Transcriptome-Based Discovery of Fusarium graminearum Stress Responses to FgHV1 Infection.

Fusarium graminearum hypovirus 1 (FgHV1), which is phylogenetically related to Cryphonectria hypovirus 1 (CHV1), is a virus in the family Hypoviridae that infects the plant pathogenic fungus F. graminearum. Although hypovirus FgHV1 infection does not attenuate the virulence of the host (hypovirulence), it results in defects in mycelial growth and spore production. We now report that the vertical transmission rate of FgHV1 through asexual spores reached 100%. Using RNA deep sequencing, we performed genome-wide expression analysis to reveal phenotype-related genes with expression changes in response to FgHV1 infection. A total of 378 genes were differentially expressed, suggesting that hypovirus infection causes a significant alteration of fungal gene expression. Nearly two times as many genes were up-regulated as were down-regulated. A differentially expressed gene enrichment analysis identified a number of important pathways. Metabolic processes, the ubiquitination system, and especially cellular redox regulation were the most affected categories in F. graminearum challenged with FgHV1. The p20, encoded by FgHV1 could induce H2O2 accumulation and hypersensitive response in Nicotiana benthamiana leaves. Moreover, hypovirus FgHV1 may regulate transcription factors and trigger the RNA silencing pathway in F. graminearum.

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes.

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion.

Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Cardioprotective effect of selenium via modulation of cardiac ryanodine receptor calcium release channels in diabetic rat cardiomyocytes through thioredoxin system.

Increased oxidative stress contributes to heart dysfunction via impaired Ca(2+) homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 µmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca(2+) ([Ca(2+)]i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca(2+) leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca(2+)]i transients compared to those of controls. Both SR-Ca(2+) leak and [Ca(2+)]i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca(2+)]i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.