Regulation Of Mitochondrial Respiration Research Articles

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

UCP2-induced hypoxia promotes lipid accumulation and tubulointerstitial fibrosis during ischemic kidney injury

Mitochondrial dysfunction leads to loss of renal function and structure; however, the precise mechanisms by which mitochondrial function can regulate renal fibrosis remain unclear. Proximal tubular cells (PTCs) prefer fatty acid oxidation as their energy source and dysregulation of lipid metabolism has been linked to tubulointerstitial fibrosis (TIF). Here, we demonstrated that mitochondrial uncoupling protein 2 (UCP2) regulates TIF through the stimulation of lipid deposition and extracellular matrix (ECM) accumulation. We show that UCP2 expression was increased in human biopsy sample and mouse kidney tissues with TIF. Moreover, UCP2-deficient mice displayed mitigated renal fibrosis in I/R-induced mouse model of TIF. Consistent with these results, UCP2 deficiency displayed reduced lipid deposition and ECM accumulation in vivo and in vitro. In UCP2-deficient PTCs, inhibition of TIF resulted from downregulation of hypoxia-inducible factor-1α (HIF-1α), a key regulator of lipid metabolism and ECM accumulation. Furthermore, we describe a molecular mechanism by which UCP2 regulates HIF-1α stabilization through regulation of mitochondrial respiration and tissue hypoxia during TIF. HIF-1α inhibition by siRNA suppressed lipid and ECM accumulation by restoration of PPARα and CPT1α, as well as suppression of fibronectin and collagen I expression in PTCs. In conclusion, our results suggest that UCP2 regulates TIF by inducing the HIF-1α stabilization pathway in tubular cells. These results identify UCP2 as a potential therapeutic target in treating chronic renal fibrosis.

Implication of Mitochondrial NO/cGMP/PKG Signaling System in the Activation and Inhibition of Mitochondrial Respiration by L-Arginine and NO Donors

The involvement of the mitochondrial calcium-dependent NO synthase (mtNOS) in the regulation of mitochondrial respiration has not been sufficiently studied. Moreover, the possible functioning of a mitochondrial signaling system involving mtNOS/guanylate cyclase (GC)/protein kinase G (PKG) and the impact of this system (mtNOS/GC/PKG-SS) on mitochondrial respiration have not yet been analyzed. To investigate this issue, we performed experiments on isolated rat liver using specific inhibitors of NOS, GC, and PKG. The high rate of mitochondrial respiration was supported by pyruvate and glutamate or by succinate in the presence of hexokinase, glucose, and ADP. It was shown that L-arginine and the NO donor sodium nitroprusside (SNP) exert concentration-dependent effects on the mitochondrial respiration rate. At low concentrations, L‑arginine (up to 200 μM) and SNP (up to100 μM) activated mitochondrial respiration. The inhibitors of NOS, GC, and PKG eliminated this effect indicating that mtNOS/GC/PKG-SS is involved in the activation of respiration. At high concentrations, L-arginine and SNP, on the contrary, inhibited respiration. Under these conditions, the inhibitors of GC and PKG enhanced the inhibition of respiration, which indicates an opposite effect of the excess of NO and PKG on the mitochondrial respiration. The results suggest that the functioning of calcium-dependent mtNOS/GC/PKG-SS can ensure the activation of respiration at low concentrations of L-arginine or SNP in the medium.

Abstract 540: Sigma 1 Receptor-dependent Regulation of Mitochondrial Respiration and Function in Cardiomyocytes

Background: Sigma 1 receptor (Sigmar1) is an abundantly expressed molecular chaperone protein in cardiomyocytes. Sigmar1 ligands have been shown to have protective function in animal models of cardiac hypertrophy, heart failure, and many other cardiovascular disorders. Despite extensive studies, the physiological and molecular functions of Sigmar1 in cardiomyocytes remains elusive. Objective: We investigated the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes. Methods and Results: To define the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes, we used small interfering RNA-mediated Sigmar1 knockdown and adenovirus-mediated overexpression in cultured neonatal rat ventricular cardiomyocytes. Sigmar1 knockdown in neonatal cardiomyocytes showed significantly decreased mitochondrial respiration measured by Seahorse Analyzer. Mitochondria stained with mitoTracker green showed a significantly increased number of hyperfused mitochondria in Sigmar1 knockdown cardiomyocytes. Similarly, mitochondria isolated from Sigmar1 global knockout mouse hearts also showed decreased mitochondrial respiration and the accumulation of larger size mitochondria measured by flowcytometry. Sigmar1 overexpression in cardiomyocytes significantly increased mitochondrial respiration and also showed increased mitochondrial fragmentation. Conclusions: Our findings suggested that Sigmar1 plays an essential role in maintaining normal mitochondrial morphology, respiration, and function in cardiomyocytes.

Sirtuin-1/Mitochondrial Ribosomal Protein S5 Axis Enhances the Metabolic Flexibility of Liver Cancer Stem Cells.

Metabolic reprogramming endows cancer cells with the ability to adjust metabolic pathways to support heterogeneously biological processes. However, it is not known how the reprogrammed activities are implemented during differentiation of cancer stem cells (CSCs). In this study, we demonstrated that liver CSCs relied on the enhanced mitochondrial function to maintain stemness properties, which is different from aerobic glycolysis playing main roles in the differentiated non-CSCs. We found that liver CSCs exhibit increased mitochondrial respiratory capacity and that complex-I of mitochondria was necessary for stemness properties of liver CSCs through regulation of mitochondrial respiration. Bioinformatics analysis reveals that mitochondrial ribosomal protein S5 (MRPS5) is closely related with the function of complex-I. Further experiments confirmed that MRPS5 promoted the production of nicotinamide adenine dinucleotide (NAD+ ), which is necessary for enhanced mitochondrial function in liver CSCs. MRPS5 played a critical role for liver CSCs to maintain stemness properties and to participate in tumor progression. Mechanistically, the acetylation status of MRPS5 is directly regulated by NAD+ dependent deacetylase sirtuin-1 (SIRT1), which is abundant in liver CSCs and decreased during differentiation. Deacetylated MRPS5 locates in mitochondria to promote the function complex-I and the generation of NAD+ to enhance mitochondrial respiration. Conversely, the acetylated MRPS5 gathered in nuclei leads to increased expression of glycolytic proteins and promotion of the Warburg Effect. Therefore, liver CSCs transform mitochondrial-dependent energy supply to a Warburg phenotype by the dual function of MRPS5. Clinical analysis of SIRT1 and MRPS5 expression in tumor tissues showed the SIRT1High /Cytoplasmic-MRPS5High profile was associated with patients with hepatocellular carcinoma with poor prognosis. Conclusion: SIRT1/MRPS5 axis participates in metabolic reprogramming to facilitate tumor progression and may serve as a promising therapeutic target of liver cancer.

Tumor protein 53-induced nuclear protein 1 deficiency alters mouse gastrocnemius muscle function and bioenergetics invivo.

Tumor protein 53‐induced nuclear protein 1 (TP53INP1) deficiency leads to oxidative stress‐associated obesity and insulin resistance. Although skeletal muscle has a predominant role in the development of metabolic syndrome, the bioenergetics and functional consequences of TP53INP1 deficiency upon this tissue remain undocumented. To clarify this issue, gastrocnemius muscle mechanical performance, energy metabolism, and anatomy were investigated in TP53INP1‐deficient and wild‐type mice using a multidisciplinary approach implementing noninvasive multimodal‐NMR techniques. TP53INP1 deficiency increased body adiposity but did not affect cytosolic oxidative stress, lipid content, and mitochondrial pool and capacity in myocyte. During a fatiguing bout of exercise, the in vivo oxidative ATP synthesis capacity was dramatically reduced in TP53INP1‐deficient mice despite ADP level (the main in vivo stimulator of mitochondrial respiration) did not differ between both genotypes. Moreover, TP53INP1 deficiency did not alter fatigue resistance but paradoxically increased the contractile force, whereas there were no differences for muscle fiber‐type distribution and calcium homeostasis between both genotypes. In addition, muscle proton efflux was decreased in TP53INP1‐deficient mice, thereby indicating a reduced blood supply. In conclusion, TP53INP1 plays a role in muscle function and bioenergetics through oxidative capacity impairment possibly as the consequence of abnormal mitochondrial respiration regulation and/or defective blood supply.

The Role of Calcium in the Regulation of Mitochondrial Respiration under Various Substrate Combinations in the Heart and Kidney

It has been recognized that the efficiency of mitochondrial oxidative phosphorylation (OxPhos) and energy metabolism depends on the choice of respiratory substrates, which feed electrons to the mitochondrial electron transport chain. In addition, mitochondrial enzymes, such as pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and alpha‐ketoglutarate dehydrogenase (AKGDH), have been shown to be stimulated by calcium ions (Ca2+) in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on the efficiency of mitochondrial OxPhos and energy metabolism in in‐situ and in‐vivo states in the heart and kidney under different substrate combinations are not well‐understood. In this study, we investigated the effect of a variety of substrate combinations and Ca2+ alternations that optimize mitochondrial respiration and ATP synthesis. We hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is both substrate dependent and tissue specific. To study mitochondrial function under a specific substrate combination and Ca2+, cardiac and renal mitochondria were isolated from Sprague‐Dawley rats, and oxygen consumption rates (JO2) were measured in the presence and absence of ADP that corresponded to leak (state 2) and ADP‐stimulated (state 3) states, respectively. We first measured the time courses of JO2 at various ADP concentrations to find the saturated ADP‐stimulated JO2. This saturated ADP concentration was then used to measure the time‐courses of JO2 under different [Ca2+]. The substrate combinations used include pyruvate + malate (PM), glutamate + malate (GM), alpha‐ketoglutarate + malate (AM), palmitoyl‐L‐carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). Different substrates led to different levels of the saturated state 3 JO2. On average, it was 9 times higher in the heart compared to the kidney with the PM substrate. Also, the alterations in the respiratory substrates led to dramatically different respiratory rates/dynamics in the isolated mitochondria of the chosen organs. The results also demonstrated that Ca2+ significantly increased mitochondrial state 3 JO2 with GM and AM substrates compared to the other substrates in both organs (Table 1). Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increase in [Ca2+] had a stimulatory effect while large increases had inhibitory effects (Figure 1). These results suggest that energized isolated mitochondria in the presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, proper [Ca2+], and specific tissue source of mitochondria to achieve an optimal level of OxPhos and energy metabolism comparable to in‐vivo conditions.Support or Funding InformationThis work was supported by the NIH grants P01‐GM066730, P01‐HL116264, R01‐HL‐122662, and U01‐HL122199.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

K+ influx triggers slow K+/H+ exchange detected by biphasic changes in matrix pH in Guinea pig cardiomyocyte mitochondria

Recent findings in cell models supports the importance of cation homeostasis by a K+/H+ exchanger (KHE) encoded by the gene LETM1, although LETM1 may also encode Ca2+/H+ exchange and or Na+/H+ exchanger (mNCE). In mitochondria, K+ influx occurs through K+ channels sensitive to Ca2+, Na+, low ATP, and other ligands. K+ influx promotes mitochondrial (m) swelling and contributes to regulation of mitochondrial respiration, membrane potential (mMP) and redox signaling. The influx of mK+ requires a mK+ efflux mechanism (mK+ cycle) paired with H+ influx, which in turn would affect mitochondrial bioenergetics, e.g. respiration, ADP phosphorylation, and mMP due effectively to a “proton leak”. We have previously reported that increased cytosolic and mCa2+ stimulate small and large Ca2+‐sensitive mK+ channels to permit mK+ influx. Cytosolic and mCa2+ increase during ischemia or hypoxia in association with an increase in mitochondrial volume so a high mK+ concentration may be a parallel effect of mCa2+ loading. Experimental conditions that modulate mKHE activity in mammalian cardiac tissue and its consequences have not been examined. Our aim was to provide evidence of mKHE activity in mitochondria isolated from Guinea pig cardiac cell mitochondria and to better understand the circumstances and kinetics of mKHE activity. We proposed that the subsequent efflux of mK+ via mKHE would re‐establish mK+ homeostasis but also provide a “H+ leak” thereby affecting bioenergetics activity. We reasoned that mKHE becomes active once K+ has accumulated in the matrix and that K+ efflux is linked to H+ influx as a marker of mKHE activity. mKHE might be affected by extra‐matrix pH (pHm) as it can change the trans‐membrane pH gradient. To test these ideas, we first added CGP37157 to the mitochondrial buffer to inhibit mNCE. Then we added 25 nM valinomycin, a K+ ionophore, and measured the time‐dependent changes in matrix pHm, using the fluorescent probe BCECF‐AM. We found that across the three extra‐matrix pHs of 6.9, 7.15, and 7.6, matrix pHm transiently increased over approximately 100 s and then transiently decreased over the next 100 s with a slower decline over 20 min. The initial increase in pHm may reflect enhanced proton pumping by respiratory complexes and the secondary decrease in pHm may reflect H+ influx in exchange for K+ efflux, via mKHE. When 500 μM quinine was given before valinomycin to attenuate mKHE, we found that the peak increase in pHm (first phase) occurred later and the subsequent decrease in pHm (second phase) also occurred later by 100 s and was slower to fall over 20 min. The changes to pHm in the presence of quinine provide evidence that the delay in pHm changes were due to block of mKHE by quinine. Interestingly the biphasic effects of valinomycin and quinine on pHm did not appear to be dependent on the extra‐matrix pH values of 6.9, 7.15, 7.6. Additional studies will determine concomitant effects of these protocols on respiration, ADP phosphorylation, mMP, and mitochondrial volume.Support or Funding InformationVA Merit BX‐002539‐01 and NIH T35 HL072483This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-21-5p regulates mitochondrial respiration and lipid content in H9C2 cells.

Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.

Assessing the role of residue E73 and lipid headgroup charge in VDAC1 voltage gating

The voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane (MOM) where it regulates transport of ions and metabolites in and out of the organelle. VDAC function is extensively studied in a lipid bilayer system that allows conductance monitoring of reconstituted channels under applied voltage. The process of switching from a high-conductance state, open to metabolites, to a variety of low-conducting states, which excludes metabolite transport, is termed voltage gating and the mechanism remains poorly understood. Recent studies have implicated the involvement of the membrane-solvated residue E73 in the gating process through β-barrel destabilization. However, there has been no direct experimental evidence of E73 involvement in VDAC1 voltage gating. Here, using electrophysiology measurements, we exclude the involvement of E73 in murine VDAC1 (mVDAC1) voltage gating process. With an established protocol of assessing voltage gating of VDACs reconstituted into planar lipid membranes, we definitively show that mVDAC1 gating properties do not change when E73 is replaced by either a glutamine or an alanine. We further demonstrate that cholesterol has no effect on mVDAC1 gating characteristics, though it was shown that E73 is coordinating residue in the cholesterol binding site. In contrast, we found a pronounced gating effect based on the charge of the phospholipid headgroup, where the positive charge stimulates and negative charge suppresses gating. These findings call for critical evaluation of the existing models of VDAC gating and contribute to our understanding of VDAC's role in control of MOM permeability and regulation of mitochondrial respiration and metabolism.

68 - Mitochondrial STAT3 serine phosphorylation by zinc reduces ROS generation at reperfusion in rat hearts

STAT3 plays a role in the regulation of mitochondrial respiration. This study tested if STAT3 serine phosphorylation by zinc could alter mitochondrial ROS generation in the setting of ischemia/reperfusion in the heart. Under normoxic conditions, treatment of isolated rat hearts with ZnCl2 increased mitochondrial STAT3 phosphorylation at Ser727. In isolated rat hearts subjected to ischemia followed by reperfusion, ZnCl2 increased mitochondrial phospho-STAT3. Moreover, ZnCl2 prevented mitochondrial ROS generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation through Ser727 phosphorylation. Finally, ZnCl2 suppression of succinate dehydrogenase (SDH) activity upon the onset of reperfusion was nullified by the Ser727 mutation. In conclusion, zinc inhibits mitochondrial ROS generation at reperfusion by increasing mitochondrial STAT3 phosphorylation at Ser727. The inhibition of SDH activity may account for the role of STAT3 in the inhibitory action of zinc on ROS generation at reperfusion.

Emerging Potential of Immediate Early Response Gene X-1 in Cardiovascular and Metabolic Diseases.

Immediate early response gene X‐1 (IEX‐1) or immediate early response 3 is an immediate early response gene that is highly expressed in epithelial, endothelial, and immune cells, including macrophages.1, 2, 3, 4 Similar to other immediate early response genes, its induction can be rapidly induced in response to several stimuli, such as UV light, inflammatory cytokines, and mechanical strain.4, 5, 6, 7, 8 Under stress, many immediate early response genes, such as c‐Fos, c‐Jun, c‐Myc, Egr‐1, and other zinc‐finger proteins, usually serve as transcription factors, activators, or repressors to regulate the transcriptional activity of several genes that are critical for a cell to appropriately respond to stress.4, 9, 10, 11, 12 However, IEX‐1 lacks a DNA binding domain and its putative transcription activity remains elusive.4, 13 Investigations from our laboratory show that IEX‐1 controls mitochondrial superoxide production by regulating the electron transport chain and oxidative phosphorylation.13, 14, 15, 16 This action of IEX‐1 might explain the transcriptional activity associated with IEX‐1 because the reactive oxygen species (ROS) at low levels can act as a signaling molecule to alter gene transcription through reduction‐oxidation–sensitive transcription factors or a signaling pathway, such as nuclear factor‐κB (NF‐κB) and nuclear factor erythroid 2‐related factor.17, 18, 19 Consistent with a key role of mitochondria in vascular and metabolic functions, IEX‐1 deficiency impairs vascular function and increases energy expenditure in mice. A key role of IEX‐1 in regulation of mitochondrial respiration and ROS production may also help to explain how IEX‐1 produces both antiapoptotic and proapoptotic actions in different cell types. Macrophages, the central player in the pathogenesis of many chronic inflammatory conditions, express high levels of IEX‐1. In agreement with the putative role of IEX‐1 in macrophage function, we recently identified a crucial role of IEX‐1 in many chronic conditions, including hypertension, sepsis, insulin resistance, obesity, and arthritis,16, 20, 21, 22 revealing previously unknown functions of IEX‐1 in cardiovascular and metabolic diseases. In this article, we reviewed the findings in IEX‐1 knockout mice from our laboratory and others and primarily focused on the roles of IEX‐1 in cardiovascular and metabolic disorders. We also alluded to the important findings from other laboratories that are relevant for its role in cardiovascular and metabolic diseases. In addition, an attempt is made to underline the involvement of IEX‐1 in inflammation and its possible link with the lipid‐mediated metabolic diseases. However, because of the limitations and scope of this article, we refrained from discussing an important role of IEX‐1 in many types of cancers that has been widely reported before.

Gas Signaling Molecules and Mitochondrial Potassium Channels.

Recently, gaseous signaling molecules, such as carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2S), which were previously considered to be highly toxic, have been of increasing interest due to their beneficial effects at low concentrations. These so-called gasotransmitters affect many cellular processes, such as apoptosis, proliferation, cytoprotection, oxygen sensing, ATP synthesis, and cellular respiration. It is thought that mitochondria, specifically their respiratory complexes, constitute an important target for these gases. On the other hand, increasing evidence of a cytoprotective role for mitochondrial potassium channels provides motivation for the analysis of the role of gasotransmitters in the regulation of channel function. A number of potassium channels have been shown to exhibit activity within the inner mitochondrial membrane, including ATP-sensitive potassium channels, Ca2+-activated potassium channels, voltage-gated Kv potassium channels, and TWIK-related acid-sensitive K+ channel 3 (TASK-3). The effects of these channels include the regulation of mitochondrial respiration and membrane potential. Additionally, they may modulate the synthesis of reactive oxygen species within mitochondria. The opening of mitochondrial potassium channels is believed to induce cytoprotection, while channel inhibition may facilitate cell death. The molecular mechanisms underlying the action of gasotransmitters are complex. In this review, we focus on the molecular mechanisms underlying the action of H2S, NO, and CO on potassium channels present within mitochondria.

Different regulation of mitochondrial respiration in human colorectal and breast cancer clinical samples

Different regulation of mitochondrial respiration in human colorectal and breast cancer clinical samples

2261 Tumor suppressor RARRES1 regulates cell survival by modulating mitochondrial energetics

OBJECTIVES/SPECIFIC AIMS: One of the driving mechanisms of cancer progression is the reprogramming of metabolic pathways in intermediary metabolism. Cancers increase their energy expenditure by increasing ATP production for utilization in anabolic pathways to increase production of proteins, nucleic acids and lipids. The Warburg effect, where cancer cells predominantly use aerobic glycolysis rather than oxidative phosphorylation to produce ATP, was long thought to be the main initiating pathway in increasing tumor burden. However, compelling new evidence shows that there exists metabolic heterogeneity among and within tumors. Mitochondrial respiration often plays a major role in tumor progression, as many different cancers contain a subpopulation of slow-cycling tumor-initiating cells that are multidrug-resistant and dependent on oxidative phosphorylation. These cells represent a target for cancer therapy. In this study, we identification a novel endogenous regulator of mitochondrial respiration, retinoic acid receptor responder 1 (RARRES1). METHODS/STUDY POPULATION: We assessed the metabolic phenotype of RARRES1-depleted normal epithelial cells through metabolomics, a flux analyzer and blotting for phosphorylation of AMP kinase, a major regulator of energy homeostasis. We further examined mitochondrial energetics by staining the mitochondria with TMRM and Mito-Tracker. We then analyzed the apoptotic phenotype of epithelial cells with depletion of RARRES1 with fluorescence-activated cell sorting analysis of annexin V-staining. RESULTS/ANTICIPATED RESULTS: Remarkably, fluorescence-activated cell sorting analysis of annexin V-stained epithelial cells with depletion of RARRES1 were resistant to all studied modes of cell death, implying an effect on a fundamental cell process. By using proteomics, metabolomics, cellular and molecular analyses, our data show that RARRES1 regulates mitochondrial membrane potential and subsequently alters 1-carbon metabolism by modulating the function of the mitochondrial voltage-dependent anion channel. We believe this is the first example of a tumor suppressor protein that functions to directly regulate mitochondrial energetics. Using an extracellular flux analyzer, our data also show that depletion of RARRES1 causes an increase in mitochondrial respiration and ATP production, thus enhancing biosynthetic pathways that drive the pathogenicity and survival of cancer. The metabolic and anti-apoptotic phenotype of RARRES1-depleted cells was reversed by treatment of metformin, a mitochondrial inhibitor. DISCUSSION/SIGNIFICANCE OF IMPACT: These data lay the foundation for metabo-therapy of the many tumor types that exhibit RARRES1 depletion and may have the added benefit of targeting drug-resistant tumor-initiating cells.

Regulation of mitochondrial respiration and ATP synthesis via cytochrome c oxidase

Cytochrome c oxidase (COX) represents the main oxygen acceptor for respiration of aerobic organisms. The energy of respiration is stored via oxidative phosphorylation (OxPhos) in ATP, the general energy intermediate in living cells, or released as heat. During evolution from bacteria to mammals the complexity of OxPhos regulation increased at COX by increasing the number of subunits, by expression of subunit isoforms which are specific for tissues, developmental stages, and oxygen concentrations, and by reversible phosphorylation. The essential function of the “supernumerary” subunits became evident in patients with mitochondrial disease related to COX-deficiency, based on mutations in these subunit genes. While the basic regulation of energy transduction in OxPhos was explained by the Mitchell theory, an additional regulation of respiration and ATP synthesis was discovered in eukaryotic organisms, based on reversible and membrane potential-independent inhibition of COX activity at high ATP/ADP ratios. So far, only some of the complex regulatory functions of nuclear-encoded subunits were uncovered.

Alkaloids, Nitric Oxide, and Nitrite Reductases: Evolutionary Coupling as Key Regulators of Cellular Bioenergetics with Special Relevance to the Human Microbiome.

Typical alkaloids expressed by prokaryotic and eukaryotic cells are small heterocyclic compounds containing weakly basic nitrogen groups that are critically important for mediating essential biological activities. The prototype opiate alkaloid morphine represents a low molecular mass heterocyclic compound that has been evolutionarily fashioned from a relatively restricted role as a secreted antimicrobial phytoalexin into a broad spectrum regulatory molecule. As an essential corollary, positive evolutionary pressure has driven the development of a cognate 6-transmembrane helical (TMH) domain μ3 opiate receptor that is exclusively responsive to morphine and related opiate alkaloids. A key aspect of “morphinergic” signaling mediated by μ3 opiate receptor activation is its functional coupling with regulatory pathways utilizing constitutive nitric oxide (NO) as a signaling molecule. Importantly, tonic and phasic intra-mitochondrial NO production exerts profound inhibitory effects on the rate of electron transport, H+ pumping, and O2 consumption. Given the pluripotent role of NO as a selective, temporally-defined chemical regulator of mitochondrial respiration and cellular bioenergetics, the expansion of prokaryotic denitrification systems into mitochondrial NO/nitrite cycling complexes represents a series of evolutionary modifications of existential proportions. Presently, our short review provides selective discussion of evolutionary development of morphine, opiate alkaloids, μ3 opiate receptors, and NO systems, within the perspectives of enhanced mitochondrial function, cellular bioenergetics, and the human microbiome.

Zinc improves mitochondrial respiratory function and prevents mitochondrial ROS generation at reperfusion by phosphorylating STAT3 at Ser727

Serine 727 (Ser727) phosphorylation of STAT3 plays a role in the regulation of mitochondrial respiration. This study aimed to test if zinc could regulate mitochondrial respiration through phosphorylation of STAT3 at Ser727 in the setting of ischemia/reperfusion in the heart. Under normoxic conditions, treatment of isolated rat hearts with ZnCl2 increased cytosolic STAT3 phosphorylation at Ser727 followed by phospho-STAT3 translocation to mitochondria. In isolated rat hearts subjected to 30 min regional ischemia followed by 20 min of reperfusion, ZnCl2 given 5 min before the onset of reperfusion also increased mitochondrial phospho-STAT3. ZnCl2 enhanced ERK phosphorylation and PD98059 reversed the effect of ZnCl2 on STAT3 phosphorylation. ZnCl2 improved the mitochondrial oxidative phosphorylation at reperfusion. This effect was abolished by STAT3S727A, a mutant in which Ser727 is replaced with alanine, in H9c2 cells subjected to hypoxia/reoxygenation. In addition, ZnCl2 increased the mRNA level of the complex I subunit ND6, which was also reversed by STAT3S727A. Moreover, ZnCl2 attenuated mitochondrial ROS generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation through Ser727 phosphorylation. Finally, ZnCl2 suppression of succinate dehydrogenase (SDH) activity upon the onset of reperfusion was nullified by the Ser727 mutation. In conclusion, zinc improves cardiac oxidative phosphorylation and inhibits mitochondrial ROS generation at reperfusion by increasing mitochondrial STAT3 phosphorylation at Ser727 via ERK. The preservation of ND6 mtDNA and the inhibition of SDH activity may account for the role of STAT3 in the beneficial action of zinc on the mitochondrial oxidative phosphorylation and ROS generation at reperfusion.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

How long noncoding RNAs enforce their will on mitochondrial activity: regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer.

The cellular transcriptome contains a wide diversity of untranslated RNAs, of which the class of regulatory long noncoding RNAs (lncRNAs) has only recently been recognized. Evidence swiftly accumulates of lncRNAs influencing mitochondrial activities of eukaryotic cells, and perturbed expression is conspicuously associated with human diseases. In this review, we describe the multifaceted effects of lncRNAs on mitochondrial function, more particularly on the balance between oxidative phosphorylation and glycolysis, on the production of reactive oxygen species, and on apoptosis in human cells. Emphasis is placed on the involvement of lncRNAs in cancer metabolism, as tumor cells rely heavily on modifications of mitochondrial functioning as an essential component for sustained tumorigenesis and cancer progression. From the nonexhaustive list of lncRNAS described in this review, ANRIL, AScmtRNA, H19, HOTAIR, LincRNA-p21, MALAT1, RMRP, SAMMSON, and VL30 have emerged as potent regulators of mitochondrial metabolism. Due to their key role in cancer progression, they represent potential targets of innovative lncRNA-based treatment strategies.