A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

AIMS: Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue. METHODS AND RESULTS: General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes. CONCLUSION: The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart

A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

AIMS: Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue. METHODS AND RESULTS: General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes. CONCLUSION: The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart

Flotillin-2 shows increased ID expression during cardiac disease.

<p>(A) Immunofluorescence imaging of N-cadherin and flotillin-2 in human left ventricular tissue. The overlay image reveals strong co-localization. (B) Co-localization studies of N-cadherin and flotillin-2 in left ventricular tissue from two patients with DCM hints at an increased level of flotillin-2 at the ID. Intensity and localization of fluorescent signals were analyzed through line scans and are plotted for both patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.s002" target="_blank">S2 Fig</a>). Robust co-localization was confirmed through correlation coefficients of R² = 0.81 and 0.86 for the upper panel and lower panel, respectively. (C) Representative Western blots of the CF and EMF in healthy human tissue, compared to left ventricular tissue of patients with either DCM or ARVC. Differences between the levels in CF and EMF were analyzed using an unpaired t-test. Differences between the groups of patients were analyzed via a one-way ANOVA. * P<0.05, ** P<0.005, ***P<0.0005.</p

Popdc2 localizes to the intercalated disk.

<p>(A) Western blot showing that POPDC2 is enriched in the EMF compared to the CF in both rat and human left ventricular tissue. (B) Immunofluorescence imaging of N-cadherin and POPDC2. Merged images (overlay, right) show clear co-localization of N-cadherin and POPDC2 in human left ventricular tissue, isolated dog cardiomyocytes and rat left ventricular tissue at the ID.</p

Molecular functions of designated ID proteins.

<p>Subnetwork GO analysis of the 97 candidate proteins observed in the membrane or integral to membrane compartment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g002" target="_blank">Fig 2</a>. Proteins were clustered according to their GO annotation in process and function. Comparable to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g002" target="_blank">Fig 2</a>, the color of the nodes represents their fold-enrichment measured (ER). The cluster in the middle contains all currently known junction proteins (based on GO and Estigoy et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.ref022" target="_blank">22</a>]). Proteins in this cluster are connected to proteins with other biological functions based on known protein-protein interactions (lines). The four candidate proteins selected for follow-up studies are enlarged.</p

Strategy to identify putative intercalated disk proteins.

<p>(A) Schematic representation of the approach followed to identify intercalated disk proteins in the enriched membrane fraction (EMF). The cytosolic fraction (CF) was used as a control to quantify enrichment levels. Enrichment was done using differential centrifugation steps. Samples were digested in-gel and subsequently analyzed by LC-MS/MS and label free quantitation using MaxQuant software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.ref025" target="_blank">25</a>]. (B) The EMF is compared to different fractions obtained during the enrichment process as visualized by connexin-43 (Cx43) Western blot. (CF = cytosolic fraction, UC&N = unbroken cells and nuclei, EMF = enhanced membrane fraction, lysate = entire cardiac tissue lysate) (C) Immunostaining of Cx43 (green) on cardiomycytes, with alpha actin staining used as reference (red). (D) Enrichment ratios (ER) of selected CF and ID markers and the log value of their standard deviations (n = 3). Depicted are Alpha-crystallin B chain (CRYAB), Creatine Kinase M (CKM), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase B chain (LDHB), L-lactate dehydrogenase A chain (LDHA), Aspartate aminotransferase, cytoplasmic (GOT1) as CF markers and Connexin-43 (Cx43, GJA1), N-Cadherin (CDH2), Beta-Catenin (CTNNB1), Tight junction protein 2 (TJP2) and Blood vessel epicardial substance (POPDC1) as known ID markers within the EMF.</p

Molecular context of proteins in the EMF.

<p>Schematic representation of protein-protein interactions and cellular localization across the 366 candidate proteins that showed enrichment (ER>10-fold) in the EMF-fraction. This highly connected network is based on protein-protein interactions currently available in the Biogrid database in mouse, rat and human (lines). The color of the nodes represents the fold-enrichment measured; darker colors mean higher enrichment ratios. All known cell junction proteins, as based on GO-terms and a recent <i>in silico</i> study by Estigoy et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.ref022" target="_blank">22</a>], are visualized by their gene names and enlarged circles. As expected, these mainly clustered in the plasma membrane region and several known ID protein complexes were readily identified at this intracellular location. These and all other (connected) proteins in this compartment were considered as most likely ID candidates and further evaluated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g003" target="_blank">Fig 3</a>.</p

Diastolic dysfunction is more apparent in STZ-induced diabetic female mice, despite less pronounced hyperglycemia

Diabetic cardiomyopathy is a distinct pathology characterized by early emergence of diastolic dysfunction. Increased cardiovascular risk associated with diabetes is more marked for women, but an understanding of the role of diastolic dysfunction in female susceptibility to diabetic cardiomyopathy is lacking. To investigate the sex-specific relationship between systemic diabetic status and in vivo occurrence of diastolic dysfunction, diabetes was induced in male and female mice by streptozotocin (5x daily i.p. 55 mg/kg). Echocardiography was performed at 7 weeks post-diabetes induction, cardiac collagen content assessed by picrosirius red staining, and gene expression measured using qPCR. The extent of diabetes-associated hyperglycemia was more marked in males than females (males: 25.8 ± 1.2 vs 9.1 ± 0.4 mM; females: 13.5 ± 1.5 vs 8.4 ± 0.4 mM, p

Atopic dermatitis characteristics and medication-use patterns in school-age children with AD and asthma symptoms

Background: Atopic dermatitis (AD) and asthma often coexist. Both diseases can have a major impact on the lives of children with AD and their caregivers. Aim: To investigate the association of patient characteristics, comorbidities and impact of AD on children who have both asthma and AD. Methods: Children with AD (n = 140) were selected from a larger cohort of children with a reported use of asthma medication. The Children's Dermatology Life Quality Index (CDLQI) was used to assess Quality of Life (QoL), and the Self-Assessed Eczema Area and Severity Index (SA-EASI) was used to measure AD severity. Characteristics assessed included: age, sex, and the number and type of atopic comorbidities. Medication use for AD was defined using the total number of AD prescriptions, the number of different topical AD prescriptions and the highest potency topical corticosteroid (TCS) used. Determinants of AD severity and QoL were evaluated using Spearman rank tests. Results: The following factors were most strongly associated with a lower QoL: characteristics of AD lesions (Spearman Rs = 0.61–0.69, P < 0.01), a higher SA-EASI score (Rs = 0.54, P < 0.01) and a larger number of different topical AD prescriptions (Rs = 0.38, P < 0.01). The following factors were correlated with more severe AD: age (Rs = −0.36, P < 0.01), larger number of different TCS preparations used (Rs = 0.27, P < 0.05) and larger number of TCS prescriptions (Rs = 0.25, P < 0.05). Conclusion: In children with asthma and AD, the number of TCS preparations used is associated with lower QoL and increased AD severity

Changes in Cx43 and Na_V1.5 Expression Precede the Occurrence of Substantial Fibrosis in Calcineurin-Induced Murine Cardiac Hypertrophy

<div><p>In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and Na_V1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and Na_V1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, Na_V1.5 and connective tissue expression) were characterized in MHC-CnA ventricles <i>versus</i> wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and Na_V1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as <i>Col1a1</i>, <i>Col1a2</i>, <i>Col3a1, Tgfb1</i>, <i>Ctgf</i>, <i>Timp1</i> and microRNA miR-21. In MHC-CnA, reduction in Cx43 and Na_V1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.</p></div