Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy

Objective: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. Methods: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. Results: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. Significance: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death

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