A rare form of LIM domain-binding protein 3 (LDB3) mutation causes hypertrophic cardiomyopathy and myofibrillar myopathy type 4.

Polymorphisms in LDB3 gene can cause various forms of cardiomyopathy and myofibrillar myopathy 4 (MM4). Patient described in this study presented with a hypertrophic cardiomyopathy (HCM) and distal myopathy suggestive of myofibrillar myopathy 4. Genetic analysis using the TruSight Cardio Sequencing Kit (Illumina) revealed suspected LDB3 variant (c.1435G>A, p.(Gly479Arg)). This is the first case in which polymorphism in LDB3 gene is likely responsible for MM4 and HCM in the same patient.

Mitochondrial unfolded protein response, mitophagy and other mitochondrial quality control mechanisms in heart disease and aged heart.

Mitochondria are involved in crucial homeostatic processes in the cell: the production of adenosine triphosphate and reactive oxygen species, and the release of pro-apoptotic molecules. Thus, cell survival depends on the maintenance of proper mitochondrial function by mitochondrial quality control. The most important mitochondrial quality control mechanisms are mitochondrial unfolded protein response, mitophagy, biogenesis, and fusion-fission dynamics. This review deals with mitochondrial quality control in heart diseases, especially myocardial infarction and heart failure. Some previous studies have demonstrated that the activation of mitochondrial quality control mechanisms may be beneficial for the heart, while others have shown that it may lead to heart damage. Our aim was to describe the mechanisms by which mitochondrial quality control contributes to heart protection or damage and to provide evidence that may resolve the seemingly contradictory results from the previous studies.

Targeted Modification of Mitochondrial ROS Production Converts High Glucose-Induced Cytotoxicity to Cytoprotection: Effects on Anesthetic Preconditioning.

Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose-driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes. We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential, and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real-time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay. High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption, and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP, and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose-induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose-induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC-induced mitochondrial depolarization, delay in mPTP opening and cytoprotection. In conclusion, high glucose-induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose-induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. J. Cell. Physiol. 232: 216-224, 2017. © 2016 Wiley Periodicals, Inc.

Cardioprotection during diabetes: the role of mitochondrial DNA.

BACKGROUND: Diabetes alters mitochondrial bioenergetics and consequently disrupts cardioprotective signaling. The authors investigated whether mitochondrial DNA (mtDNA) modulates anesthetic preconditioning (APC) and cardiac susceptibility to ischemia-reperfusion injury by using two strains of rats, both sharing nuclear genome of type 2 diabetes mellitus (T2DN) rats and having distinct mitochondrial genomes of Wistar and fawn-hooded hypertensive (FHH) rat strains (T2DN(mtWistar) and T2DN(mtFHH), respectively). METHODS: Myocardial infarct size was measured in Wistar, T2DN(mtWistar), and T2DN(mtFHH) rats with or without APC (1.4% isoflurane) in the presence or absence of antioxidant N-acetylcysteine. Flavoprotein fluorescence intensity, a marker of mitochondrial redox state, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity, a marker of reactive oxygen species generation, and mitochondrial permeability transition pore opening were assessed in isolated rat ventricular cardiomyocytes with or without isoflurane (0.5 mmol/l). RESULTS: Myocardial infarct size was decreased by APC in Wistar and T2DN(mtWistar) rats (to 42 ± 6%, n = 8; and 44 ± 7%, n = 8; of risk area, respectively) compared with their respective controls (60 ± 3%, n = 6; and 59 ± 9%, n = 7), but not in T2DN(mtFHH) rats (60 ± 2%, n = 8). N-acetylcysteine applied during isoflurane treatment restored APC in T2DN(mtFHH) (39 ± 6%, n = 7; and 38 ± 5%, n = 7; 150 and 75 mg/kg N-acetylcysteine, respectively), but abolished protection in control rats (54 ± 8%, n = 6). Similar to the data on infarct size, APC delayed mitochondrial permeability transition pore opening in T2DN(mtWistar) but not in T2DN(mtFHH) cardiomyocytes. Isoflurane increased flavoprotein and 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein fluorescence intensity in all rat strains, with the greatest effect in T2DN(mtFHH) cardiomyocytes. CONCLUSION: Differences in the mitochondrial genome modulate isoflurane-induced generation of reactive oxygen species which translates into differential susceptibility to APC and ischemia-reperfusion injury in diabetic rats.

Apolipoprotein A-1 mimetic D-4F enhances isoflurane-induced eNOS signaling and cardioprotection during acute hyperglycemia.

Acute hyperglycemia (AHG) decreases the availability of nitric oxide (NO) and impairs anesthetic preconditioning (APC)-elicited protection against myocardial infarction. We investigated whether D-4F, an apolipoprotein A-1 mimetic, rescues the myocardium by promoting APC-induced endothelial NO signaling during AHG. Myocardial infarct size was measured in mice in the absence or presence of APC [isoflurane (1.4%)] with or without AHG [dextrose (2 g/kg ip)] and D-4F (0.12 or 0.6 mg/kg ip). NO production, superoxide generation, protein compartmentalization, and posttranslational endothelial NO synthase (eNOS) modifications were assessed in human coronary artery endothelial cells cultured in 5.5 or 20 mM glucose with or without isoflurane (0.5 mM) in the presence or absence of D-4F (0.5 µg/ml). Myocardial infarct size was significantly decreased by APC (36 ± 3% of risk area) compared with control (54 ± 3%) in the absence, but not presence, of AHG (49 ± 4%). D-4F restored the cardioprotective effect of APC during AHG (36 ± 3% and 30 ± 3%, 0.12 and 0.6 mg/kg, respectively), although D-4F alone had no effect on infarct size (53 ± 3%). Isoflurane promoted caveolin-1 and eNOS compartmentalization within endothelial cell caveolae and eNOS dimerization, concomitant with increased NO production (411 ± 28 vs. 68 ± 10 pmol/mg protein in control). These actions were attenuated by AHG (NO production: 264 ± 18 pmol/mg protein). D-4F reduced superoxide generation and enhanced caveolin-1 and eNOS caveolar compartmentalization and posttranslational eNOS modifications, thus restoring NO production during isoflurane and AHG (418 ± 36 pmol/mg protein). In conclusion, D-4F restored the cardioprotective effect of APC during AHG, possibly by decreasing superoxide generation, which promoted isoflurane-induced eNOS signaling and NO biosynthesis.

Preconditioning by isoflurane elicits mitochondrial protective mechanisms independent of sarcolemmal KATP channel in mouse cardiomyocytes.

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning. Changes in mitochondrial bioenergetics influence the sarcolemmal ATP-sensitive K (sarcKATP) channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild-type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial reactive oxygen species production, and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180% ± 14% and 190% ± 15% and reactive oxygen species production to 118% ± 2% and 124% ± 6% of baseline in WT and Kir6.2 KO myocytes, respectively. Tetramethylrhodamine ethyl ester fluorescence decreased to 84% ± 6% in WT and to 86% ± 4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31% ± 1% to 21% ± 1% in WT and from 44% ± 2% to 35% ± 2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases the sensitivity of intact cardiomyocytes to oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in anesthetic-induced preconditioning by isoflurane.

Predictors of prolonged hospitalization of COVID-19 patients.

PURPOSE: Despite the importance of hospital bed network during the pandemic, there are scarce data available regarding factors predictive of prolonged length of hospitalization of COVID-19 patients. METHODS: We retrospectively analyzed a total of 5959 consecutive hospitalized COVID-19 patients in period 3/2020-6/2021 from a single tertiary-level institution. Prolonged hospitalization was defined as hospital stay > 21 days to account for mandatory isolation period in immunocompromised patients. RESULTS: Median length of hospital stay was 10 days. A total of 799 (13.4%) patients required prolonged hospitalization. Factors that remained independently associated with prolonged hospitalization in multivariate analysis were severe or critical COVID-19 and worse functional status at the time of hospital admission, referral from other institutions, acute neurological, acute surgical and social indications for admission vs admission indication of COVID-19 pneumonia, obesity, chronic liver disease, hematological malignancy, transplanted organ, occurrence of venous thromboembolism, occurrence of bacterial sepsis and occurrence of Clostridioides difficile infection during hospitalization. Patients requiring prolonged hospitalization experienced higher post-hospital discharge mortality (HR = 2.87, P < 0.001). CONCLUSIONS: Not only severity of COVID-19 clinical presentation but also worse functional status, referral from other hospitals, certain indications for admission, certain chronic comorbidities, and complications that arise during hospital stay independently reflect on the need of prolonged hospitalization. Development of specific measures aimed at improvement of functional status and prevention of complications might reduce the length of hospitalization.

Marked hyperglycemia attenuates anesthetic preconditioning in human-induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Anesthetic preconditioning protects cardiomyocytes from oxidative stress-induced injury, but it is ineffective in patients with diabetes mellitus. To address the role of hyperglycemia in the inability of diabetic individuals to be preconditioned, we used human cardiomyocytes differentiated from induced pluripotent stem cells generated from patients with or without type 2 diabetes mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of preconditioning in varying glucose conditions (5, 11, and 25 mM). METHODS: Induced pluripotent stem cells were induced to generate cardiomyocytes by directed differentiation. For subsequent studies, cardiomyocytes were identified by genetic labeling with enhanced green fluorescent protein driven by a cardiac-specific promoter. Cell viability was analyzed by lactate dehydrogenase assay. Confocal microscopy was utilized to measure opening of the mitochondrial permeability transition pore and the mitochondrial adenosine 5'-triphosphate-sensitive potassium channels. RESULTS: Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane triggered mitochondrial adenosine-5'-triphosphate-sensitive potassium channel opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM glucose; 25 mM glucose disrupted anesthetic preconditioning-mediated protection in DM-iPSC- and N-iPSC-CMs. CONCLUSIONS: The opening of mitochondrial adenosine 5'-triphosphate-sensitive potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and patients with a specific disease, such as diabetes in this study, open possibilities in studying genotype- and phenotype-related pathologies in a human-relevant model.

Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes.

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.

Activator of G protein signaling 3 promotes epithelial cell proliferation in PKD.

The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.

Human C2a and C6a iPSC lines and H9 ESC line have less efficient cardiomyogenesis than H1 ESC line: Beating enhances cardiac differentiation.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) need to be thoroughly characterized to exploit their potential advantages in various aspects of biomedicine. The aim of this study was to compare the efficiency of cardiomyogenesis of two hiPSCs and two human embryonic stem cell (hESC) lines by genetic living cardiomyocyte labeling. We also analyzed the influence of spontaneous beating on cardiac differentiation. METHODS: H1 and H9 hESC lines and C2a and C6a hiPSC lines were induced into in vitro directed cardiac differentiation. Cardiomyogenesis was evaluated by the analysis of cell cluster beating, cardiac protein expression by immunocytochemistry, ability of cells to generate calcium transients, and cardiomyocyte quantification by the myosin light chain 2v-enhanced green fluorescent protein gene construct delivered with a lentiviral vector. RESULTS: Differentiation of all cell lines yielded spontaneously beating cell clusters, indicating the presence of functional cardiomyocytes. After the cell dissociation, H1-hESC-derived cardiomyocytes exhibited spontaneous calcium transients, corresponding to autonomous electrical activity and displayed ability to transmit them between the cells. Differentiated hESC and hiPSC cells exhibited striated sarcomeres and expressed cardiac proteins sarcomeric α-actinin and cardiac troponin T. Cardiomyocytes were the most abundant in differentiated H1 hESC line (20% more than in other tested lines). In all stem cell lines, cardiomyocyte enrichment was greater in beating than in non-beating cell clusters, irrespective of cardiomyogenesis efficiency. CONCLUSION: Although C2a and C6a hiPSC and H9 hESC lines exhibited efficient cardiomyogenesis, H1 hESC line yielded the greatest cardiomyocyte enrichment of all tested lines. Beating of cell clusters promotes cardiomyogenesis in tested hESCs and hiPSCs.

Prognostic value of circulating Bcl-2 and anti-p53 antibodies in patients with breast cancer: A long term follow-up (17.5 years).

BACKGROUND: Apoptosis inhibition is a major tumorigenic factor. Bcl-2 dysregulation and TP53 mutation status, which may correlate with autoantibody generation, contribute to impaired apoptosis. OBJECTIVE: This study aimed to investigate the prognostic value of circulating Bcl-2 and anti-p53 antibodies (p53Abs) in a 17.5-year follow-up of breast cancer patients. We also analyzed the correlations of Bcl-2 and p53Abs with various clinicopathological parameters in order to assess their impact on tumor aggressiveness. METHODS: Serum Bcl-2 and p53Abs levels were analyzed by the enzyme-linked immunosorbent assay (ELISA) in 82 patients with invasive breast cancer and twenty individuals without malignancy. RESULTS: Serum Bcl-2 and p53Abs levels in breast cancer patients were significantly higher than those in controls. Patients with high levels of Bcl-2 (cut-off 200 U/ml) had a poorer prognosis (17.5-year survival) than those with lower Bcl-2 values. In combined analysis the subgroup of patients with elevated p53Abs (cut-off 15 U/ml) and elevated Bcl-2 (cut-offs 124 U/ml and 200 U/ml) had the worse prognosis in 17.5-year survival. In correlation analysis p53Abs and Bcl-2 were associated with unfavorable clinicopathological parameters. CONCLUSIONS: Our results suggest that breast cancer patients with high serum levels of p53Abs and Bcl-2 present an especially unfavorable group in a long follow-up.

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+.

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca(2+) overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca(2+), and mPTP opening. The magnitude of APC-induced decrease in DeltaPsi(m) was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in DeltaPsi(m). In cardiomyocytes, DeltaPsi(m), ROS, mPTP opening, and cytosolic and mitochondrial Ca(2+) were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca(2+) uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca(2+) uptake. However, in stressed cardiomyocytes, a similar decrease in DeltaPsi(m) attenuated both cytosolic and mitochondrial Ca(2+) accumulation. In conclusion, a partial decrease in DeltaPsi(m) underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in DeltaPsi(m) primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca(2+) uptake.

Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors.

BACKGROUND: The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. RESULTS: Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. CONCLUSIONS: Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

BACKGROUND: Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning. METHODS: H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters. RESULTS: Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening. CONCLUSIONS: APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

Mitochondrial ROS Induce Partial Dedifferentiation of Human Mesothelioma via Upregulation of NANOG.

The expression of pluripotency factors is a key regulator of tumor differentiation status and cancer stem cells. The purpose of this study was to examine the expression of pluripotency factors and differentiation status of human mesothelioma and the role of mitochondria in their regulation. We tested the expression of OCT4/POU5F1, NANOG, SOX2, PI3K-AKT pathway and BCL2 genes and proteins in 65 samples of human mesothelioma and 19 samples of normal mesothelium. Mitochondrial membrane potential, reactive oxygen species (ROS) generation and expression of pluripotency factors were also tested in human mesothelioma cell line. Human mesothelium and mesothelioma expressed SOX2, NANOG, PI3K and AKT genes and proteins and POU5F1 gene, whereby NANOG, SOX2 and phosphorylated (activated) AKT were upregulated in mesothelioma. NANOG protein expression was elevated in less differentiated samples of human mesothelioma. The expression of genes of PI3K-AKT pathway correlated with pluripotency factor genes. Mesothelioma cells had functional, but depolarized mitochondria with large capacity to generate ROS. Mitochondrial ROS upregulated NANOG and mitoTEMPO abrogated it. In conclusion, human mesothelioma displays enhanced expression of NANOG, SOX2 and phosphorylated AKT proteins, while elevated NANOG expression correlates with poor differentiation of human mesothelioma. Mitochondria of mesothelioma cells have a large capacity to form ROS and thereby upregulate NANOG, leading to dedifferentiation of mesothelioma.

Differentiating Between Malignant Mesothelioma and Other Pleural Lesions Using Fourier Transform Infrared Spectroscopy.

Histopathology, despite being the gold standard as a diagnostic tool, does not always provide a correct diagnosis for different pleural lesions. Although great progress was made in this field, the problem to differentiate between reactive and malignant pleural lesions still stimulates the search for additional diagnostic tools. Our research using vibrational spectroscopy and principal component analysis (PCA) statistical modeling represents a potentially useful tool to approach the problem. The objective method this paper explores is based on the correlation between different types of pleural lesions and their vibrational spectra. Obtained tissue spectra recorded by infrared spectroscopy allowed us to categorize spectra in different groups using a created PCA statistical model. The PCA model was built using tissues of known pathology as the model group. The validation samples were then used to confirm the functionality of our PCA model. Student's t-test was also used for comparing samples in paired groups. The PCA model was able to clearly differentiate the spectra of mesothelioma, metastasis and reactive changes (inflammation), and place them in discrete groups. Thus, we showed that Fourier transform infrared spectroscopy combined with PCA can differentiate pleural lesions with high sensitivity and specificity. This new approach could contribute in objectively differentiating specific pleural lesions, thus helping pathologists to better diagnose difficult pleural samples but also could shed additional light into the biology of malignant pleural mesothelioma.

Anesthetic-induced preconditioning delays opening of mitochondrial permeability transition pore via protein Kinase C-epsilon-mediated pathway.

BACKGROUND: Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). This study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-delta (rottlerin) and PKC-epsilon (myristoylated PKC-epsilon V1-2 peptide), and the mPTP opening time was measured by using confocal microscopy. Ca-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-epsilon was assessed in APC and control cardiomyocytes by Western blotting. RESULTS: In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261 +/- 26 s APC vs. 216 +/- 27 s control; P < 0.05), and chelerythrine abolished this delay to 213 +/- 22 s. The effect of isoflurane was also abolished when PKC-epsilon inhibitor was applied (210 +/- 22 s) but not in the presence of PKC-delta inhibitor (269 +/- 31 s). Western blotting revealed translocation of PKC-epsilon toward mitochondria in APC cells. The Ca concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45 +/- 8 microM x mg control vs. 64 +/- 8 microM x mg APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. CONCLUSION: APC induces delay of mPTP opening through PKC-epsilon mediated inhibition of mPTP opening, but not through PKC-delta. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Differences in production of reactive oxygen species and mitochondrial uncoupling as events in the preconditioning signaling cascade between desflurane and sevoflurane.

BACKGROUND: Signal transduction cascade of anesthetic-induced preconditioning has been extensively studied, yet many aspects of it remain unsolved. Here, we investigated the roles of reactive oxygen species (ROS) and mitochondrial uncoupling in cardiomyocyte preconditioning by two modern volatile anesthetics: desflurane and sevoflurane. METHODS: Adult rat ventricular cardiomyocytes were isolated enzymatically. The preconditioning potency of desflurane and sevoflurane was assessed in cell survival experiments by evaluating myocyte protection from the oxidative stress-induced cell death. ROS production and flavoprotein fluorescence, an indicator of flavoprotein oxidation and mitochondrial uncoupling, were monitored in real time by confocal microscopy. The functional aspect of enhanced ROS generation by the anesthetics was assessed in cell survival and confocal experiments using the ROS scavenger Trolox. RESULTS: Preconditioning of cardiomyocytes with desflurane or sevoflurane significantly decreased oxidative stress-induced cell death. That effect coincided with increased ROS production and increased flavoprotein oxidation detected during acute myocyte exposure to the anesthetics. Desflurane induced significantly greater ROS production and flavoprotein oxidation than sevoflurane. ROS scavenging with Trolox abrogated preconditioning potency of anesthetics and attenuated flavoprotein oxidation. CONCLUSION: Preconditioning with desflurane or sevoflurane protects isolated rat cardiomyocytes from oxidative stress-induced cell death. Scavenging of ROS abolishes the preconditioning effect of both anesthetics and attenuates anesthetic-induced mitochondrial uncoupling, suggesting a crucial role for ROS in anesthetic-induced preconditioning and implying that ROS act upstream of mitochondrial uncoupling. Desflurane exhibits greater effect on stimulation of ROS production and mitochondrial uncoupling than sevoflurane.