GPD1L-A306del modifies sodium current in a family carrying the dysfunctional SCN5A-G1661R mutation associated with Brugada syndrome.

Loss-of-function variants of SCN5A, encoding the sodium channel alpha subunit Nav1.5 are associated with high phenotypic variability and multiple cardiac presentations, while underlying mechanisms are incompletely understood. Here we investigated a family with individuals affected by Brugada Syndrome (BrS) of different severity and aimed to unravel the underlying genetic and electrophysiological basis.Next-generation sequencing was used to identify the genetic variants carried by family members. The index patient, who was severely affected by arrhythmogenic BrS, carried previously uncharacterized variants of Nav1.5 (SCN5A-G1661R) and glycerol-3-phosphate dehydrogenase-1-like protein (GPD1L-A306del) in a double heterozygous conformation. Family members exclusively carrying SCN5A-G1661R showed asymptomatic Brugada ECG patterns, while another patient solely carrying GPD1L-A306del lacked any clinical phenotype.To assess functional mechanisms, Nav1.5 channels were transiently expressed in HEK-293 cells in the presence and absence of GPD1L. Whole-cell patch-clamp recordings revealed loss of sodium currents after homozygous expression of SCN5A-G1661R, and reduction of current amplitude to ~ 50% in cells transfected with equal amounts of wildtype and mutant Nav1.5. Co-expression of wildtype Nav1.5 and GPD1L showed a trend towards increased sodium current amplitudes and a hyperpolarizing shift in steady-state activation and -inactivation compared to sole SCN5A expression. Application of the GPD1L-A306del variant shifted steady-state activation to more hyperpolarized and inactivation to more depolarized potentials.In conclusion, SCN5A-G1661R produces dysfunctional channels and associates with BrS. SCN5A mediated currents are modulated by co-expression of GDP1L and this interaction is altered by mutations in both proteins. Thus, additive genetic burden may aggravate disease severity, explaining higher arrhythmogenicity in double mutation carriers.

Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system.

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols.

Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

Epigenetic regulation of cardiac electrophysiology in atrial fibrillation: HDAC2 determines action potential duration and suppresses NRSF in cardiomyocytes.

Atrial fibrillation (AF) is associated with electrical remodeling, leading to cellular electrophysiological dysfunction and arrhythmia perpetuation. Emerging evidence suggests a key role for epigenetic mechanisms in the regulation of ion channel expression. Histone deacetylases (HDACs) control gene expression through deacetylation of histone proteins. We hypothesized that class I HDACs in complex with neuron-restrictive silencer factor (NRSF) determine atrial K+ channel expression. AF was characterized by reduced atrial HDAC2 mRNA levels and upregulation of NRSF in humans and in a pig model, with regional differences between right and left atrium. In vitro studies revealed inverse regulation of Hdac2 and Nrsf in HL-1 atrial myocytes. A direct association of HDAC2 with active regulatory elements of cardiac K+ channels was revealed by chromatin immunoprecipitation. Specific knock-down of Hdac2 and Nrsf induced alterations of K+ channel expression. Hdac2 knock-down resulted in prolongation of action potential duration (APD) in neonatal rat cardiomyocytes, whereas inactivation of Nrsf induced APD shortening. Potential AF-related triggers were recapitulated by experimental tachypacing and mechanical stretch, respectively, and exerted differential effects on the expression of class I HDACs and K+ channels in cardiomyocytes. In conclusion, HDAC2 and NRSF contribute to AF-associated remodeling of APD and K+ channel expression in cardiomyocytes via direct interaction with regulatory chromatin regions. Specific modulation of these factors may provide a starting point for the development of more individualized treatment options for atrial fibrillation.

A small secreted protein from Zymoseptoria tritici interacts with a wheat E3 ubiquitin ligase to promote disease.

Septoria tritici blotch (STB), caused by the ascomycete fungus Zymoseptoria tritici, is a major threat to wheat production worldwide. The Z. tritici genome encodes many small secreted proteins (ZtSSPs) that are likely to play a key role in the successful colonization of host tissues. However, few of these ZtSSPs have been functionally characterized for their role during infection. In this study, we identified and characterized a small, conserved cysteine-rich secreted effector from Z. tritici which has homologues in other plant pathogens in the Dothideomycetes. ZtSSP2 was expressed throughout Z. tritici infection in wheat, with the highest levels observed early during infection. A yeast two-hybrid assay revealed an interaction between ZtSSP2 and wheat E3 ubiquitin ligase (TaE3UBQ) in yeast, and this was further confirmed in planta using bimolecular fluorescence complementation and co-immunoprecipitation. Down-regulation of this wheat E3 ligase using virus-induced gene silencing increased the susceptibility of wheat to STB. Together, these results suggest that TaE3UBQ is likely to play a role in plant immunity to defend against Z. tritici.

Correction to: Genetic mapping of the labile (lab) gene: a recessive locus causing irregular spikelet fertility in labile-barley (Hordeum vulgare convar. labile).

While continuing our quest towards the identification of the labile (lab) locus in barley, we discovered that the previously assigned map location on the long arm of chromosome 5H was wrong.

Association mapping of wheat Fusarium head blight resistance-related regions using a candidate-gene approach and their verification in a biparental population.

KEY MESSAGE: Markers, located in Dicer1 and Ara6 genes, which are likely involved in cross-kingdom RNA trafficking, are associated with FHB resistance in GABI wheat population and were validated in biparental population. Association studies are a common approach to detect marker-trait associations for Fusarium head blight (FHB) resistance in wheat (Triticum aestivum), although verification of detected associations is exceptional. In the present study, candidate-gene association mapping (CG) of genes from silencing and secretory pathways, which may be involved in wheat resistance against FHB and cross-kingdom RNA trafficking, was performed. Fourteen markers, located in nine genes, were tested for association with FHB resistance in 356 lines from the GABI (genome analysis of the biological system of plants) wheat population. Three markers located in the genes Dicer1 and Ara6 were shown to be significantly associated with the studied trait. Verification of this finding was performed using the recombinant inbred lines (RILs) population 'Apache × Biscay', segregating for four of our 14 selected markers. We could show association of the Ara6 marker with plant height as well as association with FHB resistance for three markers located in Rab5-like GTPase gene Ara6 and Dicer1. These results confirmed the trait-marker associations detected also in the CG approach. Gene products of the associated genes are involved in response of the plant to pathogens, plant metabolism and may be involved in cross-kingdom RNA trafficking efficiency. The markers detected in the GABI wheat population, which were also validated in the biparental population, can potentially be used in wheat breeding.

Cloning and characterization of zebrafish K2P13.1 (THIK-1) two-pore-domain K+ channels.

Two-pore-domain potassium (K2P) channels conduct background potassium currents in the heart and other tissues. K2P currents are involved in the repolarization of action potentials and stabilize the resting membrane potential. Human K2P13.1 (THIK-1) channels are expressed in the heart and have recently been implicated in atrial fibrillation. The in vivo significance of K2P13.1 currents in cardiac electrophysiology is not known. We hypothesized that Danio rerio (zebrafish) may serve as model to elucidate the functional role of cardiac K2P13.1 channels. This work was designed to characterize zebrafish orthologs of K2P13.1. Two zkcnk13 coding sequences were identified by DNA database searches and amplified from zebrafish cDNA. Human and zebrafish K2P13.1 proteins exhibit 70% (K2P13.1a) and 66% (K2P13.1b) identity. Kcnk13 expression in zebrafish was studied using polymerase chain reaction. Zebrafish kcnk13a and zkcnk13b mRNAs were detected in brain and heart. Human and zebrafish K2P13.1 currents were analyzed in the Xenopus oocyte expression system by voltage clamp electrophysiology. Zebrafish K2P13.1a polypeptides were non-functional, while zK2P13.1b channels exhibited K+ selective, outwardly rectifying currents. Zebrafish and human K2P13.1 currents were similarly activated by arachidonic acid and reduced by barium, mexiletine, lidocaine, and inhibition of phospholipase C. In conclusion, zebrafish K2P13.1b channels and their human orthologs exhibit structural and regulatory similarities. Zebrafish may be used as in vivo model for the assessment of physiology and therapeutic significance of K2P13.1.

The C-terminal HCN4 variant P883R alters channel properties and acts as genetic modifier of atrial fibrillation and structural heart disease.

Atrial fibrillation (AF) is the most frequent sustained arrhythmia and can lead to structural cardiac changes, known as tachycardia-induced cardiomyopathy (TIC). HCN4 is implicated in spontaneous excitation of the sinoatrial node, while channel dysfunction has been associated with sinus bradycardia, AF and structural heart disease. We here asked whether HCN4 mutations may contribute to the development of TIC, as well. Mutation scanning of HCN4 in 60 independent patients with AF and suspected TIC followed by panel sequencing in carriers of HCN4 variants identified the HCN4 variant P883R [minor allele frequency (MAF): 0,88%], together with the KCNE1 variant S38G (MAF: 65%) in three unrelated patients. Family histories revealed additional cases of AF, sudden cardiac death and cardiomyopathy. Patch-clamp recordings of HCN4-P883R channels expressed in HEK293 cells showed remarkable alterations of channel properties shifting the half-maximal activation voltage to more depolarized potentials, while channel deactivation was faster compared to wild-type (WT). Co-transfection of WT and mutant subunits, resembling the heterozygous cellular situation of our patients, revealed significantly higher current densities compared to WT. In conclusion HCN4-P883R may increase ectopic trigger and maintenance of AF by shifting the activation voltage of If to more positive potentials and producing higher current density. Together with the common KCNE1 variant S38G, previously proposed as a genetic modifier of AF, HCN4-P883R may provide a substrate for the development of AF and TIC.

ANK2 functionally interacts with KCNH2 aggravating long QT syndrome in a double mutation carrier.

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation. The patient presented with a severe clinical phenotype including a markedly prolonged QTc interval (544 ms), recurrent syncope due to Torsade de Pointes tachycardias, survived cardiopulmonary resuscitation, progressive cardiac conduction defect, and atrial fibrillation. Evaluation of other family members identified a sister and a niece solely carrying the ANK2-E1813K variant, who showed age-related conduction disease. An asymptomatic second sister solely carried the KCNH2-H562R mutation. Voltage-clamp recordings in Xenopus oocytes revealed that KCNH2-H562R subunits were non-functional but did not exert dominant-negative effects on wild-type subunits. Expression of KCNH2-H562R in HEK293 cells showed a trafficking deficiency. Co-expression of the C-terminal regulatory domain of ANK2 in Xenopus oocytes revealed that ANK2-E1813K diminished currents mediated by the combination of wild-type and H562R KCNH2 subunits. Our data suggest that ANK2 functionally interacts with KCNH2 leading to a stronger current suppression and marked aggravation of long QT syndrome in the patient carrying variants in both proteins.

Abscisic acid influences tillering by modulation of strigolactones in barley.

Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8'-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

A comparative analysis of nonhost resistance across the two Triticeae crop species wheat and barley.

BACKGROUND: Nonhost resistance (NHR) protects plants against a vast number of non-adapted pathogens which implicates a potential exploitation as source for novel disease resistance strategies. Aiming at a fundamental understanding of NHR a global analysis of transcriptome reprogramming in the economically important Triticeae cereals wheat and barley, comparing host and nonhost interactions in three major fungal pathosystems responsible for powdery mildew (Blumeria graminis ff. ssp.), cereal blast (Magnaporthe sp.) and leaf rust (Puccinia sp.) diseases, was performed. RESULTS: In each pathosystem a significant transcriptome reprogramming by adapted- or non-adapted pathogen isolates was observed, with considerable overlap between Blumeria, Magnaporthe and Puccinia. Small subsets of these general pathogen-regulated genes were identified as differentially regulated between host and corresponding nonhost interactions, indicating a fine-tuning of the general pathogen response during the course of co-evolution. Additionally, the host- or nonhost-related responses were rather specific for each pair of adapted and non-adapted isolates, indicating that the nonhost resistance-related responses were to a great extent pathosystem-specific. This pathosystem-specific reprogramming may reflect different resistance mechanisms operating against non-adapted pathogens with different lifestyles, or equally, different co-option of the hosts by the adapted isolates to create an optimal environment for infection. To compare the transcriptional reprogramming between wheat and barley, putative orthologues were identified. Within the wheat and barley general pathogen-regulated genes, temporal expression profiles of orthologues looked similar, indicating conserved general responses in Triticeae against fungal attack. However, the comparison of orthologues differentially expressed between host and nonhost interactions revealed fewer commonalities between wheat and barley, but rather suggested different host or nonhost responses in the two cereal species. CONCLUSIONS: Taken together, our results suggest independent co-evolutionary forces acting on host pathosystems mirrored by barley- or wheat-specific nonhost responses. As a result of evolutionary processes, at least for the pathosystems investigated, NHR appears to rely on rather specific plant responses.

TREK-1 (K2P2.1) K+ channels are suppressed in patients with atrial fibrillation and heart failure and provide therapeutic targets for rhythm control.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Concomitant heart failure (HF) poses a particular therapeutic challenge and is associated with prolonged atrial electrical refractoriness compared with non-failing hearts. We hypothesized that downregulation of atrial repolarizing TREK-1 (K2P2.1) K+ channels contributes to electrical remodeling during AF with HF, and that TREK-1 gene transfer would provide rhythm control via normalization of atrial effective refractory periods in this AF subset. In patients with chronic AF and HF, atrial TREK-1 mRNA levels were reduced by 82% (left atrium) and 81% (right atrium) compared with sinus rhythm (SR) subjects. Human findings were recapitulated in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function. TREK-1 mRNA (-66%) and protein (-61%) was suppressed in AF animals at 14-day follow-up compared with SR controls. Downregulation of repolarizing TREK-1 channels was associated with prolongation of atrial effective refractory periods versus baseline conditions, consistent with prior observations in humans with HF. In a preclinical therapeutic approach, pigs were randomized to either atrial Ad-TREK-1 gene therapy or sham treatment. Gene transfer effectively increased TREK-1 protein levels and attenuated atrial effective refractory period prolongation in the porcine AF model. Ad-TREK-1 increased the SR prevalence to 62% during follow-up in AF animals, compared to 35% in the untreated AF group. In conclusion, TREK-1 downregulation and rhythm control by Ad-TREK-1 transfer suggest mechanistic and potential therapeutic significance of TREK-1 channels in a subgroup of AF patients with HF and prolonged atrial effective refractory periods. Functional correction of ionic remodeling through TREK-1 gene therapy represents a novel paradigm to optimize and specify AF management.

Upregulation of K(2P)3.1 K+ Current Causes Action Potential Shortening in Patients With Chronic Atrial Fibrillation.

BACKGROUND: Antiarrhythmic management of atrial fibrillation (AF) remains a major clinical challenge. Mechanism-based approaches to AF therapy are sought to increase effectiveness and to provide individualized patient care. K(2P)3.1 (TASK-1 [tandem of P domains in a weak inward-rectifying K+ channel-related acid-sensitive K+ channel-1]) 2-pore-domain K+ (K(2P)) channels have been implicated in action potential regulation in animal models. However, their role in the pathophysiology and treatment of paroxysmal and chronic patients with AF is unknown. METHODS AND RESULTS: Right and left atrial tissue was obtained from patients with paroxysmal or chronic AF and from control subjects in sinus rhythm. Ion channel expression was analyzed by quantitative real-time polymerase chain reaction and Western blot. Membrane currents and action potentials were recorded using voltage- and current-clamp techniques. K(2P)3.1 subunits exhibited predominantly atrial expression, and atrial K(2P)3.1 transcript levels were highest among functional K(2P) channels. K(2P)3.1 mRNA and protein levels were increased in chronic AF. Enhancement of corresponding currents in the right atrium resulted in shortened action potential duration at 90% of repolarization (APD90) compared with patients in sinus rhythm. In contrast, K(2P)3.1 expression was not significantly affected in subjects with paroxysmal AF. Pharmacological K(2P)3.1 inhibition prolonged APD90 in atrial myocytes from patients with chronic AF to values observed among control subjects in sinus rhythm. CONCLUSIONS: Enhancement of atrium-selective K(2P)3.1 currents contributes to APD shortening in patients with chronic AF, and K(2P)3.1 channel inhibition reverses AF-related APD shortening. These results highlight the potential of K(2P)3.1 as a novel drug target for mechanism-based AF therapy.

Metabolic and transcriptional response of central metabolism affected by root endophytic fungus Piriformospora indica under salinity in barley.

The root endophytic fungus Piriformospora indica enhances plant adaptation to environmental stress based on general and non-specific plant species mechanisms. In the present study, we integrated the ionomics, metabolomics, and transcriptomics data to identify the genes and metabolic regulatory networks conferring salt tolerance in P. indica-colonized barley plants. To this end, leaf samples were harvested at control (0 mM NaCl) and severe salt stress (300 mM NaCl) in P. indica-colonized and non-inoculated barley plants 4 weeks after fungal inoculation. The metabolome analysis resulted in an identification of a signature containing 14 metabolites and ions conferring tolerance to salt stress. Gene expression analysis has led to the identification of 254 differentially expressed genes at 0 mM NaCl and 391 genes at 300 mM NaCl in P. indica-colonized compared to non-inoculated samples. The integration of metabolome and transcriptome analysis indicated that the major and minor carbohydrate metabolism, nitrogen metabolism, and ethylene biosynthesis pathway might play a role in systemic salt-tolerance in leaf tissue induced by the root-colonized fungus.

Down-regulation of the glucan synthase-like 6 gene (HvGsl6) in barley leads to decreased callose accumulation and increased cell wall penetration by Blumeria graminis f. sp. hordei.

The recent characterization of the polysaccharide composition of papillae deposited at the barley cell wall during infection by the powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh), has provided new targets for the generation of enhanced disease resistance. The role of callose in papilla-based penetration resistance of crop species is largely unknown because the genes involved in the observed callose accumulation have not been identified unequivocally. We have employed both comparative and functional genomics approaches to identify the functional orthologue of AtGsl5 in the barley genome. HvGsl6 (the barley glucan synthase-like 6 gene), which has the highest sequence identity to AtGsl5, is the only Bgh-induced gene among the HvGsls examined in this study. Through double-stranded RNA interference (dsRNAi)-mediated silencing of HvGsl6, we have shown that the down-regulation of HvGsl6 is associated with a lower accumulation of papillary and wound callose and a higher susceptibility to penetration of the papillae by Bgh, compared with control lines. The results indicate that the HvGsl6 gene is a functional orthologue of AtGsl5 and is involved in papillary callose accumulation in barley. The increased susceptibility of HvGsl6 dsRNAi transgenic lines to infection indicates that callose positively contributes to the barley fungal penetration resistance mechanism.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the ß-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes.

Therapeutic targeting of two-pore-domain potassium (K(2P)) channels in the cardiovascular system.

The improvement of treatment strategies in cardiovascular medicine is an ongoing process that requires constant optimization. The ability of a therapeutic intervention to prevent cardiovascular pathology largely depends on its capacity to suppress the underlying mechanisms. Attenuation or reversal of disease-specific pathways has emerged as a promising paradigm, providing a mechanistic rationale for patient-tailored therapy. Two-pore-domain K(+) (K(2P)) channels conduct outward K(+) currents that stabilize the resting membrane potential and facilitate action potential repolarization. K(2P) expression in the cardiovascular system and polymodal K2P current regulation suggest functional significance and potential therapeutic roles of the channels. Recent work has focused primarily on K(2P)1.1 [tandem of pore domains in a weak inwardly rectifying K(+) channel (TWIK)-1], K(2P)2.1 [TWIK-related K(+) channel (TREK)-1], and K(2P)3.1 [TWIK-related acid-sensitive K(+) channel (TASK)-1] channels and their role in heart and vessels. K(2P) currents have been implicated in atrial and ventricular arrhythmogenesis and in setting the vascular tone. Furthermore, the association of genetic alterations in K(2P)3.1 channels with atrial fibrillation, cardiac conduction disorders and pulmonary arterial hypertension demonstrates the relevance of the channels in cardiovascular disease. The function, regulation and clinical significance of cardiovascular K(2P) channels are summarized in the present review, and therapeutic options are emphasized.

Vernakalant activates human cardiac K(2P)17.1 background K(+) channels.

Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K(+) channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K2P17.1 channels remains to be investigated. This study was designed to elucidate human K2P17.1 regulation by antiarrhythmic drugs. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. The class III antiarrhythmic compound vernakalant activated K2P17.1 currents in oocytes an in mammalian cells (EC50,CHO=40 µM) in frequency-dependent manner. K2P17.1 channel activation by vernakalant was specific among K2P channel family members. By contrast, vernakalant reduced K2P4.1 and K2P10.1 currents, in line with K2P2.1 blockade reported earlier. K2P17.1 open rectification characteristics and current-voltage relationships were not affected by vernakalant. The class I drug flecainide did not significantly modulate K2P currents. In conclusion, vernakalant activates K2P17.1 background potassium channels. Pharmacologic K2P channel activation by cardiovascular drugs has not been reported previously and may be employed for personalized rhythm control in patients with AF-associated reduction of K(+) channel function.