Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys.

Introduction: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms. Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days. Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues. Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications.

SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells.

The coronavirus transforms the cytoplasm of susceptible cells to support virus replication. It also activates autophagy-like processes, the role of which is not well understood. Here, we studied SARS-CoV-2-infected Vero E6 cells using transmission electron microscopy and autophagy PCR array. After 6-24 h post-infection (hpi), the cytoplasm of infected cells only contained double-membrane vesicles, phagophores, and phagosomes engulfing virus particles and cytoplasmic debris, including damaged mitochondria. The phagosomes interacted with the viral nucleoprotein complex, virus particles, mitochondria, and lipid droplets. The phagosomes transformed into egress vacuoles, which broke through the plasmalemma and discharged the virus particles. The Vero E6 cells exhibited pronounced virus replication at 6 hpi, which stabilized at 18-24 hpi at a high level. The autophagy PCR array tests revealed a significant upregulation of 10 and downregulation of 8 autophagic gene markers out of 84. Altogether, these results underline the importance of autophagy-like processes for SARS-CoV-2 maturation and egress, and point to deviations from a canonical autophagy response.

Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only.

BACKGROUND: The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects. METHODS: To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs. RESULTS: Cardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected. CONCLUSION: Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

Energetic Interactions Between Subcellular Organelles in Striated Muscles.

Adult striated muscle cells present highly organized structure with densely packed intracellular organelles and a very sparse cytosol accounting for only few percent of cell volume. These cells have a high and fluctuating energy demand that, in continuously working oxidative muscles, is fulfilled mainly by oxidative metabolism. ATP produced by mitochondria should be directed to the main energy consumers, ATPases of the excitation-contraction system; at the same time, ADP near ATPases should rapidly be eliminated. This is achieved by phosphotransfer kinases, the most important being creatine kinase (CK). Specific CK isoenzymes are located in mitochondria and in close proximity to ATPases, forming efficient energy shuttle between these structures. In addition to phosphotransfer kinases, ATP/ADP can be directly channeled between mitochondria co-localized with ATPases in a process called "direct adenine nucleotide channeling, DANC." This process is highly plastic so that inactivation of the CK system increases the participation of DANC to energy supply owing to the rearrangement of cell structure. The machinery for DANC is built during postnatal development in parallel with the increase in mitochondrial mass, organization, and complexification of the cell structure. Disorganization of cell architecture remodels the mitochondrial network and decreases the efficacy of DANC, showing that this process is intimately linked to cardiomyocyte structure. Accordingly, in heart failure, disorganization of the cell structure along with decrease in mitochondrial mass reduces the efficacy of DANC and together with alteration of the CK shuttle participates in energetic deficiency contributing to contractile failure.

Structural variability of dyads relates to calcium release in rat ventricular myocytes.

Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into 'compact' or 'loose' types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into 'early' and 'late' components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.

SIRT1 Protects the Heart from ER Stress-Induced Injury by Promoting eEF2K/eEF2-Dependent Autophagy.

Many recent studies have demonstrated the involvement of endoplasmic reticulum (ER) stress in the development of cardiac diseases and have suggested that modulation of ER stress response could be cardioprotective. Previously, we demonstrated that the deacetylase Sirtuin 1 (SIRT1) attenuates ER stress response and promotes cardiomyocyte survival. Here, we investigated whether and how autophagy plays a role in SIRT1-afforded cardioprotection against ER stress. The results revealed that protective autophagy was initiated before cell death in response to tunicamycin (TN)-induced ER stress in cardiac cells. SIRT1 inhibition decreased ER stress-induced autophagy, whereas its activation enhanced autophagy. In response to TN- or isoproterenol-induced ER stress, mice deficient for SIRT1 exhibited suppressed autophagy along with exacerbated cardiac dysfunction. At the molecular level, we found that in response to ER stress (i) the extinction of eEF2 or its kinase eEF2K not only reduced autophagy but further activated cell death, (ii) inhibition of SIRT1 inhibited the phosphorylation of eEF2, (iii) eIF2α co-immunoprecipitated with eEF2K, and (iv) knockdown of eIF2α reduced the phosphorylation of eEF2. Our results indicate that in response to ER stress, SIRT1 activation promotes cardiomyocyte survival by enhancing autophagy at least through activation of the eEF2K/eEF2 pathway.

Surface coating determines the inflammatory potential of magnetite nanoparticles in murine renal podocytes and mesangial cells.

Drug-induced nephrotoxicity is a frequent adverse event and a dose-limiting factor in patient treatment and is a leading cause of prospective drug attrition during pharmaceutical development. Despite the obvious benefits of nanotherapeutics in healthcare strategies, the clearance of imaging agents and nanocarriers from the body following their therapeutic or diagnostic application generates concerns about their safety for human health. Considering the potency of nanoparticles and their massive utilization in biomedicine the impact of magnetic nanoparticles (MNPs) on cells forming the filtration apparatus of the kidney was studied. Using primary mouse renal glomerular podocytes and mesangial cells, we investigated their response to exposure to magnetic nanoparticles coated with polyethylene glycol and bovine serum albumin. Cultured podocytes were more sensitive to MNPs than mesangial cells displaying signs of cell damage and stronger inflammatory response. Both types of MNPs induced the remodeling of actin fibers, affected the cell shape and triggered expression of inflammatory cytokines TNFα and IL-6 in podocytes. On the other hand, iNOS was induced in both renal cell types but only by MNPs with a polyethylene glycol coating. Our results have revealed that the type of cell and the type of nanoparticle coating might be the strongest determinants of cellular response toward nanoparticle exposure. Differences in susceptibility of cells to MNPs might be evident also between neighboring renal cell subpopulations integrally forming functional sub-units of this organ.

Kidney nanotoxicity studied in human renal proximal tubule epithelial cell line TH1.

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe3O4NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (>700 µg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 µg/ml) concentration. Fe3O4NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO2NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake.

Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats.

Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release.

Endoplasmic reticulum stress induces cardiac dysfunction through architectural modifications and alteration of mitochondrial function in cardiomyocytes.

Aims: Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. Methods and results: In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption. Conclusions: Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth.

Although in flies the atypical cadherin Fat is an upstream regulator of Hippo signalling, the closest mammalian homologue, Fat4, has been shown to regulate tissue polarity rather than growth. Here we show in the mouse heart that Fat4 modulates Hippo signalling to restrict growth. Fat4 mutant myocardium is thicker, with increased cardiomyocyte size and proliferation, and this is mediated by an upregulation of the transcriptional activity of Yap1, an effector of the Hippo pathway. Fat4 is not required for the canonical activation of Hippo kinases but it sequesters a partner of Yap1, Amotl1, out of the nucleus. The nuclear translocation of Amotl1 is accompanied by Yap1 to promote cardiomyocyte proliferation. We, therefore, identify Amotl1, which is not present in flies, as a mammalian intermediate for non-canonical Hippo signalling, downstream of Fat4. This work uncovers a mechanism for the restriction of heart growth at birth, a process which impedes the regenerative potential of the mammalian heart.

The flashlights on a distinct role of protein kinase C δ: Phosphorylation of regulatory and catalytic domain upon oxidative stress in glioma cells.

Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.

Assessing light-independent effects of hypericin on cell viability, ultrastructure and metabolism in human glioma and endothelial cells.

Cell exposure to light-independent effects of photosensitizers (PS) used in PDT is clinically relevant when PS affect the pro-apoptotic cascade. In many malignant cells, Hypericin (Hyp) has PS displayed light-dependent anti-proliferative and cytotoxic effects with no cytotoxicity in the dark. Recent studies have shown that Hyp also exhibited light-independent cytotoxic effects in a wide range of concentrations. The molecular mechanisms underlying Hyp light-independent (dark) toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and these mechanisms are not understood in detail. Here, we demonstrate that in human glioma and endothelial cells, Hyp displayed light-independent effects at several sub-cellular levels (ultrastructure, mitochondria function and metabolism, and protein synthesis). Taking together previously published and our present results, the findings strongly suggest that Hyp light independent effects: (i) depend on the cell type and metabolism; (ii) underlying molecular mechanisms are due to Hyp interaction with the multiple target molecules including Bcl2 family of proteins. In addition, the findings suggest that Hyp without illumination can be explored as an adjuvant therapeutic drug in combination with chemo- or radiation cancer therapy.

Ultrastructural remodelling of slow skeletal muscle fibres in creatine kinase deficient mice: a quantitative study.

Creatine kinase content, isoform distribution, and participation in energy transfer are muscle type specific. We analysed ultrastructural changes in slow muscle fibres of soleus due to invalidation of creatine kinase (CK) to reveal a difference in the remodelling strategy in comparison with fast muscle fibres of gastrocnemius published previously. We have employed the stereological method of vertical sections and electron microscopy of soleus muscles of wild type (WT) and CK-/- mice. The mitochondrial volume density was 1.4× higher but that of sarcoplasmic reticulum (SR) was almost 5× lower in slow CK-/- muscles fibres than in WT fibres. The volume density of terminal cisterns and of t-tubules was also lower in CK-/- than in WT fibres. The analysis of organelle environment revealed increased neighbourhood of mitochondria and A-bands that resulted from the decreased volume density of SR, from relocation of mitochondria along myofibrils, and from intrusion of mitochondria to myofibrils. These processes direct ATP supply closer to the contractile machinery. The decreased interaction between mitochondria and SR suggests reduced dependence of calcium uptake on oxidative ATP production. In conclusion, the architecture of skeletal muscle cells is under control of a cellular program that optimizes energy utilization specifically for a given muscle type.

Endosomes: guardians against [Ru(Phen)3]2+ photo-action in endothelial cells during in vivo pO2 detection?

Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO2) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(ii) hydrate ([Ru(Phen)3]2+) is a water soluble pO2 probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected [Ru(Phen)3]2+ decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of [Ru(Phen)3]2+. In vitro, we observed that [Ru(Phen)3]2+ enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination [Ru(Phen)3]2+ induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of [Ru(Phen)3]2+ leads to more marked ultrastructural changes than administration of [Ru(Phen)3]2+ only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of [Ru(Phen)3]2+ into the CAM model mostly leads to extracellular localization of [Ru(Phen)3]2+, while its topical application induces intracellular localization. We have shown in vivo that [Ru(Phen)3]2+ induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO2 measurements. This low phototoxicity is due to the fact that [Ru(Phen)3]2+ enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to [Ru(Phen)3]2+, survival or death, depends on its intracellular concentration and oxidation-reduction properties.

Mitochondrial chloride channels: electrophysiological characterization and pH induction of channel pore dilation.

Physiological and pathological functions of mitochondria are highly dependent on the properties and regulation of mitochondrial ion channels. There is still no clear understanding of the molecular identity, regulation, and properties of anion mitochondrial channels. The inner membrane anion channel (IMAC) was assumed to be equivalent to mitochondrial centum picosiemens (mCS). However, the different properties of IMAC and mCS channels challenges this opinion. In our study, we characterized the single-channel anion selectivity and pH regulation of chloride channels from purified cardiac mitochondria. We observed that channel conductance decreased in the order: Cl⁻ > Br⁻ > I⁻ > chlorate ≈ formate > acetate, and that gluconate did not permeate under control conditions. The selectivity sequence was Br⁻ ≥ chlorate ≥ I⁻ ≥ Cl⁻ ≥ formate ≈ acetate. Measurement of the concentration dependence of chloride conductance revealed altered channel gating kinetics, which was demonstrated by prolonged mean open time value with increasing chloride concentration. The observed mitochondrial chloride channels were in many respects similar to those of mCS, but not those of IMAC. Surprisingly, we observed that acidic pH increased channel conductance and that an increase of pH from 7.4 to 8.5 reduced it. The gluconate current appeared and gradually increased when pH decreased from pH 7.0 to 5.6. Our results indicate that pH regulates the channel pore diameter in such a way that dilation increases with more acidic pH. We assume this newly observed pH-dependent anion channel property may be involved in pH regulation of anion distribution in different mitochondrial compartments.

Mitochondrial dynamics in the adult cardiomyocytes: which roles for a highly specialized cell?

Mitochondrial dynamics is a recent topic of research in the field of cardiac physiology. The study of mechanisms involved in the morphological changes and in the mobility of mitochondria is legitimate since the adult cardiomyocytes possess numerous mitochondria which occupy at least 30% of cell volume. However, architectural constraints exist in the cardiomyocyte that limit mitochondrial movements and communication between adjacent mitochondria. Still, the proteins involved in mitochondrial fusion and fission are highly expressed in these cells and could be involved in different processes important for the cardiac function. For example, they are required for mitochondrial biogenesis to synthesize new mitochondria and for the quality-control of the organelles. They are also involved in inner membrane organization and may play a role in apoptosis. More generally, change in mitochondrial morphology can have consequences in the functioning of the respiratory chain, in the regulation of the mitochondrial permeability transition pore (MPTP), and in the interactions with other organelles. Furthermore, the proteins involved in fusion and fission of mitochondria are altered in cardiac pathologies such as ischemia/reperfusion or heart failure (HF), and appear to be valuable targets for pharmacological therapies. Thus, mitochondrial dynamics deserves particular attention in cardiac research. The present review draws up a report of our knowledge on these phenomena.

Morphological and functional characteristics of models of experimental myocardial injury induced by isoproterenol.

The animal models of myocardial injury induced by systemic ß-adrenergic receptor agonist administration represent an experimental approach of persisting interest. These models were found useful especially for studies of structural and functional adaptation of myocardium during the progression of cardiac adaptive response towards maladaptive hypertrophy and insufficiency. The pathological alterations induced by isoproterenol (ISO) do not develop evenly. The ISO models may contribute effectively to understanding of pathologies in signal transduction, energetics, excitability and contractility that may contribute concomitantly to cardiac dysfunction and heart failure. In this minireview we focused on the alterations in general characteristics and heart function as well as on the morphological changes of cardiomyocytes developed during ISO administration. The morphological alterations within the cellular macro- and microdomains correspond to the electrical remodelling and contractile dysfunction of ventricular myocardium that could be used to identify pathological changes ranging from hypertrophy to failing heart.

Down-regulation of OPA1 alters mouse mitochondrial morphology, PTP function, and cardiac adaptation to pressure overload.

AIMS: The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1(+/-) mice, having a 50% reduction in OPA1 protein expression in cardiac tissue. METHODS AND RESULTS: In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1(+/+). Electron and fluorescence microscopy revealed altered morphology of the Opa1(+/-) mice mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibres, mitochondrial functional properties were maintained, but direct energy channelling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction, Opa1(+/-) hearts demonstrated hypertrophy almost two-fold higher (P< 0.01) than in wild-type mice with altered ejection fraction (decrease in 43 vs. 22% in Opa1(+/+) mice, P< 0.05). CONCLUSIONS: These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.

Screening of mutant strain Streptomyces mediolani sp. AC37 for (-)-8-O-methyltetrangomycin production enhancement.

Streptomyces mediolani sp. AC37 was isolated from the root system of higher plant Taxus baccata and produced metabolite identified as (-)-8-O-methyltetrangomycin according to LC/MS/MS analysis. In our screening program for improvements of bioactive secondary metabolites from plant associate streptomycetes, mutation was used as a tool for the induction of genetic variations for selection of higher (-)-8-O-methyltetrangomycin producers of isolates. S. mediolani sp. AC37 was treated with UV irradiation and chemical mutagenic treatment (N-nitroso-N-methyl-urea). The radical scavenging and antioxidant capacity of (-)-8-O-methyltetrangomycin and extracts isolated from mutants were tested using EPR spin trapping technique and ABTS(·+) assay. Comparison of electron microscopic images of Streptomyces sp. AC37 and mutant strains of Streptomyces sp. AC37 revealed substantial differences in morphology and ultrastructure.