Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum.

BACKGROUND: Colonoscopy is a classic diagnostic method with possible complications including abdominal pain and diarrhoea. In this study, gut microbiota dynamics and related metabolic products during and after colonoscopy were explored to accelerate gut microbiome balance through probiotics. METHODS: The gut microbiota and fecal short-chain fatty acids (SCFAs) were analyzed in four healthy subjects before and after colonoscopy, along with seven individuals supplemented with Clostridium butyricum. We employed 16S rRNA sequencing and GC-MS to investigate these changes. We also conducted bioinformatic analysis to explore the buk gene, encoding butyrate kinase, across C. butyricum strains from the human gut. RESULTS: The gut microbiota and fecal short-chain fatty acids (SCFAs) of four healthy subjects were recovered on the 7th day after colonoscopy. We found that Clostridium and other bacteria might have efficient butyric acid production through bioinformatic analysis of the buk and assessment of the transcriptional level of the buk. Supplementation of seven healthy subjects with Clostridium butyricum after colonoscopy resulted in a quicker recovery and stabilization of gut microbiota and fecal SCFAs on the third day. CONCLUSION: We suggest that supplementation of Clostridium butyricum after colonoscopy should be considered in future routine clinical practice.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins.

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 µg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC-MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

RATIONALE: We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. OBJECTIVE: To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. METHODS AND RESULTS: We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. CONCLUSIONS: We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Effect of H2A.Z deletion is rescued by compensatory mutations in Fusarium graminearum.

Fusarium head blight is a destructive disease of grains resulting in reduced yields and contamination of grains with mycotoxins worldwide; Fusarium graminearum is its major causal agent. Chromatin structure changes play key roles in regulating mycotoxin biosynthesis in filamentous fungi. Using a split-marker approach in three F. graminearum strains INRA156, INRA349 and INRA812 (PH-1), we knocked out the gene encoding H2A.Z, a ubiquitous histone variant reported to be involved in a diverse range of biological processes in yeast, plants and animals, but rarely studied in filamentous fungi. All ΔH2A.Z mutants exhibit defects in development including radial growth, sporulation, germination and sexual reproduction, but with varying degrees of severity between them. Heterogeneity of osmotic and oxidative stress response as well as mycotoxin production was observed in ΔH2A.Z strains. Adding-back wild-type H2A.Z in INRA349ΔH2A.Z could not rescue the phenotypes. Whole genome sequencing revealed that, although H2A.Z has been removed from the genome and the deletion cassette is inserted at H2A.Z locus only, mutations occur at other loci in each mutant regardless of the genetic background. Genes affected by these mutations encode proteins involved in chromatin remodeling, such as the helicase Swr1p or an essential subunit of the histone deacetylase Rpd3S, and one protein of unknown function. These observations suggest that H2A.Z and the genes affected by such mutations are part or the same genetic interaction network. Our results underline the genetic plasticity of F. graminearum facing detrimental gene perturbation. These findings suggest that intergenic suppressions rescue deleterious phenotypes in ΔH2A.Z strains, and that H2A.Z may be essential in F. graminearum. This assumption is further supported by the fact that H2A.Z deletion failed in another Fusarium spp., i.e., the rice pathogen Fusarium fujikuroi.

Systematic literature review of solar-powered landfill leachate sanitation: Challenges and research directions over the past decade.

Researchers have documented the negative effects of refractory chemicals and emergent pollutants in landfill leachate (LL) that cannot be degraded using conventional methods. The propagation, invasion, and deleterious effects of several LL hazards affect aquatic species, the environment, and food outlets, causing significant safety issues. These include cancer risks, chronic exposure, and reproductive consequences. Alternatively, solar energy is a sustainable solution for treating landfill leachate to benefit humans and the environment. In this work, a thorough bibliometric and systematic analysis of studies that employed solar energy for landfill leachate remediation over the past decade was conducted in order to determine trends, and future research areas. In addition to the energy demand, the economic aspect and the advantages of using solar power to treat landfill leachate were discussed. Additionally, the study gives specific suggestions for future research purposes and important problems. The reviewed literature revealed that combining solar-based physical-chemical and biological processes has proven to be the most efficient method for landfill leachate degradation. It also appears from the bibliometric study that more collaboration and contribution are needed to develop solar-based landfill leachate treatment. This study concludes that solar-powered landfill leachate remediation techniques would considerably increase the effectiveness of treated leachate reutilization, advancing the cause of environmental sustainability.

TXN inhibitor impedes radioresistance of colorectal cancer cells with decreased ALDH1L2 expression via TXN/NF-κB signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is prevalent worldwide and is often challenged by treatment failure and recurrence due to resistance to radiotherapy. Here, we aimed to identify the elusive underlying molecular mechanisms of radioresistance in CRC. METHODS: Weighted gene co-expression network analysis was used to identify potential radiation-related genes. Colony formation and comet assays and multi-target single-hit survival and xenograft animal models were used to validate the results obtained from the bioinformatic analysis. Immunohistochemistry was performed to examine the clinical characteristics of ALDH1L2. Co-immunoprecipitation, immunofluorescence and flow cytometry were used to understand the molecular mechanisms underlying radioresistance. RESULTS: Bioinformatic analysis, in vitro, and in vivo experiments revealed that ALDH1L2 is a radiation-related gene, and a decrease in its expression induces radioresistance in CRC cells by inhibiting ROS-mediated apoptosis. Patients with low ALDH1L2 expression exhibit resistance to radiotherapy. Mechanistically, ALDH1L2 interacts with thioredoxin (TXN) and regulates the downstream NF-κB signaling pathway. PX-12, the TXN inhibitor, overcomes radioresistance due to decreased ALDH1L2. CONCLUSIONS: Our results provide valuable insights into the potential role of ALDH1L2 in CRC radiotherapy. We propose that the simultaneous application of TXN inhibitors and radiotherapy would significantly ameliorate the clinical outcomes of patients with CRC having low ALDH1L2.

Surface Modification of TiO2 Nanotubes Prepared by Porous Titanium Anodization via Hydrothermal Reaction: A Method for Synthesis High-Efficiency Adsorbents of Recovering Sr Ions.

The recycling of strontium ions (Sr2+) from sea water has been well known for its good cost-effectiveness and environment friendliness. Herein, we modified the surface of TiO2 nanotubes (TNTs) prepared by porous titanium anodization via hydrothermal (HT) reaction and synthesized a highly efficient adsorbent for the repeated recycling of Sr2+. TNTs with a high specific surface area were manufactured on porous titanium by internal anodic oxidation. The as-prepared TNTs were treated by HT method to synthesize adsorption materials with a tubular bottom and grass-type top structure loaded with Na+. The surface cracks were eliminated by annealing pretreatment, and the investigation found that the 6 h HT reaction most effectively increased the Na+ content in the adsorbent. The as-synthesized adsorbents (HT-6TNTs) were used to recover Sr2+, and the maximum adsorption efficiency (approximately 100%) and adsorption equilibrium were observed within 10 h. Meanwhile, three consecutive cycles of adsorption experiments proved the uniform behavior of the HT-6TNTs in the reproducible recycling of Sr2+. In addition, by increasing the anodization time of TNTs from 0.5 to 3 h, the maximum adsorption capacity can be increased from 4.68 to 36.15 mg·unit-1, approximately 7.7 times higher.

Bioinformatic identification of key pathways, hub genes, and microbiota for therapeutic intervention in Helicobacter pylori infection.

The pathogenic mechanisms of Helicobacter pylori infection remain to be defined, and potential interventional microbiota are just beginning to be identified. In this study, gene-set enrichment analysis (GSEA) was used to integrate three H. pylori infection microarray data sets from the gene expression omnibus database and identified ten hallmark gene sets and 35 Kyoto encyclopedia of genes and genomes (KEGG) pathways that differed between healthy and Helicobacter pylori-infected individuals. Weighted gene co-expression network analysis (WGCNA) performed on two of the data sets identified three key gene coexpression modules. These modules contained 54 enriched KEGG pathways, 25 of which overlapped with the GSEA analysis, suggesting potentially important roles in H. pylori-infection. We selected 116 hub genes from the three key modules for in vitro validation at the transcriptional level using H. pylori Sydney Strain 1 and verified the upregulation of 80. WGCNA of the microbiomes based on 20 mucosal samples and a sequence read archive data set revealed four microbiota modules correlated with H. pylori infection. The negatively correlated modules contained 11 microbiome families. These findings provide new insight into the pathogenesis of H. pylori infection and systematically identify 25 key pathways, 80 upregulated hub genes, and 11 families of candidate interventional microbiota for further research.

Association with SERCA2a directs phospholamban trafficking to sarcoplasmic reticulum from a nuclear envelope pool.

AIMS: Phospholamban (PLB) stoichiometrically regulates the cardiac Ca2+ pump (SERCA2a) in the sarcoplasmic reticulum (SR); but in the nuclear envelope (NE) of cardiomyocytes (CMs), the PLB to SERCA2a molar ratio is higher, which highlights our poor understanding of how SR proteins distribute to their functional subcompartments. By tracking newly made PLB and SERCA2a in CMs, we will elucidate underlying cellular pathways responsible for their unique intracellular distributions. METHODS AND RESULTS: Highly specific monoclonal antibodies were used to compare the subcellular distributions of SERCA2a, PLB, and junctin (JCN) in dog heart tissue. The data supported a view that both non-junctional and junctional SR proteins are all prominently enriched in transverse stretches of SR tubules, along the edges of sarcomeres (SR z-tubules). To understand the genesis of these steady state distributions, we analyzed confocal immunofluorescence images of adult rat CMs after acute expression (12-48 h) of the dog ortholog of PLB (dPLB) or dSERCA2a. Newly made dog proteins in rat CMs were detected using dog-specific monoclonal antibodies. By 12-24 h, dSERCA2a had accumulated within the NE in a punctate pattern, presumably reflecting initial sites of biosynthesis. Over the next 24-48 h, higher levels of dSERCA2a immunofluorescence accumulated in transverse/radial SR tubules, aligned along sarcolemmal transverse (T)-tubules, and extending from NE puncta. The patterns of SR tubules carrying dSERCA2a overlapped with those for newly made JCN, suggesting a common Nuclear Envelope to SR along T-tubules or NEST pathway for SR proteins. In contrast to the SERCA2a distribution pattern, dPLB accumulated uniformly in the NE, without visible puncta. With co-expression of dSERCA2a, however, PLB no longer uniformly filled the NE, but instead moved together with SERCA2a to form bright NE puncta, from which the two proteins then trafficked anterogradely. CONCLUSION: Expression of dog SR protein orthologs (dSERCA2a, dPLB, and dJCN) for as little as 48 h reproduces their characteristic steady state distributions. Detailed analyses of the time courses of protein accumulation suggest a possible mechanism by which PLB distributes to both the NE and SR, unlike SERCA2a. SERCA2a moves in SR z-tubules directly from rough ER, along pathways that are in common with those used by junctional SR proteins. A different trafficking route for PLB away the rough ER/NE led to its accumulation in the NE, a process that may account for its enrichment in NE in situ. Association of SERCA2a with PLB from this NE pool enhanced PLB trafficking along the NEST pathway, contributing to steady state stoichiometry and physiologically regulated SERCA2a.

H2A.Z and chromatin remodelling complexes: a focus on fungi.

Chromatin is a highly dynamic structure that closely relates with gene expression in eukaryotes. ATP-dependent chromatin remodelling, histone post-translational modification and DNA methylation are the main ways that mediate such plasticity. The histone variant H2A.Z is frequently encountered in eukaryotes, and can be deposited or removed from nucleosomes by chromatin remodelling complex SWR1 or INO80, respectively, leading to altered chromatin state. H2A.Z has been found to be involved in a diverse range of biological processes, including genome stability, DNA repair and transcriptional regulation. Due to their formidable production of secondary metabolites, filamentous fungi play outstanding roles in pharmaceutical production, food safety and agriculture. During the last few years, chromatin structural changes were proven to be a key factor associated with secondary metabolism in fungi. However, studies on the function of H2A.Z are scarce. Here, we summarize current knowledge of H2A.Z functions with a focus on filamentous fungi. We propose that H2A.Z is a potential target involved in the regulation of secondary metabolite biosynthesis by fungi.

Influence of bone densitometry on the anti-osteoporosis treatment after fragility hip fracture.

BACKGROUND: Fragility fracture significantly increases risk of future fracture. The fragility fracture cycle should be disrupted. The secondary fracture prevention is important for the patients with fragility hip fracture. The pharmacotherapy for osteoporosis is important for prevention of new fracture. However, many patients with hip fracture do not receive osteoporosis treatment. This retrospective study investigates the influence of bone mineral density (BMD) assessment on the initiation of anti-osteoporosis medications in the hospitalized patients with fragility hip fracture. METHODS: This retrospective research enrolled 1211 patients with fragility hip fracture 50 years of age and older. Among 1211 patients aged from 50 to 103 years with the average age of 77.83 ± 9.95 years, there were 807 females and 404 males. There were 634 fractures of femoral neck and 577 intertrochanteric fractures of femur. We examined whether patients had received bone mineral density assessment and received anti-osteoporosis therapy during the period of hospitalization. The patients were divided into BMD assessment group and no BMD assessment group. Measurement data were expressed as mean ± standard deviation and compared with t test. All parameters of groups were compared with Chi-square test. RESULTS: Of 1211 patients, 331 (27.33%) had received BMD assessment and 925 (76.38%) had received anti-osteoporosis drugs during the period of hospitalization. The rate of bisphosphonate use was lower and only 11.31% in the total patients. The anti-osteoporosis treatment rate was 93.66% in the patients receiving BMD assessment and 69.89% in the patients without BMD assessment (p < 0.01). The zoledronate use significantly increased from 6.7% in the patients without BMD assessment to 23.56% in the patients receiving BMD assessment (p < 0.01). CONCLUSIONS: BMD assessment is a good basis for communication between patients and orthopedic surgeons. BMD assessment significantly increases the initiation of osteoporosis treatment and bisphosphonate use in the patients with hip fracture during the period of hospitalization.

Phospholamban regulates nuclear Ca2+ stores and inositol 1,4,5-trisphosphate mediated nuclear Ca2+ cycling in cardiomyocytes.

AIMS: Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS: Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ±â€¯0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ±â€¯0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION: PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.

Sex-specific activation of SK current by isoproterenol facilitates action potential triangulation and arrhythmogenesis in rabbit ventricles.

KEY POINTS: It is unknown if a sex difference exists in cardiac apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ). There is no sex difference in IKAS in the basal condition. However, there is larger IKAS in female rabbit ventricles than in male during isoproterenol infusion. IKAS activation by isoproterenol leads to action potential triangulation in females, indicating its abundant activation at early phases of repolarization. IKAS activation in females induces negative Ca2+ -voltage coupling and promotes electromechanically discordant phase 2 repolarization alternans. IKAS is important in the mechanisms of ventricular fibrillation in females during sympathetic stimulation. ABSTRACT: Sex has a large influence on cardiac electrophysiological properties. Whether sex differences exist in apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ) remains unknown. We performed optical mapping, transmembrane potential, patch clamp, western blot and immunostaining in 62 normal rabbit ventricles, including 32 females and 30 males. IKAS blockade by apamin only minimally prolonged action potential (AP) duration (APD) in the basal condition for both sexes, but significantly prolonged APD in the presence of isoproterenol in females. Apamin prolonged APD at the level of 25% repolarization (APD25 ) more prominently than APD at the level of 80% repolarization (APD80 ), consequently reversing isoproterenol-induced AP triangulation in females. In comparison, apamin prolonged APD to a significantly lesser extent in males and failed to restore the AP plateau during isoproterenol infusion. IKAS in males did not respond to the L-type calcium current agonist BayK8644, but was amplified by the casein kinase 2 (CK2) inhibitor 4,5,6,7-tetrabromobenzotriazole. In addition, whole-cell outward IKAS densities in ventricular cardiomyocytes were significantly larger in females than in males. SK channel subtype 2 (SK2) protein expression was higher and the CK2/SK2 ratio was lower in females than in males. IKAS activation in females induced negative intracellular Ca2+ -voltage coupling, promoted electromechanically discordant phase 2 repolarization alternans and facilitated ventricular fibrillation (VF). Apamin eliminated the negative Ca2+ -voltage coupling, attenuated alternans and reduced VF inducibility, phase singularities and dominant frequencies in females, but not in males. We conclude that ß-adrenergic stimulation activates ventricular IKAS in females to a much greater extent than in males. IKAS activation plays an important role in ventricular arrhythmogenesis in females during sympathetic stimulation.

Ondansetron blocks wild-type and p.F503L variant small-conductance Ca2+-activated K+ channels.

Apamin-sensitive small-conductance Ca2+-activated K+ (SK) current ( IKAS) is encoded by Ca2+-activated K+ channel subfamily N ( KCNN) genes. IKAS importantly contributes to cardiac repolarization in conditions associated with reduced repolarization reserve. To test the hypothesis that IKAS inhibition contributes to drug-induced long QT syndrome (diLQTS), we screened for KCNN variants among patients with diLQTS, determined the properties of heterologously expressed wild-type (WT) and variant KCNN channels, and determined if the 5-HT3 receptor antagonist ondansetron blocks IKAS. We searched 2,306,335 records in the Indiana Network for Patient Care and found 11 patients with diLQTS who had DNA available in the Indiana Biobank. DNA sequencing discovered a heterozygous KCNN2 variant (p.F503L) in a 52-yr-old woman presenting with corrected QT interval prolongation at baseline (473 ms) and further corrected QT interval lengthening (601 ms) after oral administration of ondansetron. That patient was also heterozygous for the p.S38G and p.P2835S variants of the QT-controlling genes KCNE1 and ankyrin 2, respectively. Patch-clamp experiments revealed that the p.F503L KCNN2 variant heterologously expressed in human embryonic kidney (HEK)-293 cells augmented Ca2+ sensitivity, increasing IKAS density. The fraction of total F503L-KCNN2 protein retained in the membrane was higher than that of WT KCNN2 protein. Ondansetron at nanomolar concentrations inhibited WT and p.F503L SK2 channels expressed in HEK-293 cells as well as native SK channels in ventricular cardiomyocytes. Ondansetron-induced IKAS inhibition was also demonstrated in Langendorff-perfused murine hearts. In conclusion, the heterozygous p.F503L KCNN2 variant increases Ca2+ sensitivity and IKAS density in transfected HEK-293 cells. Ondansetron at therapeutic (i.e., nanomolar) concentrations is a potent IKAS blocker. NEW & NOTEWORTHY We showed that ondansetron, a 5-HT3 receptor antagonist, blocks small-conductance Ca2+-activated K+ (SK) current. Ondansetron may be useful in controlling arrhythmias in which increased SK current is a likely contributor. However, its SK-blocking effects may also facilitate the development of drug-induced long QT syndrome.

Phospholamban is concentrated in the nuclear envelope of cardiomyocytes and involved in perinuclear/nuclear calcium handling.

AIMS: Phospholamban (PLB) regulates the cardiac Ca2+-ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible contributions to Ca2+ cycling remain unknown. We examined the intracellular distribution of PLB and tested whether a pool of PLB exists in the nuclear envelope (NE) that might regulate perinuclear/nuclear Ca2+ (nCa2+) handling in cardiomyocytes (CMs). METHODS AND RESULTS: Using confocal immunofluorescence microscopy and immunoblot analyses of CMs and CM nuclei, we discovered that PLB was highly concentrated in NE. Moreover, the ratio of PLB levels to SERCA levels was greater in NE than in SR. The increased levels of PLB in NE were a consistent finding using a range of antibodies, tissue samples, and species. To address a possible role in affecting Ca2+ handling, we used Fluo-4 based confocal Ca2+ imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa2+ uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca2+ transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs ([Ca2+]i=400nM), we measured spontaneous Ca2+ waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100µg/ml). This highly selective immunological reagent enhanced Ca2+ uptake (faster decay times) and Ca2+ release (greater intensity) in both cytosol and across the nuclear regions. CONCLUSIONS: Besides SR, PLB is concentrated in NE of CMs, and may be involved in modulation of nCa2+ dynamics.

Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes.

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs. Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca(2+) release in normal VMs. Ca(2+) sparks and spontaneous Ca(2+) waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca(2+) fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca(2+) sparks in VMs exposed to 50 nM free [Ca(2+)]. At physiological diastolic free [Ca(2+)] (100-200 nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca(2+)], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca(2+) sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca(2+) release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca(2+) dynamics and rhythmic activity of VMs.

Competitive displacement of wild-type phospholamban from the Ca2+-free cardiac calcium pump by phospholamban mutants with different binding affinities.

Phospholamban (PLB) regulates the cardiac Ca(2+) pump (SERCA2a). To test how different species of PLB mutants compete to interact with the Ca(2+)-free, E2 conformation of SERCA2a, using the insect cell expression system, we examined how various exogenous PLB mutants regulated SER-20G-PLB, a chimeric WT-SERCA2a-Gly-WT-PLB construct, which retains a fully catalytic active Ca(2+)-pump and its intrinsically regulatory PLB-tether. Exogenous gain-of-function PLB mutants dominantly super-inhibited the WT-PLB-tethered SERCA2a. Further, in the Ca(2+)-free condition, co-expressed normal- or super-inhibitory PLB mutant with either engineered N30C or V49C residue cross-linked to Lys328 or V89C of SER-20G-PLB at the cytoplasmic or transmembrane domain, respectively, suggesting that these freely diffusing PLB mutants completely replaced the WT-PLB-tether and fit into the binding pocket previously occupied by WT-PLB. Micromolar Ca(2+) completely inhibited cross-linking, yielding a similar Ca(2+)-dependency regardless of the presence of the WT-PLB-tether. In contrast, the PLB mutant with the loss-of-function L31A mutation has decreased binding affinity for SERCA2a, thus cross-linking weakly to the WT-PLB-tethered SERCA2a, and only marginally affected the activity of SER-20G-PLB. Thus, there is a reversible equilibrium between different PLB mutants for binding to E2, in which PLB mutants possessing higher binding affinity for SERCA2a produce a more stable E2·PLB and lower Ca(2+) affinity.

The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum.

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca(2+)-ATPase), pumps contractile-dependent Ca(2+) ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca(2+) affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca(2+)-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca(2+) in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca(2+) binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca(2+) affinity and depresses cardiac contractility.