Isoprenaline modified the lipidomic profile and reduced ß-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome.

Recent studies have suggested a pathogenetic link between impaired mitochondria and Takotsubo syndrome (TTS), which is closely connected with catecholamine overstimulation, poor outcomes, and changes in lipid metabolism. We investigated the changes in lipid metabolism at the level of fatty acid ß-oxidation and changes in the intracellular lipidomic spectrum. The immortalized cell line of HL-1 cardiomyocytes was used in this study as an established in vitro model of TTS. The cells were exposed to the non-selective ß-agonist isoprenaline (ISO) for acute (2 h) and prolonged (24 h) periods. We investigated the impact on mitochondrial adenosine 5'-triphosphate (ATP) production and ß-oxidation using real-time cell metabolic analysis, total lipid content, and changes in the lipidomic spectrum using high-performance liquid chromatography (HPLC) and mass spectrometry. Furthermore, modifications of selected lipid transporters were determined using real-time - polymerase chain reaction (RT-PCR) and/or Western blot techniques. By choosing this wide range of targets, we provide a detailed overview of molecular changes in lipid metabolism during catecholamine overstimulation. The present study demonstrates that acute exposure to ISO decreased ATP production by up to 42.2%, and prolonged exposure to ISO decreased ß-oxidation by 86.4%. Prolonged exposure to ISO also increased lipid accumulation by 4%. Lipid spectrum analysis of prolonged exposure to ISO showed a reduced concentration of cardioprotective and an increased concentration of lipotoxic lipid molecules during long-term exposure. Decreased lipid utilization can lead to higher intracellular lipid accumulation and the formation of lipotoxic molecules. Changes in the lipid spectrum can induce pathophysiological signaling pathways leading to cardiomyocyte remodeling or apoptosis. Thus, changes in lipid metabolism induced by excessive doses of catecholamines may cause TTS and contribute to a progression of heart failure, which is at increased risk after a TTS episode.

Hypoxia-Induced Sarcoplasmic Reticulum Ca2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes.

BACKGROUND: To assess whether hypoxia, as can be found in obstructive sleep apnea syndrome, is causally associated with the development of heart failure through a direct effect on calcium leakage from the sarcoplasmic reticulum. METHODS: The impact of hypoxia on sarcoplasmic reticulum calcium leakage and expres- sion of RyR2 (ryanodine receptor2) and SERC2a (sarcoplasmic reticulum Ca2+ATPase 2a) was investigated together with the outcomes of JTV-519 and S107 treatment. HL-1 car- diomyocytes were cultured for 7 days on gas-permeable cultureware under control (12% O2) or hypoxic (1% O2) conditions with or without JTV-519 or S107. SRCL was assessed using a Fluo-5N probe. Gene and protein expression was analyzed using qPCR and western blotting. RESULTS: Hypoxic exposure increased sarcoplasmic reticulum calcium leakage by 39% and reduced RyR2 gene expression by 52%. No effect on RyR2 protein expression was observed. Treatment with 1µM JTV-519 reduced sarcoplasmic reticulum calcium leakage by 52% and 35% under control and hypoxic conditions, respectively. Administration of 1 µM JTV-519 increased RyR2 gene expression by 89% in control conditions. No effect on SRCL, RyR2, or SERC2a gene, or protein expression was observed with S107 treatment. CONCLUSION: Hypoxia increased sarcoplasmic reticulum calcium leakage which was ame- liorated by JTV-519 treatment independently of gene or protein expression. JTV-519 rep- resents a possible treatment for obstructive sleep apnea-associated HF.

Hypoxia Induces Saturated Fatty Acids Accumulation and Reduces Unsaturated Fatty Acids Independently of Reverse Tricarboxylic Acid Cycle in L6 Myotubes.

Obstructive sleep apnea syndrome, characterized by repetitive episodes of tissue hypoxia, is associated with several metabolic impairments. Role of fatty acids and lipids attracts attention in its pathogenesis for their metabolic effects. Parallelly, hypoxia-induced activation of reverse tricarboxylic acid cycle (rTCA) with reductive glutamine metabolism provides precursor molecules for de novo lipogenesis. Gas-permeable cultureware was used to culture L6-myotubes in chronic hypoxia (12%, 4% and 1% O2) with 13C labelled glutamine and inhibitors of glutamine uptake or rTCA-mediated lipogenesis. We investigated changes in lipidomic profile, 13C appearance in rTCA-related metabolites, gene and protein expression of rTCA-related proteins and glutamine transporters, glucose uptake and lactate production. Lipid content increased by 308% at 1% O2, predominantly composed of saturated fatty acids, while triacylglyceroles containing unsaturated fatty acids and membrane lipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositol) decreased by 20-70%. rTCA labelling of malate, citrate and 2-hydroxyglutarate increased by 4.7-fold, 2.2-fold and 1.9-fold in 1% O2, respectively. ATP-dependent citrate lyase inhibition in 1% O2 decreased lipid amount by 23% and increased intensity of triacylglyceroles containing unsaturated fatty acids by 56-80%. Lactate production increased with hypoxia. Glucose uptake dropped by 75% with progression of hypoxia from 4% to 1% O2. Protein expression remained unchanged. Altogether, hypoxia modified cell metabolism leading to lipid composition alteration and rTCA activation.

Autophagy-related proteases accompany the transition of pre-chondrogenic cells into chondroblasts.

BACKGROUND: Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium. METHODS: PCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture. RESULTS: Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression. CONCLUSIONS: The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

Multi-Hollow Surface Dielectric Barrier Discharge for Bacterial Biofilm Decontamination.

The plasma-activated gas is capable of decontaminating surfaces of different materials in remote distances. The effect of plasma-activated water vapor on Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli biofilm contamination was investigated on the polypropylene nonwoven textile surface. The robust and technically simple multi-hollow surface dielectric barrier discharge was used as a low-temperature atmospheric plasma source to activate the water-based medium. The germicidal efficiency of short and long-time exposure to plasma-activated water vapor was evaluated by standard microbiological cultivation and fluorescence analysis using a fluorescence multiwell plate reader. The test was repeated in different distances of the contaminated polypropylene nonwoven sample from the surface of the plasma source. The detection of reactive species in plasma-activated gas flow and condensed activated vapor, and thermal and electrical properties of the used plasma source, were measured. The bacterial biofilm decontamination efficiency increased with the exposure time and the plasma source power input. The log reduction of viable biofilm units decreased with the increasing distance from the dielectric surface.

Enzybiotics LYSSTAPH-S and LYSDERM-S as Potential Therapeutic Agents for Chronic MRSA Wound Infections.

Antibacterial antibiotic therapy has played an important role in the treatment of bacterial infections for almost a century. The increasing resistance of pathogenic bacteria to antibiotics leads to an attempt to use previously neglected antibacterial therapies. Here we provide information on the two recombinantly modified antistaphylococcal enzymes derived from lysostaphin (LYSSTAPH-S) and endolysin (LYSDERM-S) derived from kayvirus 812F1 whose target sites reside in the bacterial cell wall. LYSSTAPH-S showed a stable antimicrobial effect over 24-h testing, even in concentrations lower than 1 µg/mL across a wide variety of epidemiologically important sequence types (STs) of methicillin-resistant Staphylococcus aureus (MRSA), especially in the stationary phase of growth (status comparable to chronic infections). LYSDERM-S showed a less potent antimicrobial effect that lasted only a few hours at concentrations of 15 µg/mL and higher. Our data indicate that these antimicrobial enzymes could be of substantial help in the treatment of chronic MRSA wound infections.

Monitoring of kynurenine pathway metabolites, neurotransmitters and their metabolites in blood plasma and brain tissue of individuals with latent toxoplasmosis.

The aim of the presented work was to develop a highly sensitive, accurate and rapid analytical method for the determination of concentration levels of tryptophan and its metabolites of kynurenine catabolic pathway, as well as neurotransmitters and their metabolites in complex biological matrices (brain tissue and blood plasma). The developed analytical method consists of analytes separation from the biological matrices by protein precipitation (blood plasma) or solvent extraction (brain tissue), derivatization of the analytes and their detection by high-performance liquid chromatography combined with mass spectrometry. Individual steps of the whole process were optimized and the method was validated in the terms of selectivity, linearity (R2≥0.980), precision (RSD ≤ 13.3%), recovery (≥82.0%), limit of detection (1.8 ng/mL of blood plasma, 2.2 pg/mg of brain tissue) and limit of quantification (2.5 ng/mL of blood plasma, 2.8 pg/mg of brain tissue). The method was subsequently verified by an animal study, where the concentration levels of the analytes in biological matrices (blood plasma and brain tissue) of T. gondii - infected rats and control animals were compared. All the data obtained from the animal study were statistically evaluated. Increased concentration levels of kynurenine catabolic pathway metabolites (e.g. kynurenine, 3-hydroxykynurenine, quinolinic acid) were observed in the case of T. gondii - infected rats in contrast to the control group. The opposite effect was determined in the case of serotonin and its metabolite 5-hydroxyindoleacetic acid, where higher concentration levels were found in blood plasma of healthy subjects. Finally, Principal Component Analysis (PCA) was utilized for a score plot formation. PCA score plots have demonstrated the similarities of individuals within each group and the differences among the groups.