Single Splice Research Articles

A novel single nucleotide splice site mutation in FHL1 confirms an Emery-Dreifuss plus phenotype with pulmonary artery hypoplasia and facial dysmorphology

We describe a Danish family with an, until recently, unknown X-linked disease with muscular dystrophy (MD), facial dysmorphology and pulmonary artery hypoplasia. One patient died suddenly before age 20 and another was resuscitated from cardiac arrest at the age of 28. Linkage analysis pointed to a region of 25 Mb from 123.6 Mb to 148.4 Mb on chromosome X containing over 100 genes. Exome sequencing identified a single nucleotide splice site mutation c.502-2A > T, which is located 5' to exon 6 in the gene encoding four and a half LIM domain 1 (FHL1) protein. FHL1 expresses three main splice variants, known as FHL1A, FHL1B and FHL1C. In healthy individuals, FHL1A is the predominant splice variant and is mainly found in skeletal and cardiac muscle. The FHL1 transcript profiles from two affected individuals were investigated in skin fibroblasts with quantitative real-time PCR. This demonstrated loss of isoform A and B, and an almost 200-fold overexpression of isoform C confirming that lack of FHL1A and overexpression of FHL1C results in an extended phenotype of EDMD as recently shown by Tiffin et al. [2013].

Modelling the Behaviour of Single Stage Splicing Language: A Yusof Goode Computational Approach

Yusof-Goode (Y-G) splicing system is a formal characterization of the generative capacity of specified enzymatic activities acting on DNA molecules with new extension symbolization of representing rule. The output of Y-G splicing system can be categorized into three types of single stage splicing language namely active persistent, transient and inert persistent language. It is both money and time consuming to conduct laboratory experiments to determine the behaviour of splicing language. Hence, research has been conducted to predict the characteristic of single stage splicing language based on limit adjacency matrix computational modelling in order to optimize time and money. The utilization of software programming has been developed through Visual Basic Software for scientists to determine the behaviour of single stage splicing language as well as the number types of resulted DNA molecules restricted to at most two strings and two rules with one cutting site. The output from the program was found to match the outcomes of wet lab experiments.

Nano-displacement sensor based on photonic crystal fiber modal interferometer.

A stable nano-displacement sensor based on large mode area photonic crystal fiber (PCF) modal interferometer is presented. The compact setup requires simple splicing of a small piece of PCF with a single mode fiber (SMF). The excitation and recombination of modes is carried out in a single splice. The use of a reflecting target creates an extra cavity that discretizes the interference pattern of the mode interferometer, boosting the displacement resolution to nanometer level. The proposed modal interferometric based displacement sensor is highly stable and shows sensitivity of 32 pm/nm.

Abstract 4185: Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy

Abstract INTRODUCTION Next generation sequencing (NGS) of patients has significantly changed our ways to study cancer genomics as it provides precise estimates of gene expression, fusion transcripts, expressed single nucleotide variants (eSNVs), splice variants and copy number variants. The Breast Cancer Genome Guided Therapy (BEAUTY) is an ongoing clinical study in which RNA sequencing (RNAseq) and whole exome sequencing (WES) are performed prior to, during and after neoadjuvant chemotherapy. Here we report the use of these sequencing technologies to investigate gene expression levels and mutational profiles in a triple negative breast cancer (TNBC) patient enrolled in BEAUTY whose disease did not respond to neoadjuvant paclitaxel and anthracycline/cycphosphamide. METHODS Computational approaches were used to integrate WES and RNAseq data obtained before therapy (V1T), after 12 weekly paclitaxel treatments (V2T) and after anthracycline-based regimen at surgical resection (V3T) to study a single patient with persistent TNBC disease after neoadjuvant chemotherapy. RESULTS Using RNA-Seq data, we identified an inter-chromosomal fusion transcript between chromosome 20 and 22 (GNAS-TTC38) that was highly expressed at V1T, V2T and V3T. We also identified intra-chromosomal fusion transcripts that were expressed at two time points, such as fusion transcript (KANSL1-ARL17A) on chromosome 17 for V1T and V2T and fusion transcript (RBM12B-LINC00535) on chromosome 8 for V2T and V3T time points. Several gene expression changes were also observed. Gene expression analysis of V1T, V2T and V3T tumors was performed. Differential temporal gene expression profiles of 9884 genes that were significant and varying at different time points were obtained for pathway analysis. Pathway analysis of 9884 genes identified up regulation and down regulation of several transcription factors with a fold change of 2x or more. When compared to blood, DNA tumor and RNA-Seq data, we identified 81 common somatic eSNVs that were expressed in both V1T and V2T time points and we are in the process of investigating V3T data. We found alterations of key transporter domains (CD225, Coatamer_beta_C, DUF2435, Dynamitin, EI24, GLTP, LMF1, Porin_3, V-ATPase_C) in our V1T and V2T SNV data. Similar to gene expression analysis, we are in the process of obtaining the list of mutations at various time points to identify driver and passenger mutation candidate genes for this specific TNBC patient. CONCLUSIONS Our initial time-series analysis of eSNV, fusion transcripts and gene expression data demonstrate that intensive analysis for individual patients is feasible. Further investigation of drug transporters and transcription regulators may help develop personalized treatment strategies for patients with disease resistant to current regimens. Citation Format: Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Douglas W. Mahoney, Poulami Barman, Jason P. Sinnwell, Hugues Sicotte, Peter Vedell, Steven N. Hart, Travis J. Dockter, Katie N. Jones, Amy L. Conners, Ann M. Moyer, Daniel W. Visscher, Jia Yu, Bowen Gao, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Jean-Pierre A. Kocher, Eric D. Wieben, Gianrico Farrugia, Cloann G. Schultz, James N. Ingle, Richard Weinshilboum, Matthew P. Goetz, Liewei Wang, Judy C. Boughey. Analysis of sequencing data to identify potential drug targets for an individual newly diagnosed with basal breast cancer who failed to respond to current standard neoadjuvant chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2014-4185

Inelastic Buckling Analysis of Steel X-Bracing with Bolted Single Shear Lap Connections

AbstractAnalytical models are proposed to predict the compressive resistance of discontinuous bracing members in steel X-braced frames when governed by flexure of the connecting plates at the mid-connections. The connections are bolted single shear splice connections commonly used for HSS bracing members. An incremental analysis procedure is developed to predict the axial load-deformation response of the brace-connection assembly including geometric and material nonlinearities. The buckling strength depends on the thickness of the connecting plates, the length of the connection, the clear distance between the continuous and discontinuous bracing members at the brace intersection, and the length of the discontinuous brace segments. The method is validated against test results from full-scale X-bracing specimens. It is also used to verify the design procedure proposed in the AISC Design Guide 24 for Hollow Structural Section Connections for a series of 17 X-braces covering a wide range of properties. The AI...

Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

Recent advances in proteomics: towards the human proteome.

After the successful completion of the Human Genome project in 2003, the next major challenge was to understand when and where the encoded proteins were expressed, and to generate a map of the complex, interconnected pathways, networks and molecular systems (the human proteome) that, taken together, control the workings of all cells, tissues, organs and organisms. Proteomics will be fundamental for such studies. This review summarizes the key discoveries that laid down the foundations for proteomics as we now know it, and describes key recent technological advances that will undoubtedly contribute to achieving the initial goal of the Human Proteome Organization of identifying and characterizing at least one protein product and representative post-translational modifications, single amino acid polymorphisms and splice variant isoforms from the 20,300 human protein-coding genes within the next 10 years. Successful unraveling of the human proteome will undoubtedly improve our understanding of human biology at the cellular level and lay the foundations for improved diagnostic, prognostic, therapeutic and preventive medical outcomes as we enter the era of personalized medicine.

Quantitative imaging of single mRNA splice variants in living cells.

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

Antisense Oligonucleotide-Based Therapy in Human Erythropoietic Protoporphyria

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

Stabilization of the μ-Opioid Receptor by Truncated Single Transmembrane Splice Variants through a Chaperone-like Action

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

Widespread splicing changes in human brain development and aging

While splicing differences between tissues, sexes and species are well documented, little is known about the extent and the nature of splicing changes that take place during human or mammalian development and aging. Here, using high-throughput transcriptome sequencing, we have characterized splicing changes that take place during whole human lifespan in two brain regions: prefrontal cortex and cerebellum. Identified changes were confirmed using independent human and rhesus macaque RNA-seq data sets, exon arrays and PCR, and were detected at the protein level using mass spectrometry. Splicing changes across lifespan were abundant in both of the brain regions studied, affecting more than a third of the genes expressed in the human brain. Approximately 15% of these changes differed between the two brain regions. Across lifespan, splicing changes followed discrete patterns that could be linked to neural functions, and associated with the expression profiles of the corresponding splicing factors. More than 60% of all splicing changes represented a single splicing pattern reflecting preferential inclusion of gene segments potentially targeting transcripts for nonsense-mediated decay in infants and elderly.

Mechanical Analysis on the Splicing Form of Cross-Section Multiple Angle Steel for Transmission Line

The stress inside and outside the traditional cross-section angle steel in transmission lines are uneven, resulting in the deviation of the actual carrying capacity and the theoretical design value. This paper proposes the use of improved splicing form of the cross-section angle steel to improve the structure’s mechanical properties. Two spliced forms of cross-section multiple angle steel are analyzed by finite element simulation analysis, so as to compare two different forms’ coordination of component displacement, uniform of force, and stress status on the maximum stress control section. It is pointed out that double angle splicing forms’ displacement compatibility and bearing capacity are better than single angle splicing forms. Especially for towers with different height legs, the maximum stress of the main members of the two splicing forms in the control section is respectively 4.7% and 13.7%.

From single splicing events to thousands: the ambiguous step forward in splicing research

Since the discovery of RNA splicing in 1977 the knowledge of this important biological process has increased steadily following the identification of many of the mechanistic features of splicing: from the basic cis-acting splicing signals, through the detail composition and dynamics of the spliceosome, to the role played by accessory splicing factors and their interactions. Moreover, the realization that most genes undergo alternative splicing has had a strong impact in the overall cell proteome and metabolism research fields and also in better appraising the fundamental role played by splicing defects in human disease. This robust growth of knowledge is due in particular to the development of new powerful technical tools that range from methodologies useful to focus on single events in extreme detail to microarray and high-throughput RNA sequencing approaches that aim at providing a global vision of splicing changes. Here, we will discuss how these techniques relate to each other in terms of their respective strengths and weaknesses. In particular, we will focus on their value for evaluating the biological significance of splicing events. Finally, we provide some views on how these methodologies should move forward to improve our basic and applied knowledge of RNA splicing.

Expression and regulation of mouse selenoprotein P transcript variants differing in non-coding RNA

Selenoprotein P (Sepp1), a glycoprotein rich in selenium, is thought to function in selenium transport throughout the body. The sepp1 gene locus potentially produces three alternative transcripts that differ only in their 5′ untranslated regions (5′UTRs) and not in their protein coding regions, as indicated by transcript information in genomic databases. Here we investigated the distribution, relative expression, and biological significance of these transcript variants. We confirmed the expression of Sepp1 transcript variants using PCR and sequencing. Using 5′-RACE, we identified multiple 5′-termini upstream from three different splice donor sites, and a single splice acceptor site for exon 2. We found regional and temporal changes in variant expression in select adult and neonate murine tissue and brain regions. Distribution of variants in heart and kidney varied with stage of development. Notably, the Sepp1b variant was localized specifically to the hippocampus in brain. Targeted silencing of individual variants using RNAi demonstrated the biological importance for all transcript variants in cell viability. Additionally, we determined that the Sepp1b variant is a specific target for the miR-7 microRNA by means of its unique 5′UTR structure. Our results emphasize the importance of non-coding transcript variations as a regulatory means for Sepp1 expression in different tissues and stages of development. The presence of a variant localized in the hippocampus and regulated by a microRNA may have implications for the known deficits in synaptic function caused by genetic deletion of Sepp1.

Muscle-specific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans.

Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.

Analysis of the Novel Fanconi Anemia GeneSLX4/FANCPin Familial Breast Cancer Cases

SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers--BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2-negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C-induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.

Analysis of MDM2 and MDM4 Single Nucleotide Polymorphisms, mRNA Splicing and Protein Expression in Retinoblastoma

Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

Nitric oxide-dependent bradycardia in mutant analbuminemic rats

Nagase analbuminemic rats (NAR) are natural mutant Sprague-Dawley rats which do not express albumin due to a single splice mutation in the albumin gene. We accidentally discovered that NAR have a significant bradycardia compared with wild type Sprague-Dawley rats, and the present study was carried out to investigate the mechanism of bradycardia in analbuminemic rats. In vitro studies showed that the basal spontaneous beating rate of isolated atria is similar in NAR compared with wild type animals. However, the chronotropic responsiveness of isolated atria to cholinergic stimulation was markedly increased in NAR, an effect which was prevented by incubation with a nitric oxide synthase (NOS) or guanylyl cyclase inhibitor. NAR had a significant increase in plasma nitrite/nitrate concentrations. Administration of a NOS inhibitor for 5 days normalized heart rate in NAR. The level of NOS isoforms, caveolin-1 and caveolin-3 expression in the atria was assessed by real time PCR. There was no significant difference in the expression of NOS isoforms or caveolin-3 in NAR compared with wild type controls. However, NAR exhibited a significant decrease in caveolin-1 expression in the atria. Since caveolin-1 is known to inhibit endothelial NOS activity in cardiomyocytes, we suggest that decreased caveolin-1 levels may have a role in increased nitric oxide production in NAR. Our data suggest that a NOS/cGMP-dependent mechanism might be involved in increased responsiveness to vagal stimulation and bradycardia in analbuminemic condition.