14 research outputs found

    Anchored protein kinase A signalling in cardiac cellular electrophysiology

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    The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is an elementary molecule involved in both acute and chronic modulation of cardiac function. Substantial research in recent years has highlighted the importance of A-kinase anchoring proteins (AKAP) therein as they act as the backbones of major macromolecular signalling complexes of the β-adrenergic/cAMP/PKA pathway. This review discusses the role of AKAP-associated protein complexes in acute and chronic cardiac modulation by dissecting their role in altering the activity of different ion channels, which underlie cardiac action potential (AP) generation. In addition, we review the involvement of different AKAP complexes in mechanisms of cardiac remodelling and arrhythmias

    Anchored protein kinase A signalling in cardiac cellular electrophysiology

    No full text
    The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is an elementary molecule involved in both acute and chronic modulation of cardiac function. Substantial research in recent years has highlighted the importance of A-kinase anchoring proteins (AKAP) therein as they act as the backbones of major macromolecular signalling complexes of the β-adrenergic/cAMP/PKA pathway. This review discusses the role of AKAP-associated protein complexes in acute and chronic cardiac modulation by dissecting their role in altering the activity of different ion channels, which underlie cardiac action potential (AP) generation. In addition, we review the involvement of different AKAP complexes in mechanisms of cardiac remodelling and arrhythmias

    A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

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    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

    No full text
    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

    Profile and triage validity of trauma patients triaged green: a prospective cohort study from a secondary care hospital in India

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    Objectives To evaluate the profile of non-urgent patients triaged ‘green’, as part of a triage trial in the emergency department (ED) of a secondary care hospital in India. The secondary aim was to validate the triage trial with the South African Triage Score (SATS).Design Prospective cohort study.Setting A secondary care hospital in Mumbai, India.Participants Patients aged 18 years and above with a history of trauma defined as having any of the external causes of morbidity and mortality listed in block V01–Y36, chapter XX of the International Classification of Disease version 10 codebook, triaged green between July 2016 and November 2019.Primary and secondary outcome measures Outcome measures were mortality within 24 hours, 30 days and mistriage.Results We included 4135 trauma patients triaged green. The mean age of patients was 32.8 (±13.1) years, and 77% were males. The median (IQR) length of stay of admitted patients was 3 (13) days. Half the patients had a mild Injury Severity Score (3–8), with the majority of injuries being blunt (98%). Of the patients triaged green by clinicians, three-quarters (74%) were undertriaged on validating with SATS. On telephonic follow-up, two patients were reported dead whereas one died while admitted in hospital.Conclusions Our study highlights the need for implementation and evaluation of training in trauma triage systems that use physiological parameters, including pulse, systolic blood pressure and Glasgow Coma Scale, for the in-hospital first responders in the EDs

    Flotillin-2 shows increased ID expression during cardiac disease.

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    <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 R<sup>2</sup> = 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.

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    <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.

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    <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

    Molecular context of proteins in the EMF.

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    <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
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