Among invertebrates, specific pheromones elicit inherent (fixed) behavioural responses to coordinate social behaviours such as sexual recognition and attraction. By contrast, the much more complex social odours of mammals provide a broad range of information about the individual owner and stimulate individual-specific responses that are modulated by learning. How do mammals use such odours to coordinate important social interactions such as sexual attraction while allowing for individual-specific choice? We hypothesized that male mouse urine contains a specific pheromonal component that invokes inherent sexual attraction to the scent and which also stimulates female memory and conditions sexual attraction to the airborne odours of an individual scent owner associated with this pheromone.Using wild-stock house mice to ensure natural responses that generalize across individual genomes, we identify a single atypical male-specific major urinary protein (MUP) of mass 18893Da that invokes a female's inherent sexual attraction to male compared to female urinary scent. Attraction to this protein pheromone, which we named darcin, was as strong as the attraction to intact male urine. Importantly, contact with darcin also stimulated a strong learned attraction to the associated airborne urinary odour of an individual male, such that, subsequently, females were attracted to the airborne scent of that specific individual but not to that of other males.This involatile protein is a mammalian male sex pheromone that stimulates a flexible response to individual-specific odours through associative learning and memory, allowing female sexual attraction to be inherent but selective towards particular males. This 'darcin effect' offers a new system to investigate the neural basis of individual-specific memories in the brain and give new insights into the regulation of behaviour in complex social mammals.http://www.biomedcentral.com/1741-7007/8/71See associated Commentary  Pheromones are specific chemical signals, produced for communication between individuals of the same species, that trigger a specific natural behaviour or physiological process . SignalsScents play an integral role in mediating reproductive interactions in many mammals. This includes not only the recognition and location of opposite sex conspecifics but also assessment of the suitability and, thus, the attractiveness of different individuals as potential mates ,7. In coC. elegans to elephants [Sexual attraction which causes an animal to spend more time in the vicinity of chemical signals from the opposite sex, and much greater interest in opposite sex scents, is common to many animals, from lephants -15. Althlephants -18. Contlephants  as this lephants . Howeverlephants . Althouglephants , attractlephants . This suMus musculus domesticus) were presented with a choice between a male versus a female urine stimulus. These were streaked onto filter papers in the shape of scent marks, placed 28 cm apart on the Perspex ceiling of a clean test chamber, such that females could make nasal contact with each stimulus by stretching upright on their hind legs ; wild mice normally spend very little time in this central area of the arena, preferring to stay close to the outer walls. We used a large number of wild-derived female house mice in oestrus or proestrus as subjects throughout this study (bred in captivity to control experience). This ensures that any attraction to male scent is a general response across normal genetically-variable female mice rather than a response that is specific to a laboratory strain, and has not been influenced by many generations of artificial selection for successful breeding in the laboratory. It also avoids the problem of unnatural genetic similarity between subjects and stimulus donors that would be implicit with inbred laboratory mice [In order to identify the molecule(s) that elicit inherent sexual attraction to male mouse urine, female house mice  androgen-dependent volatiles detected through V1R receptors in vomeronasal sensory neurons -25. In ae Figure .Mup genes that show sex-specific expression in the B6 mouse strain [As the main components of the HMW fraction consisted of a mixture of different MUP isoforms and their bound ligands ,33, thesA mixture of these four MUP-containing fractions  presented at concentrations similar to that in intact urine was highly effective in eliciting much greater time near the male stimulus Figure . The difMup genes in the central region of the Mup cluster on chromosome 4 where there has been recent rapid expansion of gene-pseudogene pairs [Mup genes encode the urinary MUPs expressed by both sexes as well as the 18645Da isoform in male fraction I. While expression of the 18645Da MUP is male-specific among laboratory strains [Mup cluster where there is considerably greater divergence between Mup genes [sec-butyl 4,5 dihydrothiazole (thiazole) in mouse urine [Pride and Prejudice).In house mice, most urinary MUP isoforms are encoded by ne pairs . Variati strains , it is eup genes . This prup genes  and unpuse urine . In ordeThe tight binding of thiazole to darcin considerably extends the release of this volatile pheromone from scent marks over many hours; when artificially displaced from MUPs, thiazole evaporates from scent marks within minutes . Thus, gEscherichia coli  but is unlikely to be a metabolic pathway that is essential for life. Second, a comprehensive search of the metabolic pathways of E. coli reveals that the only thiazole known in E. coli metabolism is that related to biotin synthesis, an essential metabolic function that has been lost in mammals (hence it is a vitamin). Third, as one of the other recombinant MUPs tested (18694Da MUP) also binds thiazole in mouse urine [E. coli, and clearly stimulated no response. Thus the response appears to be to darcin alone rather than to a darcin-ligand complex.Thus, darcin acts as a sex pheromone that reliably elicits female sexual attraction to a male's urine scent, doubling the time spent near a male compared to a female urine mark. Unlike most mammalian scents that have been termed 'pheromones', we have shown that darcin meets the criteria widely accepted for the definition of the term pheromone ,5: it isse urine  it proviAlthough the response to darcin is inherent and highly consistent, this does not mean that female sexual attraction to male mice is inflexible. Nor does this mean that other components of male urine scent have no role to play in sexual attraction. Importantly, female attraction is to the individual owner of the scent encountered, not to males in general ,31. As dTo test whether it is contact with darcin in male urine that stimulates this learned attraction to individual-specific airborne volatiles, we provided females with the opportunity to learn odours by pre-exposing them to a male and female urine stimulus prior to testing. We then tested female attraction to airborne volatiles from a male versus female urine stimulus when they were unable to contact the stimuli during the test. Using urine from males that expressed normal adult levels of darcin, females learned an attraction to airborne urinary volatiles if they had prior physical contact before the test either with urine from the same individual male [using stimuli from genetically diverse wild males, Figure By contrast, prior contact with urine from BALB/c laboratory males, which express only trace levels of darcin (see above), failed to elicit any learned attraction to the males' airborne volatiles Figure . HoweverWe have shown that female sexual attraction to spend time near male urine scent is an inherent response to a single male-specific protein pheromone, darcin, detected through physical contact. This involatile pheromone also stimulates females to remember the associated airborne volatile profile and, subsequently, also show attraction to this individual-specific airborne stimulus. Contact with darcin, whether presented alone or combined with urinary volatiles, consistently doubled the time spent near a male compared to a female scent mark. Response to the airborne volatiles learned in association with darcin was even stronger, tripling the time spent near to the source of the learned individual male's scent mark relative to female airborne scent. The consistency of response is all the more remarkable in view of our deliberate use of a large random selection of genetically heterogeneous wild-stock mice to ensure that responses reflect normal functional behaviour in this species, mirroring the attraction to male urine shown by females from seminatural populations . Adult mInherent attraction to darcin in male urine ensures that females naturally discriminate between conspecific male and female scents, without any need for social learning. However, females fail to show the same inherent attraction to male airborne urinary volatiles, despite the prevalence of many androgen-dependent volatiles in male mouse urine -41 that Most importantly, contact with the male-specific pheromone darcin also stimulates females to learn and become attracted to the individual-specific airborne volatiles associated with this pheromone. Thus, this pheromone signal stimulates both inherent attraction on contact and the learned individual-specific attraction that is essential for choosiness in females. Intuitively, it has been assumed that attractant pheromones in terrestrial animals will be small airborne volatiles that can be detected at a distance from the scent source , with inThe learning of individual odours during prior contact with scents from the opposite sex appears to be common across many mammals, resulting in memory and selective attraction to that scent owner -54. HoweThe involatile protein darcin is most likely to be detected through the accessory olfactory subsystem which detects scents that are actively pumped to the vomeronasal organ on nasal contact with the scent source ,59. WhilThere has been much debate about whether the original concept of a 'pheromone'  has any Mus musculus domesticus (F0-F2) aged 3-12 months, from a colony derived from wild ancestors captured from five different populations in the northwest of England, UK. Most females (77%) were used in only a single test over a total of 614 trials, but a small number were used in two (17%), three (4%) or four (2%) tests, each involving different scent stimuli, with 2-16 weeks between successive tests. Females were housed in 45 × 28 × 13 cm cages  in single-sex small family groups (2-4 sisters per cage during the test period) in a different room from the urine donors. As we have previously shown that females show very similar attraction responses to urine stimuli from unfamiliar males whether completely naïve to adult male scents or after natural social and sexual experience [The subjects were 469 captive bred adult female d in two %, three The urine donors were 17 male and 14 female C57BL/6 laboratory mice  aged 60 days - 9 months, 12 male and 17 female BALB/c mice  aged 4-9 months, and 33 captive bred adult male house mice derived from the same colony as the female subjects but unrelated. Males were housed singly in 43 × 11.5 × 12 cm cages  to ensure that there were no subordinate males whose scent was unattractive to females. Females were housed in single sex small groups (2-4) in 45 × 28 × 13 cm cages. Urine was collected by holding the donor mouse by the scruff of the neck over a clean 1.5 ml Eppendorf tube. Urine from 5-8 individual donors of the same strain and sex was pooled for testing, using different combinations of donors to create each stimulus pool. Pooling also provided a standard female control stimulus that combined urine from donors in different stages of the oestrus cycle rather than reflecting a particular stage. Urine samples from individual captive-bred wild donor males were not pooled as each has a different individual genetic signature. Urine was collected up to 1-2 weeks prior to testing and stored at -20°C until use. A single large pool was used in each fractionation experiment to ensure that fractions were identical between behavioural replicates and biochemical analyses.ad libitum access to water and food . Cardboard tubes and red plastic mouse houses  were provided for home cage enrichment.Throughout, all animals were housed on a reversed 12:12 h light cycle with lights off at 0800 h, and were maintained on Corn Cob Absorb 10/14 substrate with paper wool nest material and Three days prior to each trial, soiled nest material and substrate from wild-derived males was introduced into the subject female's home cage to induce oestrus during the preference test . PreviouTests were conducted in a clean 45 × 28 × 13 cm arena (MB1 cage base fitted with a perforated Perspex lid ) to whicIn order to test sexual attraction to airborne urinary volatiles alone, and the influence of prior contact with male-specific urine components, females were first pre-exposed for 30 min in their home cage to a 55 mm GMF cut in half and streaked with 10 μL of each test stimulus (or water). Filter papers were held outside a mesh sphere that allowed full nasal contact with the test stimuli, or inside the mesh sphere for pre-exposure to airborne volatiles only, following the procedure in . Femaless+1) of behaviour durations approximated normality  and allowed parametric analyses . Non-parametric tests were used when log transformation did not approximate normality (Wilcoxon matched pair signed-ranks test). As it is widely established that females are more attracted to male than to female urine stimuli, and we needed to be conservative in concluding that manipulation of a urine signal eliminates this typical attraction to male urine, one-tailed statistical tests of greater attraction to male than to female or control stimuli were used throughout. In tests with scent contact, we also confirmed that the response observed after the first close sniff at a stimulus  was the same as the response over the whole trial, and that time under a stimulus did not differ between the two stimuli prior to this first close contact sniff (data not presented).In order to assess sexual attraction, we tested whether females spent more total time under the male rather than the female or control stimulus (nose within the 55 mm diameter circle in which a test stimulus was presented) during the 10 min trials. Time under a stimulus consisted of time spent sniffing or not sniffing the stimulus, each of which were recorded separately to provide further insight into the female's response. Time spent sniffing up at the cage lid within each 55 mm circle indicates the female's interest in gaining further information from a scent source and is part of the information gathering process, thus most sniffing occurs in the first few minutes of a test. Time under the stimulus when not directly investigating the stimulus (defined as the subject's nose being within the 55 mm diameter circle but not sniffing up at the lid) reflects non-investigatory attraction to spend time close to the scent source, and is thus likely to be a more robust measure of attraction ,31. In m2O (500 μL added and centrifuged at 13800 rpm for 15 min). Vivaspins were emptied of water and urine (500 μL) was added and centrifuged at 13800 rpm for 15 min. The LMW component was then pipetted into a clean capped 1.5 mL Eppendorf tube, labelled and stored at -20°C until use. Urine was added to the Vivaspin to make up to 500 μL and the process repeated. The HMW and LMW components were pipette into separate capped Eppendorf tubes, labelled and stored at -20°C until use. Protein concentrations of each fraction were determined with the Coomassie plus protein assay reagent kit .Samples were separated into HMW and LMW components using Vivaspin 500 centrifugal concentrators  with a 3000Da molecular weight cut-off. Vivaspins were washed twice using ddHt = 6 mL), equilibrated with several column volumes of 50 mM MES buffer, pH 5.0. Pooled urine was desalted on 5 mL Sephadex G-25 columns previously equilibrated with MES buffer, pH 5.0, then passed through a 0.45 μm filter. Typically 1 mL (~10 mg) of desalted protein was applied to the column. Bound protein was eluted from the column using a linear salt gradient of 0 - 1 M NaCl. Fractions (3 mL) were collected and fractions corresponding to individual MUP peaks were pooled then concentrated and desalted to deionised water  using Vivaspin centrifugal concentrators . Final protein concentrations were obtained by Coomassie dye binding assay. Purity and mass was confirmed by native and SDS-PAGE as well as electrospray ionisation mass spectroscopy.Purification of individual MUP peaks from C57BL/6 urine was achieved by high-resolution strong anion exchange chromatography on either the Duo-Flow  or the Äkta  chromatography platforms. Both systems were fitted with a Resource Q column  coupled to a QToF micro mass spectrometer , fitted with an ESI source. Samples (10 μL) were desalted and concentrated on a C4 reverse phase trap (LC packings). MUPs were eluted at a flow rate of 0.8 μL/min using repeated 0 - 100% acetonitrile gradients. Data were gathered between 800 and 1600 Th and were processed and transformed to a true mass scale using MaxENT 1 maximum entropy software . All data sets were processed at 0.25 Da/channel over a mass range of 18400 -19000 Da, and a peak width of 0.75 Da was used to construct the damage model. The instrument was calibrated initially using a 500 fmol/μL solution of Glu fibrinopeptide  in 50% acetonitrile and 0.2% formic acid and calibrated during runs with a 1 pmol/μL solution of horse heart myoglobin (Sigma), 2 mM DTT in 0.2% formic acid. All water used was HPLC grade Hexane extraction of urine and analysis of volatiles was performed according to the methodology in , using ade novo gene synthesis for maximal expression in E. coli. The gene was codon optimised for expression in E. coli and cloned into pET28b via NcoI and XhoI restriction sites . The plasmid was used to transform BL21(λ)DE3 cells and darcin expressed in Luria Broth containing kanamycin (30 μg/mL). At OD600 nm of between 0.6-0.8, the expression of recombinant darcin (r-darcin) was induced by the addition of isopropyl β-D-1 thiogalactopyranoside to a final concentration of 1 mM. Five hours post-induction, cells were harvested by centrifugation at 2000 × g and the cell pellets stored at -20°C prior to further purification. Harvested cells were lysed using Bugbuster protein extraction reagent  containing Complete™ EDTA-free protease inhibitor cocktail . Darcin, present in the soluble fraction of the bacterial cell lysate was purified by virtue of the hexahistidine tag on nickel affinity columns according to manufacturers protocols (Novagen). Column fractions containing r-darcin were pooled and dialysed against 50 mM phosphate, 20 mM NaCl pH7.4. This preparation was used without further processing. The purity of r-darcin was assessed by SDS-PAGE analysis and protein concentration was determined by protein assay. Similar workflows were applied to express the 18645Da MUP (accession GB/AAH91744.1) and 18694Da MUP (accession NP_001157998.1).The primary sequence of darcin was obtained from the sequence located under accession numbers NP_001012323/XP_355497 and was used to direct Recombinant darcin and other recombinant MUPs were tested alone (11 μg in 10 μL buffer) or added to stimulus urine samples  in order to mimic the natural concentration of darcin observed in B6 males  and in wild male mice ,32. To eESI: electrospray ionization; ESP: exocrine-gland secreting proteins; GMF: glass microfibre filter; HMW: high molecular weight; HPLC: high performance liquid chromatography; LMW: low molecular weight; MGI: Mouse Genome Informatics Database; MS: mass spectroscopy; MUP: major urinary protein; MHC: major histocompatibility complex; B6: C57BL/6 strain mice.JLH and RJB conceived the idea and gained funding for the study; SAR and JLH designed and carried out all the behavioural analyses; RJB guided all the molecular analyses. Urine fractionations and characterization were performed by SDA, AJD, DHLR and SAR; DMS and LM expressed the recombinant MUPs; JLH drafted the manuscript to which other authors contributed.Supplementary figures. Figure S1: Female sexual attraction to male urine: time sniffing and not sniffing. Figure S2: Female sexual attraction to MUP urine fractions: time sniffing and not sniffing. Figure S3: Female sexual attraction to male urine is elicited by darcin: time sniffing and not sniffing. Figure S4: Expression of recombinant darcin. Figure S5: Expression of 18645Da and 18694Da recombinant MUPs.Click here for file


Ctgf expression is associated with fibrosis in chronic organ injury. Studying the role of CTGF in fibrotic disease in vivo, however, has been hampered by perinatal lethality of the Ctgf null mice as well as the limited scope of previous mouse models of Ctgf overproduction. Here, we devised a new approach and engineered a single mutant mouse strain where the endogenous Ctgf-3′ untranslated region (3′UTR) was replaced with a cassette containing two 3′UTR sequences arranged in tandem. The modified Ctgf allele uses a 3′UTR from the mouse FBJ osteosarcoma oncogene (c-Fos) and produces an unstable mRNA, resulting in 60% of normal Ctgf expression . Upon Cre-expression, excision of the c-Fos-3′UTR creates a transcript utilizing the more stable bovine growth hormone (bGH) 3′UTR, resulting in increased Ctgf expression . Using the Ctgf Lo and Hi mutants, and crosses to a Ctgf knockout or Cre-expressing mice, we have generated a series of strains with a 30-fold range of Ctgf expression. Mice with the lowest Ctgf expression, 30% of normal, appear healthy, while a global nine-fold overexpression of Ctgf causes abnormalities, including developmental delay and craniofacial defects, and embryonic death at E10-12. Overexpression of Ctgf by tamoxifen-inducible Cre in the postnatal life, on the other hand, is compatible with life. The Ctgf Lo-Hi mutant mice should prove useful in further understanding the function of CTGF in fibrotic diseases. Additionally, this method can be used for the production of mouse lines with quantitative variations in other genes, particularly with genes that are broadly expressed, have distinct functions in different tissues, or where altered gene expression is not compatible with normal development.Connective tissue growth factor (CTGF) is a signaling molecule that primarily functions in extracellular matrix maintenance and repair. Increased  Many of the diseases that have a major impact on human health and pose a major burden on healthcare costs have a fibrosis-related component. Coronary heart disease (CHD) was the single largest killer in the United States in 2003, accounting for 1 in every 5 deaths Ctgf is widely expressed during development, and in adulthood it is expressed in all major organs including the vasculature and the skeletal system Ctgf expression Ctgf expression is clearly associated with fibrosis in multiple tissues including skin, liver, heart, lung, and kidney , a primary fibrosis-related signaling molecule in vivo, several animal models have been created. The first is a traditional knockout, which when homozygous, is perinatally lethal. The pups are born, but do not survive due to skeletal deformity, including a malformed rib cage which causes an inability of pups to breathe properly. The heterozygous knockouts survive and have little discernable fibrosis-related phenotype Ctgf overproduction in bone and cartilage, cardiomyocytes, kidney podocytes, hepatocytes, lung epithelia and fibroblasts have also been made using promoter-enhancers constructs specific to each cell type Ctgf gene expression is disrupted and the mechanism by which altered steady state Ctgf gene expression influences non-target tissues has not been described. Thus, the role of Ctgf expression in fibrosis remains unclear and there is a need for animal models with variable Ctgf expression in which there is minimal alteration in the natural transcriptional regulation to clarify how altered Ctgf expression levels may predispose or change the progression of heart disease and other fibrotic diseases.To examine the role of CTGF in vivo from low to high. The endogenous 3′-untranslated region (3′UTR) is replaced with a cassette containing the 3′UTR from the FBJ osteosarcoma oncogene  and the 3′UTR from the bovine growth hormone (bGH) gene, also known as somatotropin (ENSBTAG00000017220), placed in tandem. Each 3′UTR modulates the stability of the mRNA transcript and can respectively decrease (c-Fos) or increase (bGH) steady state levels of mRNA in both cells and whole animals Here we describe an innovative method of modulating gene expression by making one animal that can express either low or high levels of a gene. This is made possible by use of Cre-recombinase to alter expression Ctgf when it has reduced expression, increased expression, or when expression is altered in a tissue-specific manner. Our results show that Ctgf expression reduced to 30% of wild type is survivable, while expression nine fold higher than wild type leads to embryonic lethality by day E12.5. Thus, we have created a 30-fold range of Ctgf expression in vivo, which should prove useful in further understanding the function of CTGF in fibrotic disease.Using this method, we have created a single construct that allows the examination of the role of Ctgf gene, we have made a targeting construct to delete exons 3 through 5 of the Ctgf gene in the genome in ES cells as illustrated in Ctgf KO allele appear normal and express the Ctgf gene at approximately 50% levels. As reported by Ivkovic et al, homozygous mice completely lacking CTGF are born but die within 16 hours In order to inactivate the Ctgf transcripts in vivo using 3′UTR replacement with the bGH-3′UTR Ctgf expression and generate adult mice overexpressing the Ctgf gene, we devised a novel strategy using a low-high cassette (Lo-Hi) that allows us to change gene expression levels from low to high by altering mRNA stability , the neomycin resistant gene (neo), a second lox P site, and the bGH-3′UTR. The modified allele, Ctgf-Lo, uses the c-Fos-3′UTR and so its transcripts are less stable than those from the wild type Ctgf allele. However, a Cre-mediated recombination between the two loxP sequences in the Lo-Hi cassette excises the c-Fos-3′UTR and the neo gene and generates the Ctgf-Hi allele that uses bGH-3′UTR  allele are born and appear normal. Ctgf Lo/+ mice were first intercrossed to determine if the Ctgf Lo/Lo phenotype is viable. Pups were born from this cross at a 10∶18∶5 genotypic ratio (+/+: Lo/+: Lo/Lo). The number of Ctgf Lo/Lo pups was about half the expected Mendelian ratio of 1∶2∶1 for the cross, but the Chi square test was not significant . The Ctgf Lo/+ and Ctgf Lo/Lo pups are healthy, reproduce normally, and are indistinguishable from their wild type and heterozygous littermates.Heterozygotes for the Ctgf Lo/Lo mice are expected to have reduced Ctgf mRNA expression throughout the body. Tail snips from 10 day old pups were used to assay for Ctgf mRNA expression. Ctgf Lo/Lo mice express about 60% of wild type levels of Ctgf. The Ctgf Lo/Lo mice are similar in Ctgf expression level to the Ctgf heterozygous knockout (Ctgf KO/+) mice which express 50% of wild type Ctgf. Ctgf Lo/+ heterozygotes have an intermediate Ctgf expression level that is 85% of wild type mice  to generate mice with very low Ctgf expression (Ctgf Lo/KO). Ctgf Lo/KO mice were born at the expected Mendelian ratio of 1∶1 (17 Lo/+: 15 Lo/KO). Tissues from adult siblings  were assayed for Ctgf mRNA expression by RT-PCR. Ctgf Lo/KO heart tissue had significantly lower Ctgf expression /3 = 28.7%). The Ctgf Lo/KO animals reproduce adequately, are healthy, phenotypically normal in appearance, and display no overt structural phenotypes.Ctgf-Lo allele excises the c-Fos-3′UTR and the neo gene, this generates the Ctgf-Hi allele that uses the bGH-3′UTR. To generate mice carrying a Ctgf-Hi allele, Ctgf Lo/+ mice on a B6.129 mixed genetic background were crossed to a heterozygous whole body Cre expressing mouse line 129.Tg(Ella-Cre) (from B6.FVB-Tg(EIIa-cre) C5379Lmgd/J stock that was backcrossed to 129/SvEv for 18 or more generations). Heterozygotes for the Ctgf Hi allele (Ctgf Hi/+ mice) were defined as presence of the bGH-3′UTR, presence of a Cre allele and low Neo gene copy number. After seven litters and 40 pups, no live mice carrying both the modified Ctgf allele and the EIIa-Cre transgene were recovered, suggesting the Ctgf-Hi allele causes an embryonic lethal phenotype.When Cre is present, a Cre-mediated recombination between the two loxP sequences within the Ctgf Hi/+ embryos were recovered at a ratio of 10∶15∶11∶7  which was not different from the expected Mendelian genotypic ratio of 1∶1∶1∶1 .To examine the timing and nature of the lethality, we used timed matings and recovered embryos between E13.5 and E14.5. At this embryonic age, Ctgf Hi/+ embryos were present among 43 embryos at E13.5-E14.5 . Small atypical embryos were developmentally delayed, macerated, or poorly vascularized with a body weight below two standard deviations of the litter average. By this classification method, no normal .5-E14.5 . All of sorption . This suCtgf Lo/Lo mice on a 129/SvEv background were crossed with 129.Tg(Ella-Cre) mice. After genotyping three litters, no Ctgf Hi/+ embryos were recovered at E13.5, suggesting an earlier lethality in the fully inbred background. Embryos on a 129/SvEv background were then harvested at E10.5  Cre+ males and the embryos were harvested at E11.5 and assayed for whole body Ctgf gene expression using RT-PCR. Ctgf expression varied greatly in Ctgf Hi/+ embryos, ranging between two and fifty times greater than the mean of Ctgf Lo/+ embryos. Most likely this is due to variations in developmental stage, embryo condition, and variations in Cre expression. On average the Ctgf Hi/+ embryos had a greater than ten fold increase in Ctgf mRNA levels compared to the Ctgf Lo/+ littermate controls, which is a nine fold increase in Ctgf expression over WT .To validate that excision of the Ctgf mRNA levels may be causing changes in the levels or pattern of CTGF protein in Ctgf Hi/+ embryos. Thin sections of embryos were stained with a CTGF antibody and a fluorescent seconday then counterstained with DAPI to visualize the level and pattern of CTGF protein. CTGF was visible in almost evey tissue of the embryos and appeared to be primarily staining the extracellular matrix and the cell surface  escaped embryonic lethality and survived to adulthood . One male and one female of the surviving animals were siblings from a mixed B6.129 mating. Two additional male survivors came one year later, one from a mixed B6.129 mating and one from a 129/SvEv mating. All four mice exhibited the same phenotype: a shortened face, small ears, a shortened and kinked or curled tail, and a shortened overall body length  to assess whole body: bone mineral density (BMD), bone mineral content (BMC) and body composition . Although the number of samples was small and the animals were in different genetic backgrounds, all of the Ctgf Hi/+ animals had significantly smaller bone area and lean mass than controls . Organ weights tended to be smaller with both kidney and lung significantly smaller in the controls . The orgCtgf Hi/+ survivors and siblings or age-matched controls were harvested and assayed for Ctgf gene expression using RT-PCR. There was a significant increase in Ctgf expression in each tissue .Heart, kidney and lungs from h tissue . The heaCtgf expression suggests that while high Ctgf expression is embryonic lethal, in an adult it is compatible with survival. To test this hypothesis we crossed Ctgf Lo/Lo mice with CAG-Cre mice, which are a tamoxifen-inducible global Cre expresser strain Lo-Hi allele functioned as predicted with excision of the c-fos-3-UTR and neo  to generate the smaller the Hi allele in the presence of tamoxifen and the inducible Cre gene  was approximately four-fold higher than in Ctgf Lo/+ mice without the Cre gene  are born but die within a day.Previous studies have shown that altering or interchanging the 3′UTR of a gene is an effective method for altering gene expression in cells and in animals Ctgf Hi allele had increased Ctgf mRNA levels that were nine-fold higher than WT in embryos and the increase in Ctgf expression was embryonic lethal, depending on genetic background, between day E11 and E13.5. The lethality of the Ctgf Hi allele, made from the Lo-Hi allele, recapitulates the lethality observed in our previous attempts to directly generate a Ctgf Hi allele with the bGH-3′UTR. Embryos from both 129/SvEv and B6.129 mixed backgrounds display craniofacial defects consistent with alterations to the first and second pharyngeal arches. The fissures between the arches in the mutant E10.5 embryo in Ctgf Hi/+ embryos, suggesting that this defect was a result of high Ctgf gene expression. Defect of the pharyngeal arches, rostrum and midline in Ctgf Hi/+ embryos are in congruence with previous reports that Ctgf is highly expressed in these regions at E9.5-E10.5 We found that embryos with a Ctgf Hi/+ embryos by themselves are not necessarily lethal Ctgf Hi allele is embryonic lethal. Craniofacial defects are often observed in conjuction with cardiac defects and both phenotypes are thought to be caused by improper migration and differentiation of neural crest cells TGFβ) family knockout mutant mouse strains and were thought to be caused by defective neural crest cell migration TGFβ isoforms are most commonly thought to increase Ctgf expression Tgfβ2 can inhibit Ctgf expression in utero, loss of Tgfβ2 could lead to increased levels of CTGF, creating a similar phenotype to our model of increased Ctgf expression. Therefore, dysfunction of neural crest cells, which likely causes the craniofacial defects in Ctgf Hi/+ embryos, may be the ultimate reason for the embryonic lethality. No overt defects were observed in the heart in our limited histological analysis, and immunohistochemical staining revealed that Ctgf expression may be generally increased in Hi/+ embryos but is not consistent. Despite the large increase in mRNA, immunohistochemistry (IHC) does not show large differences in CTGF levels suggesting that IHC may not be sufficiently sensitive or that translational regulation may be occurring. Future experiments using in situ hybridization and/or tissue-specific overexpression of Ctgf should focus on defects in neural crest cells, especially in the heart, as a possible cause for the Ctgf Hi/+ embryonic lethality.The generalized developmental delay and craniofacial defects like those seen in Ctgf Hi/+ embryos include failures of vasculogeneis or erythropoeisis, both are crucial developmental milestones of E10-E12 embryos Ctgf Hi/+ embryos recovered appeared generally pale and may have lacked proper circulation. Vascular endothelial growth factor (VEGF) is known to be an essential factor in vasculogenesis and is thought to have a complex regulatory relationship with CTGF Ctgf Hi/+ embryos Alternative causes for lethality in Ctgf Lo and Ctgf Hi alleles and likely escaped lethality due to low Cre expression during embryogenesis. Like the embryos, the survivors express significantly increased Ctgf as well as exhibit craniofacial defects in the form of a shortened rostrum. In addition, similar to previously reported Ctgf high expression transgenics, Ctgf Hi/+ survivors are true high expressers.An unexpected benefit of the uneven penetrance of the EIIa-Cre has been the few animals that have escaped the embryonic lethality. Genotyping showed that the survivors do not have complete neo excision in tail DNA suggesting they are mosaics for the Ctgf expression is moderately increased in heart (two-fold) and substantially increased in the kidney and lung .The EIIa-Cre line is known to have a mosaic pattern of Cre expression with varying excision efficiency in different tissues as well as large variability between animals in the amount of Cre-mediated excision. Previous reports have shown that with the Ella-Cre as few as 50% of animals undergo high levels of Cre-mediated excision in the first generation Ctgf Hi/+ allele is embryonic lethal. Therefore, due to their rarity and lack of direct transmission of the Ctgf Hi/+ allele, these animals are not a tenable model for Ctgf overexpression.These surviving animals are rare with only four animals surviving the embryonic lethality in two years of breeding. Matings of survivor males never produced offspring that carried a fully floxed (neo negative) high expressing allele, reinforcing that the Ctgf expression in an adult is not lethal. With only a moderate dose of tamoxifen, these animals express a four-fold increase in Ctgf mRNA and a nearly two fold increase in CTGF protein abundance in plasma and do no appear to suffer any immediate ill effects. Unlike the low Ctgf expression models where decreases in protein levels paralleled that of mRNA, in the Ctgf Hi/+ there is a two-fold difference between the protein abundance in plasma (1.7-fold increased) and the tissue mRNA gene expression (3.6-fold increased). The discrepancy may be due to the inaccuracy of the method used to quatitate the protein or to unknown regulation at the translational level. The increase in Ctgf mRNA was not as high as in the embryos so a higher tamoxifen dose, or treatment at a younger age, may increase Ctgf expression to a level closer to the nine-fold increase we observe in embryos.We then used a mouse strain that expresses tamoxifen-inducible Cre gene and showed that high Ctgf expression by treatment with a drug or a monoclonal antibody may be a worthwhile therapeutic strategy in treating fibrotic disease Ctgf Hi/+ survivors and the externally normal phenotype of the tamoxifen treated Ctgf Hi/+ animals suggests that genetically increasing Ctgf expression by itself is not sufficient to cause fibrosis. This is consistent with a report which showed that exogenous application of CTGF to the skin alone is not sufficient to cause fibrosis Ctgf in cardiomyocytes Ctgf in lung respiratory epithelial cells in vivo level of Ctfg expression was not established Ctgf expression or that the transgene is not responsive to normal transcriptional regulation of the Ctgf allele at the endogenous locus. Altogether, the current literature does not preclude a role for CTGF in fibrotic disease, but it suggests that activation of other factors or the application of stress may be required in addition to Ctgf overexpression in order to cause fibrosis.Previous reports have implicated CTGF as a profibrotic signaling molecule leading to the suggestion that reducing Ctgf expression has been observed in conjunction with fibrotic pathology in a number of organs and disease conditions. However, an unequivocal role for Ctgf as a causal factor in pathological fibrosis remains questionable. An allelic series with a wide range of genetically altered Ctgf gene expression provides a tool to test, the effectiveness of reducing Ctgf expression, in vivo, to prevent fibrotic disease and determining if increased expression of Ctgf is causal in fibrotic disease. In addition, by generating mouse lines with varying Ctgf expression using the endogenous locus, the function of Ctgf in fibrotic disease can be tested more effectively.Increased Lo-Hi allele in combination with existing resources allows the production of an allelic series of quantitative variants. We also note that, the construct can be arranged to produce a Hi-Lo allele which high expression allele can be switched to low expression allele. Altogether, this method will likely be particularly useful for genes with multiple separate functions, genes expressed in many tissues, as well as in cases where altered gene expression causes embryonic lethality.We note that this method of altering gene expression, through altering a gene's 3′UTR, avoids many of the issues of random insertion transgenes including insertional mutagenesis. Additionally, a single founder with the All animals were cared for in accordance with guidelines set forth by the Association for Assessment and Accreditation of Laboratory Animal Care. The University of North Carolina – Chapel Hill's “Institutional Animal Care and Use Committee” (IACUC) approved all studies (protocol #08-045 and #07-228).Ctgf gene, we have made a targeting construct to delete exons 3 through 5 of the Ctgf gene in the genome in ES cells as illustrated in Ctgf-Lo allele , neo, a second lox P site, and the 3′UTR from the bovine growth hormone gene (bGH-3′UTR). A 1060 bp fragment of DNA including the Ctgf-3′UTR sequence, 510 bp of 3′flanking and the polyA addition signal were removed from the endogenous locus leaving the 20 bp immediately following the TAA stop codon intact.In order to inactivate the o allele  was madeGene targeting in mouse ES cells (TC1) from 129S6/SvEvTac (129/SvEv) was carried out using the procedures described previously Ctgf KO and Ctgf Lo-Hi mice were established and maintained on a 129S6/SvEvTac (129/SvEv) background. The Ctgf Lo-Hi mice were also backcrossed to C57BL6/J (B6) then intercrossed two to three generations to create a mixed B6.129 background. Initial experiments were with these mixed background animals as noted.Both the Ctgf Lo-Hi mice.The B6.FVB-TgN(EIIa-Cre)C5379Lmgd/J (EIIa-Cre) constitutive global Cre expressing mice were a gift from Dr. Westphal at the NIH chicken beta actin promoter/enhancer coupled with a cytomegalovirus enhancer (CAG) under control of a mutant mouse estrogen receptor ligand binding domain. The latter receptor ligand domain is unresponsive to estrogen but sensitive to the synthetic ligand 4-hydroxytamoxifen (tamoxifen). Therefore, the CAG-Cre allele produces robust expression of Cre only in the presence of tamoxifen The inducible global Cre expressing mice are B6.Cg-Tg(CAG-cre/Esr1)5Amc/J from The Jackson Laboratory, Bar Harbor, ME stock number: 4682 (CAG-Cre) Ctgf KO animals were genotyped by PCR with the primers: 5′-TCG AGT TCA GAA CCA GAG CT-3′ (common), 5′-TCC GAT TCC TAC CAG GAA GT-3′ (endogenous), and 5′-TTA TGG CGC GCC ATC GAT CT-3′ (neo) with the conditions  for 35 cycles. Because amplification is passing through the highly repetitive 3′UTR region the amplification step is done at a lower temperature to improve Taq fidelity. The endogenous band is 550 bp and the KO band is 320 bp.Animals were genotyped as appropriate using DNA from either toe or tail snips using standard protocols. Ctgf Lo alleles were genotyped by PCR with the primers: 5′-CAC TCT GCC AGT GGA GTT CA-3′ (common), 5′-TAA TTT CCC TCC CCG GTT AC-3′ (endogenous), and 5′-CAC AGC CTG GTG TGT TTC AC-3′ (c-Fos) and the conditions  for 35 cycles. The endogenous band is 525 bp and the Ctgf KO band is 375 bp.Ctgf Hi allele was performed using fluorescent primer probe sets in real time PCR with ABI 7500 Fast Real Time PCR 3 system . The Ctgf Lo allele was defined as the presence of the bGH-3′UTR and neo and the absence of Cre (where applicable). The Ctgf Hi allele was defined as the presence of both the bGH-3′UTR and Cre as well as a the absence or low neo gene copy number. Primer sets for bGH-3′UTR: TGC CAG CCA TCT GTT GTT TG (forward), ACA GTG GGA GTG GCA TCT T (reverse), and FTC TCC CCC GTG CCT TCC TTG AQ (probe), for neo: GAC GGC GAG GAT CTC GTC G (forward), TAT GTC CTG ATA GCG GTC CG (reverse), and FAC CCA TGG CGA TGC CTG CTT GCC GQ (probe), and for Cre: GGC AGT AAA AAC TAT CCA GCA (forward), GCC GCA TAA CCA GTG AAA CA (reverse), FAT TGC TGT CAC TTG GTC GTG GCA GCQ (probe). For all probes F is 5′ fluorescein (FAM) and Q is the 3′ quencher (TAMRA).Genotyping for c-fos-3′UTR and neo) portion of the Ctgf Lo-Hi allele.When genotyping by real time PCR relative gene copy number for the allele of interest, in this case the neo gene, is quantitatively measured. We have used the relative neo gene copy number information from RT-PCR genotyping as a proxy for determining the efficiency of excision of the Lo  mothers were sacrificed and embryos were recovered live. The embryonic sac (amnion) was separated from each embryo and used for genotyping. Each intact embryo was rinsed in PBS then weighed whole (when possible) and assessed for phenotype. At recovery, embryos were phenotypically classified based on body weight and gross appearance before genotype was determined. “Normal” embryos were nondysmorphic in appearance and at the expected developmental stage for the embryonic age and were within two standard deviations (heavier or lighter) of the mean embryo weight for that litter. “Small” embryos were nondysmorphic in appearance and at the expected developmental stage for the embryonic age, but were small in size and had a body weight below two standard deviations of the litter average . “Small atypical” embryos were developmentally delayed, macerated, or poorly vascularized and had a body weight below two standard deviations of the litter average. Embryos were fixed with 4% paraformaldehyde or gluteraldehyde as needed for further studies.Ctgf mRNA expression were: AGT CGC CTC TGC ATG GTC A (forward), GCG ATT TTA GGT GTC CGG AT (reverse), and FCC TGC GAA GCT GAC CTG GAG GAA AQ (probe). All samples were normalized to β-actin using primer set: AAG AGC TAT AGA CTG CCT GA (forward), ACG GAT GTC AAC GTC ACA CT (reverse), and FCA CTA TTG GCA ACG AGC GGT TCC GQ (probe). Additional samples for gene expression were either tail samples from 10–12 day old pups or whole embryos harvested from timed matings at embryonic day E10-12 (days post-coitum). For tamoxifen treated animals, three weeks after tamoxifen treatment ear tissue biopsies were taken by small punch on lightly anesthetized  mice at 3–4 months of age.Mice were euthanized with an overdose of 2,2,2-tribromoethanol and mRNA was harvested from tissues for gene expression profiling. Assays were performed by real-time quantitative RT-PCR as previously described Embryos were recovered and sectioned as described above. Sections were deparrafinned then stained with Rabbit anti-CTGF primary antiboday at 1∶100 or 1∶500 dilution  and a anti-Rabbit Alexa Fluor 594 at 1∶200 dilution . Sections were counterstained with DAPI to visualize nuclei. To correct for autofluorescence, for each section there was an adjacent section stained with secondary antibody only. Sections were examined on a Nikon Eclipse 80i and photographned with Nikon DS-QiMc 12mp digital camera and NIS Elements BR 3.0, SP6 (build 539) software.Ctgf Hi/+ or controls .Using Dual Energy X-ray Absorptiometry (DEXA) we measured body mass composition and bone density. Mice were lightly anesthetized with isofluorane then scanned, excluding the head, using PIXImus DEXA . Measurements and calculations were performed as previously described Ctgf Lo/Lo mice were crossed to the tamoxifen inducible CAG-Cre mice to produce Ctgf Lo/+ and +Ctgf Lo/+; Cre mice. At six weeks of age mice were treated with tamoxifen at a dose of 3 mg/kg of body weight for four consecutive days +Ctgf Lo/+; Cre mice are designated with the genotype Ctgf Hi/+. Mice were monitored for any overt phenotypic changes.The Ctgf-Lo was allele amplified with primer1  and primer2  and the Ctgf-Hi allele amplified with primer1 and primer3  10% polyacrylamide gels were loaded with 1 µL of plasma mixed with loading buffer. Gels were run, transferred and blotted by standard methods. Membranes were blocked using 10% nonfat milk in tris-buffered saline with Tween (TBST). Primary CTGF antibody  was applied overnight 1∶2000 dilution. Secondary  was applied for 3 hours 1∶5000 dilution. Chemiluminescence  was applied per manufacturer instructions. Band density was quantitated with ImageJ software.Adult mice were lightly anesthetized with 2,2,2-tribromoethanol then photographed live . Whole fixed E13.5 embryos were pictured using a dissecting macroscope  with a QImaging camera . Whole unfixed E10.5 embryos were pictured with embryos immersed in buffered saline using light microscopy . Embryos were then fixed and prepared for scanning electron microscopy (EM) as previously described Data are presented as mean ± standard error of the mean and p-values are from paired Student t-test performed in Microsoft Excel. Additional statistics including Chi Squares were performed by hand. Any p-value less than 0.05 was considered statistically significant.


X<0 kcal/mol) around a proteinlike motif (X) are capable of inducing antibodies with similar affinity for both peptide and native protein, more weakly stable peptides  trigger antibodies recognizing full protein but not peptide, and unstable peptides  fail to generate antibodies against either peptide or protein. Immunization experiments involving peptides derived from the autoantigen histidyl-tRNA synthetase verify that selected peptides with varying relative stabilities predicted by molecular dynamics simulations induce antibody responses consistent with this theory. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the development/evolution of humoral immune responses.In the conventional paradigm of humoral immunity, B cells recognize their cognate antigen target in its native form. However, it is well known that relatively unstable peptides bearing only partial structural resemblance to the native protein can trigger antibodies recognizing higher-order structures found in the native protein. On the basis of sound thermodynamic principles, this work reveals that stability of immunogenic proteinlike motifs is a critical parameter rationalizing the diverse humoral immune responses induced by different linear peptide epitopes. In this paradigm, peptides with a minimal amount of stability (ΔG In the current paradigm of immune system recognition, T cell receptors bind to relatively short peptide sequences complexed with major histocompatibility complex proteins on the surface of antigen presenting cells, while B cell receptors recognize unprocessed protein structures. Yet, ample data exist showing that peptide immunization can trigger B cell responses targeting both the immunizing peptide and peptidelike motifs contained within intact protein—despite the fact that the folding stability of such peptides is often quite low. Using thermodynamic modeling and the technique of molecular dynamics simulations, this work provides a cogent framework for understanding the relative capacity of inherently unstable peptide structures to faithfully trigger B cell antibody production against specific conformational motifs found in native/intact proteins. In the conventional paradigm of humoral immune responses, B cells recognize conformational epitopes of protein antigens through interactions with surface expressed immunoglobulin receptors Neisseria meningitidis protein PorA correlates with immunogenicity. However, because typical linear peptides are inherently unstable, with stabilities that are virtually impossible to assess due to the lack of a well defined folded (reference) state, more complete elucidation of the molecular mechanism(s) underlying these empirical observations remains elusive. Underscoring the complexity of this problem, an analysis involving a helical motif of the enzyme barnase represents the only published measurement of peptide folding free energy  Based on this mechanism of B cell activation and immunoglobulin production, native protein should be highly immunogenic relative to short peptide sequences less than 20 amino acids in length. While this concept may hold true for many antigens, the existing literature does provide examples of peptides capable of stimulating antibody production not only against the immunizing peptide, but also against corresponding regions of the native protein In the current study, we have reexamined this issue through detailed analysis of serologic profiles generated in mice immunized with overlapping 18 amino acid peptides comprising the amino terminal portion of histidyl-tRNA synthetase (HRS = Jo-1), an autoantigen implicated in the pathogenesis of idiopathic inflammatory myopathy and the anti-synthetase syndrome Beyond the definition of immunodominant peptides dictating B cell recognition of HRS peptide/protein combinations, this analysis has permitted correlation of the humoral immune response with structural and thermodynamic determinants of peptide immunogenicity. Of note, molecular modeling calculations indicate that although peptides are intrinsically disordered and therefore less stable than full protein, they are capable of adopting relevant structural “mimetopes” with enough stability to trigger humoral responses against corresponding regions of native protein. Immunization experiments verify that selected peptides predicted to form higher order structures similar to those existing in parent proteins induce significant antibody responses against intact protein. Moreover, competition experiments show that several of these immunogenic peptides are able to bind to stimulated antibodies with similar affinity to that of the full protein. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as a tool to probe the development/evolution of humoral immune responses.X) of their protein-like motifs defines three classes of peptides possessing very different immunogenic properties. These categories include: (a) “stable” peptides  that can form the same number of peptide-antibody ([XAb]) complexes as stable protein despite a wide range of folding free energy values; (b) “weakly-stable” peptides with ΔGX>0 kcal/mol  that have a drastic decrease in antibody binding events relative to the full protein; and, (c) “unstable” or “non-immunogenic” peptides with ΔGX>8 kcal/mol and resulting unfolding rates of 109 s−1 or higher that preclude any effective binding To establish a thermodynamic basis for previous observations linking peptide immunization with humoral immune responses against native protein structural motifs, we examined the relationship between peptide folding stability and antibody-antigen binding. Although the capacity of intrinsically disordered peptides to generate and effectively bind antibodies recognizing three-dimensional epitopes appears counterintuitive, the kinetic scheme in Idd3/5 mice. As shown in As an example of the epitope classification scheme derived from this thermodynamic analysis, we have mapped relevant B cell epitopes of histidyl-tRNA synthetase (HRS) through peptide immunization of NOD.protein  by two weeks, most notably peptides 1 (a.a. 1–18), 4 (a.a. 31–48), 6 (a.a. 51–68), 7 (a.a. 61–78), 8 (a.a. 71–88), and 9 (a.a. 81–98). Temporal assessment of anti-HRS protein antibody responses induced by these peptides and comparison to antibody responses against the immunizing peptide  triggers affinity maturation towards MA/MBP and/or peptide, whereas peptides with less stable backbone structures  typically do not promote this temporal pattern of increasing antibody titer. Of note, MD simulations indicate that for those peptides capable of adopting higher order structure, the identified motifs can persist for several nanoseconds—a time period sufficient for antibody recognition A more detailed analysis of the hydrogen bond (HB) networks X values allow maximal peptide-antibody complex formation. In contrast, this combined analysis indicates that peptides 4 and 6 are weakly stable, with ΔGX values that favor diminished antibody binding of peptide relative to full protein. This classification is fully consistent with ELISAs  might be responsible for the weak anti-peptide response shown in Coupled with the thermodynamic modeling of h ELISAs  showing h ELISAs , and no h ELISAs  is flankCollectively, these studies show that several peptides corresponding to the amino terminal portion of murine HRS are capable of inducing anti-protein antibodies of varying affinity and temporal persistence. As shown by molecular dynamics simulations, sequences of the most immunogenic peptides correspond to highly ordered structural motifs in the parent protein. Competitive ELISAs provide direct evidence that these peptides share structural determinants with native protein by demonstrating the relative equivalence of antibody affinity for HRS protein (MA/MBP) and selected peptides . Of greater significance, first principle calculations and molecular dynamics simulations underscore the thermodynamic and structural basis of these experimental observations.Among the most interesting findings emerging from the experiments summarized in f values  in which peptides are capable of triggering an immune response similar to full protein. Hence, as long as the peptide fold resembles that of the full protein, this class of peptides , this instability does not translate into an equivalent drop in affinity towards the repertoire of B cells receptors. Indeed, thermodynamic calculations in From a modeling point of view, molecular dynamics (MD) simulation in explicit solvent represents the most accurate approach to assess peptide stability. Although this technique has time limitations that prevent a full thermodynamic analysis of each peptide, the 10 nanosecond period used here is sufficient to assess the stability of protein-like conformations relevant to the comparison of peptide- versus protein-targeted antibody responses. Clearly, the MD simulations demonstrate a wide range of structural stabilities over 10 nanosecond runs; in the case of peptides 1 and 9, however, the composite structural motifs greatly resemble those presented by full protein, confirming that helical as well as some proline-based linear motifs can preserve their structural integrity over a time frame that is fully compatible with molecular recognition Perhaps the differences in stability and antibody binding affinity between overlapping sequences of peptide 9 (amino acids 81–98) and peptide 10 (amino acids 91–108) best illustrate the power as well as predictive potential of MD simulation. While competition ELISAs demonstrate that sera derived from peptide 9-immunized mice recognize both peptide 10 and peptide 9 (consistent with the immunodominant proline-containing epitope suggested by MD that encompasses amino acids 93–96), the relative affinity for peptide 9 exceeds that for peptide 10 by a log order of magnitude (data not shown)—a result that again correlates with MD simulations showing that the same proline-containing motif is significantly destabilized by surrounding sequence in peptide 10, but not in peptide 9 . Second, identification of immunodominant peptide epitopes will permit more detailed categorization of disease subsets and correlation with disease activity. Finally, this computational tool will facilitate the prediction and design of immunodominant peptide epitopes that can be used to define novel autoantibody specificities in patients with underlying autoimmune diseases. Through such identification of autoantigen panels, this approach may provide insight regarding more general epigenetic shifts that generate multiple autoantigens and ultimately lead to autoimmunity.In situ mutagenesis  with insertion of a stop codon after base pair 453 yielded a construct encoding the amino terminal 151 amino acids of murine HRS fused to MBP (MA/MBP). Expressed proteins were purified with amylose resin per the manufacturer's protocol , filter sterilized, and then subjected to additional column purification for endotoxin removal  prior to use in ELISAs.Overlapping peptides (18–20 mers) comprising the amino terminal 108 amino acids of murine histidyl-tRNA synthetase (HRS) were synthesized and HPLC purified by the University of Pittsburgh Molecular Medicine Institute using Fmoc chemistry. As previously described, recombinant murine HRS was generated as a maltose binding protein (MBP) fusion protein following subcloning of the appropriate sequence (derived from RT-PCR amplification of C57BL/6 myocyte RNA) into the bacterial expression vector pMALc2  Idd3/5 (C57BL/6 Insulin dependent diabetes Idd3/5 non-MHC loci transgressed onto the NOD background) mice were bred in our animal facility. Eight to ten week old mice were used in immunization protocols approved by the University of Pittsburgh IACUC. PBS containing 90 µg of the indicated peptides was emulsified with CFA in a 1∶1 ratio and then injected at the base of the tail in a total volume of 200 µl. Pertussis toxin  was administered intraperitoneally (200 ng/mouse in 100 µl PBS) at the time of immunization and 48 hours later. Mice were tail-bled 2 and 4 weeks after immunization. 8 weeks post immunization, mice were sacrificed, and additional blood was collected from the heart.NOD.2SO4. Color development was measured at 450 nm by a Wallac 1420 multilabel counter , and values were plotted as OD450 substrate antigen - OD450 no antigen. All assays were performed in triplicate wells.Standard solid phase ELISAs provided measurements of IgG anti-MA/MBP and anti-HRS peptide antibody levels in the sera of mice immunized with different HRS peptides Plates were coated and blocked as described above. Diluted serum samples (1∶250) were mixed in a 1∶1 ratio with serially diluted MA/MBP or HRS peptide solutions in microtubes and preincubated for 30 minutes at room temperature. Preincubated samples  were then applied to the plates and incubated for another 2 hours at room temperature. ELISAs were completed using the same protocol as described above.Mus musculus histidyl-tRNA synthetase (HRS = Jo-1) in Thermoplasma acidophilum HRS . With more than 25% sequence identity, including perfect matching of prolines and glycines in the domains of peptides 1 to 9 listed in The structural model of f. The results in 6 M−1s−1 and 10−1 s−1 , respectively f ([Xf]) than on peptide stability. For simplicity, we assume a concentration of antibody ([Ab]) and protein ([X]) equal to 1 µM. However, the overall shape of the curve does not change significantly with a higher or lower [Ab]. For [Ab]>1 µM, the maximum amount of complex [XAb] remains the same, but the stability thresholds  Folding of protein X and binding of X with antibody Ab. (B) Alignment of amino terminal 1–151 Histidyl tRNA synthetase from Mus musculus (MA) and Thermoplasma acidophilum (PDB code 1WU7)(0.54 MB TIF)Click here for additional data file.


We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, 51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56bright NK cells represented 28% and 45%, respectively, of all NK cells . The patients also displayed a higher percentage of CD56dimCD16− NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56 They constitute a heterogeneous blood cell population and subsets can be defined depending on their level of expression of CD16 and CD56. In healthy individuals, CD56dimCD16bright cells represent at least 90% of all NK cells bright subset that can be further subdivided in a CD16− (30–50% of CD56bright) and a CD16dim (50–70% of CD56bright) fraction dimCD16− and conversely CD56−CD16+ phenotype have been described Human natural killer (NK) cells are phenotypically defined as CD3The major functional properties of NK cells are cytotoxicity and cytokine production, which are governed by a balance between activating messages transmitted by activating receptors (AR) and inhibitory signals transmitted by inhibitory receptors (IR), respectively + T cells. Newly synthesized HLA class I molecules acquire their peptidic ligands in the endoplasmic reticulum (ER) HLA class I molecules present endogenous peptides to cytotoxic CD8+ T cell responses to TAP-independent antigens are sufficiently efficient Nineteen human cases of TAP deficiency have to date been described + normal NK cells but not by autologous resting NK cells. Nevertheless, resting TAP− NK cells are able to perform antibody-dependent cellular cytotoxicity (ADCC) to some extent, and after stimulation with interleukin (IL) 2, they become cytotoxic not only towards tumor cell lines, but also to two types of autologous cells, namely Epstein-Barr virus (EBV)-transformed B lymphoblasts − NK cells in cytotoxicity tests against allogeneic normal cells. Thus, the mechanism responsible for the hyporesponsiveness of resting TAP-deficient NK cells is still unknown.The symptomatic patients have a very large (30- to 100-fold) reduction in the cell surface expression levels of HLA class I molecules, as compared to healthy individuals bright cells among the patients' NK cells bright NK cells and the over-expression of NKG2A were constant findings in TAP deficiency, and if these features could also be observed in diseases of other origins. Therefore, we compared by flow cytometry peripheral blood NK cells from the symptomatic TAP-deficient patients to those from two asymptomatic patients Previous investigations also revealed a higher than normal percentage of CD56Blood samples were obtained from volunteers or patients attending the clinics of Strasbourg University Hospitals and were collected during routine clinical (diagnostic/prognostic/therapeutic) procedures prescribed by two co-authors, Pr. E. Andrès and Dr. L. Donato. Before blood samples were collected for the study, all healthy donors, all patients and, for those patients who were minor at the time of blood sampling, their parents, gave written informed consent in agreement with the Helsinki Declaration and French legislation, under which no approval by an Ethics Committee was required in this case.We included four patients with a known TAP deficiency Peripheral blood mononuclear cells (PBMC) were isolated from citrated venous blood by Ficoll-Hypaque density gradient centrifugation and either used directly for flow cytometry or frozen in RPMI 1640 culture medium with 20% fetal calf serum (FCS) and 10% dimethylsulfoxyde (DMSO).6 cells/ml and stained in aliquots of 100 µl with various purified or fluorochrome-conjugated Ab at optimal concentrations previously determined by titration. The Ab and fluorochromes used are listed in For flow cytometry stainings, PBMC were washed at 4°C, once in RPMI 1640/10% FCS and once in phosphate buffered saline (PBS) with 3% FCS (staining buffer). Cells were then resuspended in ice-cold staining buffer at 5×1051Chromium release assays were performed as previously described Activated NK cells and phytohemagglutinin (PHA)-induced T cell blasts were obtained in culture as previously described bright NK cells in two TAP-deficient patients −CD19− lymphocytes corresponding to NK cells, we analyzed CD16 versus CD56 expression. As shown in bright NK cells compared to normal donors. CD56bright NK cells were distributed quite equally between the CD16− and CD16dim subsets in patient EMO, whereas the CD16dim population was predominant in patient EFA. In addition, both patients also had a higher percentage of CD56dimCD16− NK cells . Should this be true, higher than normal percentages of CD56bright NK cells might also be found in patients with no TAP deficiency but with acute or chronic infections and/or inflammatory diseases of other origins.In the absence of a systematic expansion of the CD56bright population clearly above 10% of all peripheral blood NK cells in five of the seventeen patients (29.41%) and even in three of the ten healthy donors (30%). Regarding the CD56dimCD16− NK cell subtype, it was higher than 10% in four patients (23.53%), of whom only one also displayed a high percentage of CD56bright NK cells in parallel . Much more inter-individual differences were observed for CD56dim NK cells, of whom a mean of 49.37% were NKG2A+ (range: 20–77%). To compare MFI, we randomly designed a healthy donor whose values of NKG2A MFI on both NK cell subsets were arbitrarily considered to be 100%. The values measured in all the other control donors and in the patients were then expressed relative to this reference value, and we calculated means of MFI for the different subject groups . Interestingly, it appeared that NK cells from EFA and EMO, but not from SFH and DFH, dramatically over-expressed NKG2A on both CD56bright and CD56dim NK cells . In accordance with the literature bright, compared to CD56dim NK cells, was clearly confirmed in each individual except EFA and EMO with comparable MFI on both subsets (data not shown). Thus, in contrast to the proportion of CD56bright NK cells that might increase in several different pathologic conditions, over-expression of NKG2A was found exclusively in the two symptomatic TAP-deficient patients.The previously reported over-expression of the IR CD94/NKG2A by NK cells from patients EFA and EMO NK cells . The difch lower . When lodim NK cells, and percentages of ILT2+ NK cells considerably differed between donors. Regarding the expression levels of ILT2, that we considered only on CD56dim NK cells, they were strongly augmented on CD56dim NK cells from EFA, EMO and DFH, although not as importantly as for NKG2A. SFH NK cells also over-expressed ILT2 to some extent compared to the mean of MFI from healthy donors and patients other than those with TAP deficiency , and only moderately in patient EMO. Reductions of MFI of ILT2 were modest. The percentage of CD56dimCD16− NK cells strongly decreased with time in patient EFA, but slightly increased in patient EMO. At least regarding CD56bright NK cells, the reduced percentage that parallels clinical improvement could be in favor of a link between the proportion of this subset among total NK cells and the intensity and severity of respiratory infections.The results shown for patients EFA and EMO were obtained with PBMC harvested at the time when their TAP deficiency was diagnosed and their respiratory symptoms were very severe. Clinical status progressively improved over the following years due to an intense therapeutic approach with repeated intravenous administration of antibiotics and chest physiotherapy. In parallel, we observed a reduction of the percentages of CD56bright NK cells among total peripheral blood NK cells is very high in comparison to healthy donors. To our knowledge, NK cell subset distribution has not yet been investigated in detail in TAP-deficient patients studied by other groups, so that it is currently unknown if the distribution of CD56bright and CD56dim NK cells is similar or not in these other cases. The two asymptomatic TAP-deficient individuals bright and CD56dim NK cells and thus the increased proportion of CD56bright NK cells is not a constant hallmark of human TAP deficiency.In this paper, we confirm and extend our previous findings bright NK cells could be a consequence of the chronic infectious and inflammatory state of the patients. Indeed, among patients with chronic inflammatory diseases other than TAP deficiency, approximately 30% displayed CD56bright percentages above 10% of total NK cells, although their values never reached those of the TAP-deficient individuals. Due to the small number and the heterogeneity of our series that was intended as part of a preliminary study, we cannot conclude if CD56bright NK cell percentages are preferentially increased in certain well-defined disease stages or subtypes, or if this might rather occur, irrespective of the underlying condition, in response to infections with particular but not all microorganisms. These points would be very interesting to address in future large scale studies, as well as the question if CD56bright NK cell expansions could be used as a prognostic marker for the short- or long-term outcome of some diseases.Based on this finding, we reasoned that the high percentage of CD56bright NK cell percentages also among 30% of the healthy control donors we tested, as it is generally admitted that in this population, CD56bright NK cells do not exceed 10% of all peripheral blood NK cells We were somehow surprised to observe high CD56bright NK cells are known during reconstitution of the immune system after bone marrow grafts, where the first lymphocytes to reappear in peripheral blood usually are CD56bright NK cells, and these cells also expand in patients daily treated with a low dose of IL-2 bright type and only a minor subset was CD56dim. In an atypical case of X-SCID with Omenn syndrome-like manifestations, the patient displayed an increased number of peripheral blood NK cells, half of them being of the CD56bright subtype Expansions of CD56bright NK cells are immature precursors of CD56dim NK cells, or that (ii) both NK cell types are functionally different, terminally differentiated effectors that arise from separate hematopoietic precursors. Cited arguments in favor of the former hypothesis are the small number of CD56bright NK cells in peripheral blood, their proliferation in the presence of picomolar concentrations of IL-2, the expression of the hematopoietic stem cell marker CD117, the low content of cytotoxic granules and the low cytotoxic activity bright and CD56dim NK cells display very different repertoires of adhesion molecules and chemokine receptors bright NK cells produce much higher amounts of cytokines than their CD56dim counterparts, so that both subtypes might well be distinct and specialized effector cell populations bright NK cells would again be precursors of CD56dim NK cells. On the other hand, comparative microarrays between purified CD56bright and CD56dim NK cells are strongly in favor of the hypothesis of two distinct subtypes The latter observations raise the question of the relationship between both peripheral blood NK cell subsets, which is quite a controversial issue still not clearly resolved. The two main views are that either (i) CD56bright NK cells are increased during infectious and/or inflammatory processes because the immune system is globally activated and high amounts of NK cell-derived cytokines are needed as part of the immune response. The corresponding NK cell subset would therefore expand in these situations before going back to baseline levels once the stimuli have disappeared. In support of this idea, a child with human herpes virus 6 (HHV-6)-associated acute necrotizing encephalopathy had a very high percentage of CD56bright NK cells in peripheral blood bright NK cells dim NK cells might be very high and the number of new NK cells generated from bone marrow precursors would increase through a feedback mechanism. The consequence would then be a high percentage of immature CD56bright NK cell precursors that appear in peripheral blood to continuously replace CD56dim NK cells.The findings of our study do not allow to strengthen one rather than another of these models. If one considers that both NK cell types are distinct effectors, it could be conceivable that CD56bright NK cells, over-expression of the IR NKG2A and ILT2 was exclusively observed in the TAP-deficient patients. The former molecule was dramatically up-regulated on both CD56bright and CD56dim NK cell subsets of the two symptomatic individuals, whereas ILT2 was over-expressed, although to a lesser extent than NKG2A, by CD56dim NK cells from all four TAP-deficient patients. Interestingly, the over-expression only implicates IR with a broad spectrum of HLA class I ligands, but no receptors of the KIR family In contrast to the increased percentages of CD56d or H-2Dk through a cis interaction cis interaction has important functional consequences and actually governs the inhibitory potential of Ly49A as well as its accessibility for tetramer and Ab staining cis interaction would render them simply more accessible for specific Ab. At least for NKG2A, this explanation seems rather unlikely, as (i) over-expression of NKG2A is not a constant finding in TAP deficiency, and (ii) expression levels diminish in parallel to clinical improvement. Regarding ILT2, the possibility still exists but would have to be tested in the context of the crystal structure of this receptor.In the mouse, it has been shown that the inhibitory NK cell receptor Ly49A interacts with its ligands H-2DAlthough the over-expression of some IR might be an adaptive mechanism to the low levels of HLA class I expression, it does not seem to have any functional role in the regulation of the cytotoxic activity of IL2-activated TAP-deficient NK cells, as revealed by the absence of killing of autologous PHA-induced T cell blasts even in the presence of anti-IR masking Ab. These data, in accordance with our previous hypothesis bright NK cells might exceed 10% of all peripheral blood NK cells in a substantial fraction of patients with chronic diseases other than TAP deficiency and even in donors considered as immunologically normal. Therefore, it might be recommendable to check more frequently the NK cell subset distribution in infectious and inflammatory diseases, in order to precise the conditions under which CD56bright NK cells expand in vivo, and what is the pathophysiological significance of this phenomenon.In conclusion, in this paper, we provide data that extend our knowledge on the phenotype of peripheral blood NK cells from TAP-deficient patients. In addition, we show that percentages of CD56


Calmodulin kinase II (CaMKII) mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ). These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart. Calmodulin kinase II (CaMKII) is a multifunctional serine/threonine kinase that regulates diverse functions in heart. Recently, a novel pathway for CaMKII activation was discovered where oxidation of the kinase at specific methionine residues produces persistent activity. This alternative oxidation-dependent pathway has important implications for heart disease where oxidative stress is increased . We hypothesized that myocardial infarction caused by occlusion of a coronary artery would increase levels of oxidized CaMKII. Moreover, we hypothesized that oxidative CaMKII activation represents an important mechanistic link between increased oxidative stress and life-threatening heart rhythm disturbances (arrhythmias) in heart disease. We report a dramatic increase in levels of oxidized CaMKII following myocardial infarction in the canine. Based on these experimental data, we developed a novel mathematical model of CaMKII activity to study the role of oxidation-dependent CaMKII activation in regulating cardiac cell excitability. Our findings identify a novel role for oxidation-dependent CaMKII activation following myocardial infarction and provide a mechanistic link between oxidative stress and lethal cardiac arrhythmias in heart disease. Calmodulin kinase II (CaMKII) mediates diverse roles in the heart, including excitation-contraction coupling, sinus node automaticity, apoptosis, hypertrophy, and gene transcription 2+/calmodulin and may undergo inter-subunit autophosphorylation that allows the kinase to retain activity even upon dissociation of Ca2+/calmodulin (autonomy) 2O2 and superoxide are elevated following myocardial infarction + channel remodeling and conduction slowing following MI.CaMKII is activated by binding of CaIn this study, we describe a dramatic increase in levels of oxidized CaMKII in a well-validated large animal model of arrhythmias following MI Myocardial infarction (MI) was produced by total coronary artery occlusion, as described previously This investigation used adult mongrel dogs  and conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health .axI is the axial current, a is the fiber radius (0.0011 cm), iR is the axial resistance per unit length  and gR (1.5 Ωcm2)), mC is the membrane capacitance (1 µF/cm2), and ionI is the transmembrane current density. A discretization element of Δx = 0.01 cm corresponding to one cell length was used in all simulations. An adaptive time step (Δt) was implemented that solves for transmembrane currents and mV along the fiber with Δt = 5 µs during AP wavefront propagation, Δt = 10 µs during repolarization, and Δt = 50 µs during diastole. Solutions using the adaptive timestep were verified to be within 1% of those using a constant Δt = 5 µs.A multicellular fiber comprised of 200 cells in a serial arrangement was used to simulate action potential (AP) propagation through normal and border zone tissue as described previously Transmembrane currents and ion concentration changes at each cell in the fiber are described by the Hund-Rudy dynamic (HRd) model of the canine epicardial myocyte The fiber was paced at one end to steady state using a conservative current stimulus When appropriate, differences between groups were analyzed with ANOVA and least squares difference post-hoc test. A value of p<0.05 was considered statistically significant. Values are expressed as mean ± SD.Based on the recent discovery of a novel oxidation-dependent pathway for CaMKII activation 2+/CaM bound active state and an autophosphorylated active state. Inclusion of an additional autonomous active state (Ca2+/CaM dissociates from phosphorylated subunit) was found to have no impact on model behavior  and was therefore not included in the final model. Consistent with experimental observations 2+/CaM must bind to a subunit before oxidation may occur (no direct transition from inactive to oxidized active state). Rate constants for state transitions were taken from the literature or chosen to fit experimental data  at a varying S1S2 interval and plotting channel availability  vs. recovery interval . INa steady-state inactivation is shifted to more hyperpolarized potentials , by increasing [K+]o incrementally from 5.4 to 13 mM. Conduction velocity was measured across the central 100 cells  . Making CaMKII resistant to oxidation increased conduction velocity at all Vm,dia in the BZ with a greater effect at more depolarized Vm,dia . In contrast, the BZ model resistant to CaMKII autophosphorylation showed very little improvement in conduction  . Further to 1 µM . To deted Vm,dia . Moreovenduction . Furthernduction , indicatnt model , indicatg) from 1.5 to 60 Ωcm2 produced a similar decrease in conduction velocity in NZ and BZ fibers . Successful conduction occurred in the NZ fiber for Rg up to 300 Ωcm2, while conduction block was observed in the BZ fiber for Rg>76 Ωcm2. Eliminating oxidation-dependent CaMKII activity increased conduction velocity in the BZ fiber at all Rg and restored conduction for Rg up to 150 Ωcm2  of the action potential is dramatically prolonged in BZ compared to NZ, despite comparable action potential durations tivation , we hypotivation . ERP is tivation . Small don (INa . Making on (INa  by elimion (INa . Likewistoriness . These rm and INa availability (h*j) spatial profiles as the wavefront reaches the BZ margin indicate that INa availability is a critical determinant of whether or not a premature wavefront blocks at the BZ margin . VW shows a monophasic increase as Vm,dia is depolarized from −85 mV to −72 mV. Vm,dia greater than −72 mV results in transient block at the BZ margin even at the basic cycle length of 500 ms. Making CaMKII resistant to oxidative activation greatly reduces VW at all Vm,dia .l Vm,dia . FurtherINa) also likely play an important role. To address the role of CaMKII-independent remodeling in conduction block, we measured VW in the heterogeneous fiber with normal INa conductance throughout. Eliminating differences in INa conductance successfully reduced vulnerability to transient conduction block across a wide range of Vm,dia  or CaMKII-independent  had very little effect on VW for a moderate degree of uncoupling (Rg up to 38 Ωcm2). However, VW increased sharply for Rg>38 Ωcm2 until block occurred at the BZ margin even at the basic cycle length (Rg>60 Ωcm2). Eliminating oxidation-dependent CaMKII activation or normalizing INa conductance reduced VW at all Rg  indicating that CaMKII-dependent and CaMKII-independent effects on INa regulate VW even in the presence of cellular uncoupling.We next determined whether cellular uncoupling in the BZ region would alter the role of CaMKII-dependent or CaMKII-independent Z margin . Increas+ channel kinetics due to oxidative CaMKII activation are sufficient to impact conduction in the BZ.Our data provide the first evidence for oxidation of CaMKII as an important component of the remodeling process following MI. Furthermore, our simulation results provide the following insight into regulation of CaMKII signaling by this novel oxidative pathway: 1) Significant oxidative activation of the kianse occurs under pathophysiological conditions; 2) Oxidative stress may activate the kinase not only through direct oxidation but also through a secondary increase in autophosphorylation; and 3) Changes in Na+ channel dysfunction contributes to reduced action potential upstroke and action potential amplitude in myocytes isolated from the infarct border zone + channel availability contributes to prolonged refractoriness in BZ cells despite AP duration comparable to NZ + channel recovery from inactivation or reducing Na+ channel conductance increases the vulnerable period for unidirectional block in cardiac tissue + channel kinetics. Furthermore, by introducing gradients in INa availability and refractoriness, CaMKII activation, due in part to oxidation, increases vulnerability to conduction block at the BZ margin, a prerequisite for reentrant excitation. Moreover, our simulations suggest that CaMKII inhibition improves conduction (particularly in depolarized tissue) and reduces ERP in the BZ, thereby reducing the risk for conduction block and reentrant excitation. These results are significant in light of experimental mapping studies showing premature excitation block in areas of large gradients in refractoriness 2+ cycling that itself may promote arrhythmias Conduction in the canine infarct border zone is highly irregular with areas of very slow and discontinuous conduction during sinus rhythm + channel recovery (slowing) with the measured effects on refractoriness in transgenic mice. It is possible that the decrease in ERP measured in transgenic mice is due to secondary effects of chronic CaMKII over-expression rather than acute signaling effects. Regardless, further studies are needed to define the role of CaMKII in regulating refractoriness in the heart.Our findings regarding the effects of CaMKII on conduction are consistent with experimental studies in mice that over-express CaMKIIδ. Specifically, consistent with our simulations, CaMKIIδ over-expression results in prolonged QRS intervals (marker of slowed intraventricular conduction) and increased arrhythmia susceptibility + channels influence conduction in the infarct BZ. Specifically, alterations in cell-to-cell coupling due to gap junction remodeling and/or fibrosis undoubtedly play an important role in abnormal conduction. In fact, studies have shown a close correlation between location of the central common pathway of the reentrant circuit and connexin43 redistribution suggesting that gap junction remodeling is required for maintenance of reentrant excitation INa kinetics regulate conduction in the BZ independent of the degree of cell coupling. Moreover, we report that CaMKII inhibition may restore conduction in the BZ even in the setting of very poor coupling (Rg up to 150 Ωcm2).Clearly, many factors besides remodeling of voltage-gated Na+ channels expressed in HEK cells to ROS recapitulates the remodeling phenotypes observed in BZ myocytes. Our results suggest that the newly discovered oxidation-dependent pathway for CaMKII activation serves as a critical link between oxidative stress, enhanced CaMKII activity, Na+ channel dysfunction, and abnormal conduction in the infarct BZ. Of course, CaMKII is unlikely to be the only pathway through which oxidative stress alters cell excitability as oxidation affects many proteins in the heart, including kinases, transcription factors, ion channels, pumps, transporters, Ca2+ handling proteins, and contractile machinery Previous studies have shown increased levels of ROS in five-day infarct BZ regions + channel remodeling and increased susceptibility to reentrant arrhythmias after MI.While studies from our group and others demonstrate an important role for activated CaMKII in remodeling following MI, the upstream signaling pathways responsible for enhanced CaMKII activity remain to be fully elucidated . In this2+/calmodulin, oxidative activation is likely controlled by a delicate balance of oxidase/reductase activity, mitochondrial function and local calcium signaling. Furthermore, degree of crosstalk between oxidative and autophosphorylation pathways and their relative importance in response to the complex set of upstream stressors in heart disease remain to be determined. These details, as they emerge, may be incorporated into our model to analyze the functional consequences of upstream signals that converge through distinct pathways to alter CaMKII activity.Finally, it is important to note that activation of CaMKII through direct oxidation has only recently been discovered. Consequently, much remains unknown regarding the signaling mechanisms that regulate this pathway. Analogous to regulation of autophosphorylation by local kinase/phosphatase activity and local concentrations of CaWhile our mathematical model accounts for central aspects of the newly identified oxidation-dependent pathway for CaMKII activation, it has important limitations based on the available experimental data. Many questions remain to be answered regarding the function of CaMKII oxidation in the normal and diseased heart. For example, how do oxidation and autophosphorylation of CaMKII interact to control regulation of the holoenzyme? Do these pathways interact synergistically to activate the kinase and what are the unique/shared targets for each activation pathway? Finally, what is the relative importance of oxidized versus autophosphorylated CaMKII in normal and diseased hearts? Answers to these questions will not only facilitate the development of more comprehensive models but also will provide critical information necessary to design novel cell-specific therapies for regulating cardiac excitability.2+ that reaches concentrations somewhere between cytosolic and dyadic concentrations (peak concentration 10–20 µM). Previous modeling studies have shown that the dynamic response of CaMKII may vary greatly between dyadic and cytosolic pools based on variability in concentrations of Ca2+ and CaM 2+ transients do not significantly activate CaMKII activity at baseline or even in the presence of 1 µM H2O2 . However 10 µM H2O2 was able to activate CaMKII , suggesting that a sufficiently high level of oxidative stress may be able to activate even cytosolic CaMKII. Clearly, local regulation of CaMKII in well-defined subcellular domains is an exciting area for future research with important implications for human disease. As we learn more about CaM and CaMKII signaling in the vicinity of Na+ channels, it will be important to incorporate these data into the model.It is important to note that CaMKII in the model detects a subspace pool of CaFigure S1Cell excitability as a function of ROS levels in the BZ. (A) Conduction velocity and (B) refractoriness in the infarct BZ as a function of ROS concentration. Conduction velocity is determined across the middle 100 cells in the BZ fiber. Postrepolarization refractoriness is calculated as the difference between effective refractory period and action potential duration at 90% repolarization.(2.17 MB TIF)Click here for additional data file.Figure S2m,dia). [K+]o is increased incrementally from 5.4 mM to 13 mM to Vm,dia from −87 to −63 mV. Elimination of autophosphorylation-dependent CaMKII activation has a small effect on conduction velocity compared to elimination of oxidation-dependent activation.Role of autophosphorylation-dependent CaMKII activation in regulating conduction in the infarct BZ. Conduction velocity is determined across the middle 100 cells in the BZ fiber over a range of end diastolic membrane potentials (V(2.87 MB TIF)Click here for additional data file.Table S1Model definitions and abbreviations(0.02 MB PDF)Click here for additional data file.Table S2Mathematical model initial conditions(0.01 MB PDF)Click here for additional data file.Table S3CaMKII transition rate parameters(0.01 MB PDF)Click here for additional data file.Text S1Model equations for NZ and BZ fiber.(0.06 MB PDF)Click here for additional data file.