SR Ca2+-leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure

Aims: Ethanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction cou- pling in atrial and ventricular cardiomyocytes. Methods and results: Isolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol in- duced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 ± 0.45 mF/F0 vs 1.86 ± 0.26 in control, n ≥ 80). This effect was dose-dependent, while spontaneous arrhythmogenic Ca2+- waves increased ~5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scaven- ging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardi- omyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. Conclusion: We show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation- contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+- release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII)

Classification and naming of polymethine dyes used as staining agents for microscopy. A short guide for biomedical investigators

The scientific literature contains many accounts of application of polymethine dyes, including cyanine dyes, as imaging agents, i.e., “biological stains,” for microscopic investigation of biological materials. Currently, many such dyes are used as probes for living cells, i.e., “fluorescent probes.” Polymethine dyes are defined here by two criteria. First, they possess a conjugated chain of (2n + 1) sp2-hybridized carbon atoms that connect a terminal π-electron-accepting (π-electron withdrawing) group with a terminal π-electron-donating group. Second, they have an odd number (2n + 3) of π-centers and an even number (2n + 4) of π-electrons in this chain, where n equals the number of –CR2=CR3– groups, usually vinylene groups –CH=CH–. Commercialization of diverse chemical types of many polymethine dyes has been attempted. The dyes that have achieved wide application, however, are limited in number and it is these dyes that are emphasized here. Because these polymethine dyes sometimes have been described by confusing, and sometimes confused, names, we clarify here the chemical categories and names of such dyes for the nonchemist, biomedical end user of such imaging agents. Nevertheless, the nomenclature presented here is not intended to replace the traditional “chromophore” categories of dyestuff chemistry, because the latter are held in place both by wide usage and by venerable authorities, such as the Colour Index

Transcriptional Response of Two Brassica napus Cultivars to Short-Term Hypoxia in the Root Zone

Waterlogging is one major stress for crops and causes multiple problems for plants, for example low gas diffusion, changes in redox potential and accumulation of toxic metabolites. Brassica napus is an important oil crop with high waterlogging sensitivity, which may cause severe yield losses. Its reactions to the stress are not fully understood. In this work the transcriptional response of rapeseed to one aspect of waterlogging, hypoxia in the root zone, was analyzed by RNAseq, including two rapeseed cultivars from different origin, Avatar from Europe and Zhongshuang 9 from Asia. Both cultivars showed a high number of differentially expressed genes in roots after 4 and 24 h of hypoxia. The response included many well-known hypoxia-induced genes such as genes coding for glycolytic and fermentative enzymes, and strongly resembled the hypoxia response of the model organism Arabidopsis thaliana. The carbohydrate status of roots, however, was minimally affected by root hypoxia, with a tendency of carbohydrate accumulation rather than a carbon starvation. Leaves did not respond to the root stress after a 24-h treatment. In agreement with the gene expression data, subsequent experiments with soil waterlogging for up to 14 days revealed no differences in response or tolerance to waterlogging between the two genotypes used in this study. Interestingly, using a 0.1% starch solution for waterlogging, which caused a lowered soil redox potential, resulted in much stronger effects of the stress treatment than using pure water suggesting a new screening method for rapeseed cultivars in future experiments

Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.Instituto de Biotecnologia y Biologia Molecula

Cross-Kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differ-entially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.Instituto de Biotecnologia y Biologia Molecula

Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation

Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s