Decrease In Intracellular ATP Levels Research Articles

Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

Glucose Induces Resistance to Polymyxins in High-Alcohol-Producing Klebsiella pneumoniae via Increasing Capsular Polysaccharide and Maintaining Intracellular ATP.

High-alcohol-producing K. pneumoniae (HiAlc Kpn) causes nonalcoholic fatty liver disease (NAFLD) by producing excess endogenous alcohol in the gut of patients with NAFLD, using glucose as the main carbon source. The role of glucose in the response of HiAlc Kpn to environmental stresses such as antibiotics remains unclear. In this study, we found that glucose could enhance the resistance of HiAlc Kpn to polymyxins. First, glucose inhibited the expression of crp in HiAlc Kpn and promoted the increase of capsular polysaccharide (CPS), which promoted the drug resistance of HiAlc Kpn. Second, glucose maintained high ATP levels in HiAlc Kpn cells under the pressure of polymyxins, enhancing the resistance of the cells to the killing effect of antibiotics. Notably, the inhibition of CPS formation and the decrease of intracellular ATP levels could both effectively reverse glucose-induced polymyxins resistance. Our work demonstrated the mechanism by which glucose induces polymyxins resistance in HiAlc Kpn, thereby laying the foundation for developing effective treatments for NAFLD caused by HiAlc Kpn. IMPORTANCE HiAlc Kpn can use glucose to produce excess endogenous alcohol for promoting the development of NAFLD. Polymyxins are the last line of antibiotics and are commonly used to treat infections caused by carbapenem-resistant K. pneumoniae. In this study, we found that glucose increased bacterial resistance to polymyxins via increasing CPS and maintaining intracellular ATP; this increases the risk of failure to treat NAFLD caused by multidrug-resistant HiAlc Kpn infection. Further research revealed the important roles of glucose and the global regulator, CRP, in bacterial resistance and found that inhibiting CPS formation and decreasing intracellular ATP levels could effectively reverse glucose-induced polymyxins resistance. Our work reveals that glucose and the regulatory factor CRP can affect the resistance of bacteria to polymyxins, laying a foundation for the treatment of infections caused by multidrug-resistant bacteria.

Toxicity mechanism of patulin on 293T cells and correlation analysis of Caspase family.

Patulin (PAT), a kind of mycotoxin, is a widely disseminated mycotoxin found in agricultural products. Although the existing research results show that PAT can cause nerve, immune, and skin toxicities, resulting in heart, liver, and kidney damages. However, evidence on the underlying mechanisms of PAT is still lacking. Present study aims to investigate the renal toxicity and related mechanisms of PAT on 293T cells. Cell Counting Kit-8 method was used to reveal the dose-effect relationship and the time-effect relationship of PAT toxicity. Trypan blue staining and Hoechst 33342 staining were used to analyze PAT, which induced apoptosis on 293T cells. Superoxide-dismutase (SOD), GSH, and malondialdehyde (MDA) were used to measure the changes of oxidative stress status of 293T cells induced by PAT. The changes of reactive oxygen species (ROS) and ATP in mitochondria indicate the role of mitochondria when PAT induced cell damage and apoptosis. Through Cyt-C release assay analysis, caspase activity change, and correlation analysis, the potential mechanism of mitochondrial apoptosis pathway was proved. Results demonstrated that PAT significantly induced cell injury, and with the increase of time and concentration, the cell survival rate decreased significantly. Hoechst 33342 staining and Trypan blue staining showed that apoptosis rate was elevated by PAT. As PAT concentration increased, intracellular SOD, glutathion peroxidase activities were decreased and the MDA content was increased. The decrease of intracellular ATP level and accumulation of ROS content indicated an increased permeability of the mitochondrial membrane. Overexpression of Cyt-C activated the cascade reaction of caspase enzyme, leading to apoptosis. The results of enzyme activity assay and correlation analysis indicated that caspase 3 was the most critical caspase in the cascade system and that it was most correlated with caspase 8 and caspase 9.

Molecular and Cellular Response of the Myocardium (H9C2 Cells) Towards Hypoxia and HIF-1α Inhibition.

IntroductionOxidative phosphorylation is an essential feature of Animalian life. Multiple adaptations have developed to protect against hypoxia, including hypoxia-inducible-factors (HIFs). The major role of HIFs may be in protecting against oxidative stress, not the preservation of high-energy phosphates. The precise mechanism(s) of HIF protection is not completely understood.Materials and MethodsTo better understand the role of hypoxia-inducible-factor-1, we exposed heart/myocardium cells (H9c2) to both normoxia and hypoxia, as well as cobalt chloride (prolyl hydroxylase inhibitor), echniomycin (HIF inhibitor), A2P (anti-oxidant), and small interfering RNA to beclin-1. We measured cell viability, intracellular calcium and adenosine triphosphate, NADP/NADPH ratios, total intracellular reactive oxidative species levels, and markers of oxidative and antioxidant levels measured.ResultsHypoxia (1%) leads to increased intracellular Ca2+ levels, and this response was inhibited by A2P and echinomycin (ECM). Exposure of H9c2 cells to hypoxia also led to an increase in both mRNA and protein expression for Cav 1.2 and Cav 1.3. Exposure of H9c2 cells to hypoxia led to a decrease in intracellular ATP levels and a sharp reduction in total ROS, SOD, and CAT levels. The impact of hypoxia on ROS was reversed with HIF-1 inhibition through ECM. Exposure of H9c2 cells to hypoxia led to an increase in Hif1a, VEGF and EPO protein expression, as well as a decrease in mitochondrial DNA. Both A2P and ECM attenuated this response to varying degrees.ConclusionHypoxia leads to increased intracellular Ca2+, and inhibition of HIF-1 attenuates the increase in intracellular Ca2+ that occurs with hypoxia. HIF-1 expression leads to decreased adenosine triphosphate levels, but the role of HIF-1 on the production of reactive oxidative species remains uncertain. Anti-oxidants decrease HIF-1 expression in the setting of hypoxia and attenuate the increase in Ca2+ that occurs during hypoxia (with no effect during normoxia). Beclin-1 appears to drive autophagy in the setting of hypoxia (through ATG5) but not in normoxia. Additionally, Beclin-1 is a powerful driver of reactive oxidative species production and plays a role in ATP production. HIF-1 inhibition does not affect autophagy in the setting of hypoxia, suggesting that there are other drivers of autophagy that impact beclin-1.

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.

Physiological, Morphological and Antioxidant Responses of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 Isolated from Harbin Dry Sausages to Oxidative Stress.

As functional starter cultures and potential probiotics, the ability of lactic acid bacteria to resist oxidative stress is essential to maintain viability and functional properties. This study investigates the effects of H2O2 at different concentrations (0, 1, 2, and 3 mM) on the physiological, morphological, and antioxidant properties of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 isolated from Harbin dry sausages. The increase in H2O2 concentration induced a significant increase in reactive oxygen species and a decrease in intracellular ATP levels (p < 0.05). Based on scanning electron microscopy, transmission electron microscopy, and electric conductivity analysis, H2O2 stress caused cell deformation, the destruction of cell membrane integrity, partial loss of the cytoplasm, and an increase in the cell conductivity of both strains. H2O2 stress with 1 mM or 2 mM concentrations could effectively improve the scavenging rates of free radicals, the activities of superoxide dismutase and glutathione peroxide, and the total antioxidant capacity of both strains (p < 0.05). In conclusion, an appropriate oxidative stress contributed to the activation of the antioxidant defense system of both strains, conferred strains a better effect in inhibiting the oxidation of fermented foods, and improved the health of the host.

The bitter taste receptor agonist denatonium influences mouse tracheal epithelial ion transport

In the airway epithelium, mucociliary clearance is strictly regulated by ciliary beat and transepithelial ion transport to provide homeostasis. During recent years it was shown that ciliated cells and specialized chemosensory cells in the airway epithelium express bitter taste receptors (Tas2R). We have previously shown that the Tas2R agonist denatonium stimulates particle transport speed in murine trachea via acetylcholine (ACh) release from brush cells. However, it remains to be elucidated if Tas2R stimulation also influences transepithelial ion transport processes. To address this question, we here performed measurements of the transepithelial ion current (ISC) of murine tracheas in Ussing chamber experiments. Apical application of denatonium changed ISC in a dose dependent manner (EC50 132.6 µM). Application of the Gai subunit inhibitor pertussis toxin (500 ng/ml) significantly reduced the denatonium-effect (1 mM), while the Gbg subunit inhibitor gallein (50 µM) had no effect. In the presence of TPPO (100 µM) an inhibitor of the transient receptor potential melastatin-5 (TRPM5) ion channel, a member of the canonical bitter signalling cascade, and in TRPM5 knock-out mice, the denatonium-effect was significantly reduced. Inhibition of an increase of intracellular Ca2+ with the phospholipase C inhibitor U-73122 (10 µM) did not change the denatonium-effect. Furthermore, the denatonium-effect was not dependent on cholinergic signalling, as blocking ACh receptors with mecamylamine (100 µM) and atropine (50 µM) had no influence. Interestingly application of the pannexin1 cannel inhibitor probenecid (1 mM) also reduced the denatonium-effect, pointing towards a release of ATP via pannexin channels. A decrease in intracellular ATP-levels leads to an activation of KATP potassium channels as shown with the inhibitor glibenclamide (100 µM). Additionally, blocking ATP-receptors with suramin (100 µM) significantly reduced the denatonium-effect, indicating that the released ATP acts in a paracrine way on the airway epithelial cells. Taken together we showed that activation of Tas2R in brush cells might represent important regulators of ion transport activity via ATP-dependent signalling. Selectively targeting Tas2R in the trachea might represent a useful target for stimulation of the mucociliary clearance.

Nitrogen-doped fluorescence carbon dots as multi-mechanism detection for iodide and curcumin in biological and food samples

Iodine ion is one of the most indispensable anions in living organisms, particularly being an important substance for the synthesis of thyroid hormones. Curcumin is a yellow-orange polyphenol compound derived from the rhizome of Curcuma longa L., which has been commonly used as a spice and natural coloring agent, food additives, cosmetics as well as Chinese medicine. However, excess curcumin may cause DNA inactivation, lead to a decrease in intracellular ATP levels, and trigger the tissue necrosis. Therefore, quantitative detection of iodine and curcumin is of great significance in the fields of food and life sciences. Herein, we develop nitrogen-doped fluorescent carbon dots (NCDs) as a multi-mechanism detection for iodide and curcumin in actual complex biological and food samples, which was prepared by a one-step solid-phase synthesis using tartaric acid and urea as precursors without adding any other reagents. An assembled NCDs-Hg2+ fluorescence-enhanced sensor for the quantitative detection of I− was established based on a fluorescence “turn-off-on” mechanism in a linear range of 0.3–15 μM with a detection limit of 69.4 nM and successfully quantified trace amounts of I− in water samples and urine sample. Meanwhile, the as-synthesized NCDs also can be used as a fluorescent quenched sensor for curcumin detection based on the synergistic internal filtration effect (IFE) and static quenching, achieving a good linear range of 0.1–20 μM with a satisfactory detection limit of 29.8 nM. These results indicate that carbon dots are potential sensing materials for iodine and curcumin detection for the good of our health.

Trans, trans-2,4-decadienal (tt-DDE), a composition of cooking oil fumes, induces oxidative stress and endoplasmic reticulum stress in human corneal epithelial cells

Indoor pollution with cooking oil fumes (COF) as one of the main components is closely related to ocular surface disorders. However, as the most abundant aldehyde in COF, the toxicity of trans, trans-2,4-decadienal (tt-DDE) on human cornea has not been explored before. In the present study, we observed a time- and dose-dependent cytotoxicity induced by tt-DDE in human corneal epithelial (HCE) cells, as evidenced by decreased cell viability, altered cell morphology, and increased proportion of apoptotic cells. Exposure to tt-DDE also led to an increase in reactive oxygen species (ROS) production, MMP loss, and a decrease in intracellular ATP levels. In addition, after exposure to tt-DDE, the expression of endoplasmic reticulum (ER) stress-related proteins (Bip, pIRE1, XBP1, pPERK, peIF2α, ATF4, and CHOP) increased, indicating that ER stress was activated. Moreover, pretreatment of HCE cells with two ER stress inhibitors (200 nM ISRIB or 1 mM 4-PBA) effectively attenuated oxidative stress induced by tt-DDE. These results suggested that tt-DDE could cause damage to HCE cells by triggering oxidative stress and ER stress. Furthermore, regulation of ER stress can be considered as a potential protective method for tt-DDE-induced ocular surface disorders.

Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis.

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 μM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.

Imidazo[2,1-a]isoindole scaffold as an uncharted structure active on Leishmania donovani

The human protozoan parasites Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis, as such, responsible for approximately 30,000 deaths annually. The available chemotherapeutic treatments are reduced to a few drugs whose effectiveness is limited by rising drug resistance/therapeutic failure, and noxious side-effects. Therefore, new therapeutic hits are needed. Compounds displaying the imidazo[2,1-a]isoindole skeleton have shown antichagasic, anti-HIV, antimalarial and anorectic activities. Here, we report the leishmanicidal activity of thirty one imidazo[2,1-a]isoindol-5-ol derivatives on promastigotes and intracellular amastigotes of L. donovani. Eight out of thirty one assayed compounds showed EC50 values ranging between 1 and 2 μM with selectivity indexes from 29 to 69 on infected THP-1 cells. Six compounds were selected for further elucidation of their leishmanicidal mechanism. In this regard, compound 29, the imidazoisoindolol with the highest activity on intracellular amastigotes, induced an early decrease of intracellular ATP levels, as well as mitochondrial depolarization, together with a partial plasma membrane destructuration, as assessed by transmission electron microscopy. Consequently, the inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound, even when other additional targets cannot be ruled out. In all, the results supported the inclusion of the imidazoisoindole scaffold for the development of new leishmanicidal drugs.

4-Aminoquinoline-based compounds as antileishmanial agents that inhibit the energy metabolism of Leishmania

Among neglected tropical diseases, leishmaniasis is one of the most relevant with an estimated 30,000 deaths annually. Existing therapies have serious drawbacks in safety, drug resistance, field-adapted application and cost; therefore, new safer and shorter treatments are needed for this disease. Here we report on the synthesis of novel 4-amino-7-chloroquinoline-based compounds with leishmanicidal activity, together with deeper insight into the mechanism of action of our previously published hit compound 1. New derivatives showed comparable activity to 1 against both promastigote and intracellular amastigote forms of Leishmania infantum, with IC50 < 1 μM. Furthermore, we have determined that compound 1 induced a decrease of intracellular ATP levels, as well as a mitochondrial depolarization, together with an alteration of plasma membrane permeability and a significant ROS production. The inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound. In all, these results support the consideration of compound 1 for the future development of new leishmanicidal drugs.

Mitochondrial miR-762 regulates apoptosis and myocardial infarction by impairing ND2

Mitochondrial dysfunction plays a major role in the pathogenesis of cardiovascular diseases. MicroRNAs (miRNAs) are small RNAs that act as negative regulators of gene expression, but how miRNAs affect mitochondrial function in the heart is unclear. Using a miRNA microarray assay, we found that miR-762 predominantly translocated in the mitochondria and was significantly upregulated upon anoxia/reoxygenation (A/R) treatment. Knockdown of endogenous miR-762 significantly attenuated the decrease in intracellular ATP levels, the increase in ROS levels, the decrease in mitochondrial complex I enzyme activity and the increase in apoptotic cell death in cardiomyocytes, which was induced by A/R treatment. In addition, knockdown of miR-762 ameliorated myocardial ischemia/reperfusion (I/R) injury in mice. Mechanistically, we showed that enforced expression of miR-762 dramatically decreased the protein levels of endogenous NADH dehydrogenase subunit 2 (ND2) but had no effect on the transcript levels of ND2. The luciferase reporter assay showed that miR-762 bound to the coding sequence of ND2. In addition, knockdown of endogenous ND2 significantly decreased intracellular ATP levels, increased ROS levels, reduced mitochondrial complex I enzyme activity and increased apoptotic cell death in cardiomyocytes, which was induced by A/R treatment. Furthermore, we found that the inhibitory effect of miR-762 downregulation was attenuated by ND2 knockdown. Thus, our findings suggest that miR-762 participates in the regulation of mitochondrial function and cardiomyocyte apoptosis by ND2, a core assembly subunit of mitochondrial complex I. Our results revealed that mitochondrial miR-762, as a new player in mitochondrial dysfunction, may provide a new therapeutic target for myocardial infarction.

Hypoxia-inducible factor-1 α deletion in myeloid lineage attenuates hypoxia-induced pulmonary hypertension.

Hypoxemia is seen in patients with pulmonary hypertension and hypoxic pulmonary vasoconstriction worsens their clinical condition. However, vasoconstriction is not the only aspect through which hypoxia induces the progression to pulmonary hypertension. Hypoxia‐inducible factor‐1α (HIF‐1α) is a transcription factor responding to hypoxic conditions by regulating hundreds of genes involved in angiogenesis, erythropoiesis, inflammation, and proliferation. We sought to determine the contribution of HIF‐1α in myeloid lineage cells to the pulmonary vascular response to chronic exposure to hypoxia. We generated myeloid‐specific HIF‐1α knockout (MyeHIF1KO) mice by using Cre‐lox P system, and exposed them to hypoxic conditions for 3 weeks to induce pulmonary hypertension. Macrophages from MyeHIF1KO and control mice were used for western blotting, RT‐qPCR, chemotaxis assay, and ATP assay. MyeHIF1KO mice exposed to hypoxia for 3 weeks exhibited a significant reduction in the right ventricular systolic pressure accompanied by a decrease in the ratio of the right ventricular weight to left ventricular weight, muscularization of the small pulmonary arteries, and infiltration of macrophages into the lung and right ventricle compared with control mice. HIF‐1α‐deficient peritoneal macrophages showed less migration toward monocyte chemoattractant protein‐1 and a decrease in intracellular ATP levels. These results indicate that HIF‐1α in macrophages contributes to the progression of pulmonary vascular remodeling and pulmonary hypertension induced by chronic exposure to hypoxic conditions. The inhibition of myeloid‐specific HIF‐1α may be a novel therapeutic strategy for the treatment of pulmonary hypertension.

Nitrogen and Sulfur Co-doped Fluorescent Carbon Dots for the Detection of Morin and Cell Imaging

Background: Morin has many pharmacological functions including antioxidant, anticancer, anti-inflammatory, and antibacterial effects. It is commonly used in the treatment of antiviral infection, gastropathy, coronary heart disease and hepatitis B in clinic. However, researches have shown that morin is likely to show prooxidative effects on the cells when the amount of treatment is at high dose, leading to the decrease of intracellular ATP levels and the increase of necrosis process. Therefore, it is necessary to determine the concentration of morin in biologic samples. Method: Novel water-soluble and green nitrogen and sulfur co-doped carbon dots (NSCDs) were prepared by a microwave heating process with citric acid and L-cysteine. The fluorescence spectra were collected at an excitation wavelength of 350 nm when solutions of NSCDs were mixed with various concentrations of morin. Results: The as-prepared NSCDs were characterized by transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The fluorescence intensity of NSCDs decreased significantly with the increase of morin concentration. The fluorescence intensity of NSCDs displayed a linear response to morin in the concentration 0.10-30 μM with a low detection limit of 56 nM. The proposed fluorescent probe was applied to analysis of morin in human body fluids with recoveries of 98.0-102%. Conclusion: NSCDs were prepared by a microwave heating process. The present analytical method is sensitive to morin. The quenching process between NSCDs and morin is attributed to the static quenching. In addition, the cellular toxicity on HeLa cells indicated that the as-prepared NSCDs fluorescent probe does not show obvious cytotoxicity in cell imaging. Our proposed method possibly opens up a rapid and nontoxic way for preparing heteroatom doped carbon dots with a broad application prospect.

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention.

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

NV gene variations of viral hemorrhagic septicemia virus decrease cellular energy metabolism

The viral hemorrhagic septicemia virus (VHSV) is a viral pathogen to cause severe losses in farming industry of olive flounder. Among six-encoding VHSV proteins, NV protein has been shown to have an impact on virulence. Thirteen VHSV strains are isolated from olive flounders showing VHS symptoms and mortality in Korea, and then molecular variation of nucleotide and amino acid sequences of the NV genes were compared. Although common mutations were detected at four sites in nucleotide sequences of NV gene, there was no difference in the amino acid sequences among 13 VHSV variants. We identified substitution of one base of 34 positions within the NV ORF of 13 VHSV isolates and these point mutations resulted in changes in amino acid sequences at 6 residues of NV (8, 56, 81, 88, 117, and 119). Based on finding of NV-mediated decrease of intracellular ATP level, protein expression of six NV variants in flounder cells decreased more than control NV protein. The strongest decreasing activity on cellular ATP level was shown by No. 56 (serine→leucine) of NV variation. The variation changes the hydropathy index significantly. The results imply that we can use appearance of some amino acid variations as virulence markers in VHSV-derived mortality. Statement of relevance1. Recently VHSV has infected >80 kinds of fish species. The number of infection host of VHSV is rapidly increased. Among the VHSV-infected fishes, olive flounder is one of the most important fish of aquaculture in Korea, Japan, and China. In every year, we found the massive VHSV-derived mortality of olive flounder aquaculture in Korea. To overcome this VHSV pathogenesis, we need to identify high-pathogenic VHSV appearance at aquaculture in earlier time.2. Since VHSV gene is highly mutated in time-dependent manner, the efficient vaccine for VHSV-derived disease prevention is not well achieved. Therefore, the other approaches for prevention of epidemics by VHSV should be required. Our research goal is to develop prevention of epidemics propagation of VHSV infection by high pathogenic VHSV prediction with gene variation analysis.3. In this study, we identified several gene variations of VHSV NV protein from 13 VHSV specimens of severe VHS-showing olive flounders in Korea. The gene variations are associated with amino acid changes of VHSV NV proteins. The amino acid variants of NV protein show the inhibitory activity of fish cellular energy (ATP) generation. The cellular energy deprivation might give a big impact on fish health and mortality. The DNA mutation on specific amino acid changes is possibly applied to develop the PCR diagnosis system for early detection of high pathogenic VHSV appearance. The early pathogenic gene diagnosis of VHSV gives more efficient prevention of epidemics.

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.

Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.

Drp1 Loss-of-function Reduces Cardiomyocyte Oxygen Dependence Protecting the Heart From Ischemia-reperfusion Injury

Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.