart-enriched microprotein that localizes to the inner mitochondrial membrane where it interacts with complex III of the ETC. Our data indicate that Mtlbn serves as a critical regulator of mitochondrial ETC activity through a direct role in affecting complex III function.

Peroxynitrite decomposition catalyst enhances respiratory function in isolated brain mitochondria.

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Hydrolytic activity of mitochondrial F1Fo ATP synthase as a target for myocardial ischemia: discovery and in vitro evaluation of novel inhibitors

Abstract Background/Introduction F1Fo ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyse ATP leading to cellular energetic deficit, contracture and lethal cardiomyocyte injury. Therefore, inhibition of ATP hydrolase is of major significance and the discovery of selective hydrolase inhibitors could be of particular interest in terms of cardiac response to ischemia. Purpose We performed an in vitro screening in order to identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase. Methods Initially, we identified inhibitors of the hydrolytic activity of ATP synthase using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase of mus musculus that was modelled for the first time. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on our in-house library of 2000 compounds, Pharmalab, plus 266,151 compounds of the National Cancer Institute (NCI) database. The best candidates were evaluated in vitro on isolated murine heart mitochondria to verify their inhibitory effect on ATP synthase hydrolytic activity. Moreover, we confirmed their action at a cellular level. H9C2 cells were treated with the three best candidates in the presence of rotenone and their ability to maintain membrane potential was monitored. The experiment was repeated in absence of rotenone in order to detect toxic or non-specific effects (n=5 independent experiments). Finally, the compounds were tested using an assay in which primary adult rat ventricular cardiomyocytes (ARVCs) were challenged with the mitochondrial uncoupler CCCP to induce reverse ATPase activity, and time until contracture was determined. For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls. Results Different steps of filtering through the virtual screening provided 38 molecules from Pharmalab and 15 candidates form NCI database. From the 53 candidates in total, five compounds displayed in vitro inhibitory activity at 200μM on isolated mitochondria and their IC50 values were determined. Among them, three synthetic derivatives possessing a central pyrazolopyridine core displayed the best IC50 values (81.7±1.3 μM, 99.8±1.2 μM, 144.8±1.3μM) and were evaluated on the H9C2 cells. They exhibited significant inhibitory activity at 50μM (p<0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the final novel inhibitors significantly extended (p<0.001) the time until shortening of the ARVCs. Conclusion We have discovered two novel agents that act as selective inhibitors of ATP hydrolase and can be tested against myocardial ischemia. Funding Acknowledgement Type of funding source: None

Temperature sensitivity differs between heart and red muscle mitochondria in mahi-mahi (Coryphaena hippurus)

Maintaining energy balance over a wide range of temperatures is critical for an active pelagic fish species such as the mahi-mahi (Coryphaena hippurus), which can experience rapid changes in temperature during vertical migrations. Due to the profound effect of temperature on mitochondrial function, this study was designed to investigate the effects of temperature on mitochondrial respiration in permeabilized heart and red skeletal muscle (RM) fibres isolated from mahi-mahi. As RM is thought to be more anatomically isolated from rapid ambient temperature changes compared to the myocardium, it was hypothesized that heart mitochondria would be more tolerant of temperature changes through a greater ability to match respiratory capacity to an increase in temperature and to maintain coupling, when compared to RM mitochondria. Results show that heart fibres were more temperature sensitive and increased respiration rate with temperature increases to a greater degree than RM. Respiratory coupling ratios at the three assay temperatures (20, 26, and 30 °C), revealed that heart mitochondria were less coupled at a lower temperature (26 °C) compared to RM mitochondria (30 °C). In response to an in vitro acute temperature challenge, both tissues showed irreversible effects, where both heart and RM increased uncoupling whether the assay temperature was acutely changed from 20 to 30 °C or 30 to 20 °C. The findings from this study indicate that mahi-mahi heart mitochondria were more temperature sensitive compared to those from RM.

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO− production during oxidative stress. We monitored generation of ONOO− from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO− production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO− to form dityrosine (diTyr) in proportion to the ONOO− present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO− production via NO• and O2•−. Changes in mitochondrial ONOO− production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO− production, PTIO decreased production of ONOO−, and TEMPOL decreased ONOO− levels by converting more O2•− to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO− into the buffer is dependent both on O2•− released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

Abstract 250: Acting Instructor

Rational: Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment. Methods and Results: Using the pH indicator cpYFP in the model of naturally aging mice and rats, we show direct evidence of increased mitochondrial proton leak in aged heart mitochondria following a pH gradient stress. Furthermore, we identified Adenine Nucleotide Translocator 1 (ANT1) as mediating the increased proton permeability of old cardiomyocytes. Most importantly, acute (2 hours) in vitro treatment with the tetra-peptide drug SS-31 (elamipretide) reverses age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function. Moreover, we show that SS-31 benefits the old mitochondria by direct association with ANT1 and stabilization of the mitochondrial ATP synthasome, leading to substantial reversal of diastolic dysfunction. Conclusion: Our results uncover excessive mitochondrial proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging.

Aging is associated with a decline in Atg9b-mediated autophagosome formation and appearance of enlarged mitochondria in the heart.

Advancing age is a major risk factor for developing heart disease, and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age‐associated defects in autophagy remain poorly characterized. Here, we have investigated how the autophagic process is altered in aged mouse hearts. We report that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. Atg9 proteins are critical in delivering membrane to the growing autophagosome, and siRNA knockdown of Atg9b in cells confirmed a reduction in autophagosome formation. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We also found increased levels of Parkin‐positive mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. In contrast, Nrf1, a major transcriptional regulator of mitochondrial biogenesis, was significantly reduced in aged hearts. Additionally, our data showed reduced Drp1‐mediated mitochondrial fission and formation of enlarged mitochondria in the aged heart. Overall, our findings suggest that cardiac aging is associated with reduced autophagosome number, decreased mitochondrial turnover, and formation of megamitochondria.