7 results found
Brook J, Kim M-Y, Koutsoftidis S, et al., 2020, Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling, Pflügers Archiv European Journal of Physiology, Vol: 472, Pages: 1435-1446, ISSN: 0031-6768
We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap-junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting Porcine (n=9) and human (n=4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. 1mM of carbenoxolone was administered at 5ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed.We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9±4.1ms to 67.2±2.7ms) indicating conduction slowing. The features with the largest percentage change between baseline to Carbenoxolone were Fractionation +185.3%, Endpoint amplitude -106.9%, S-Endpoint Gradient +54.9%, S Point, -39.4%, RS Ratio +38.6% and (-dV/dt)max -20.9%.The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically-induced proarrhythmic substrates.
Handa B, Li X, Baxan N, et al., 2020, Ventricular fibrillation mechanism and global fibrillatory organisation are determined by gap junction coupling and fibrosis pattern, Cardiovascular Research, ISSN: 0008-6363
AimsConflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganised multiple-wavelet activation to organised rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism.Methods and ResultsOptical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact (CF), diffuse (DiF) and patchy (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum.Enhanced GJ coupling with rotigaptide (n = 10) progressively organised fibrillation in a concentration-dependent manner; increasing FDI (0nM: 0.53±0.04, 80nM: 0.78±0.03, p < 0.001), increasing RA sustained VF time (0nM:44±6%, 80nM: 94±2%, p < 0.001) and stabilised RAs (maximum rotations for a RA; 0nM:5.4±0.5, 80nM: 48.2±12.3, p < 0.001). GJ uncoupling with carbenoxolone progressively disorganised VF; the FDI decreased (0µM: 0.60±0.05, 50µM: 0.17±0.03, p < 0.001) and RA-sustained VF time decreased (0µM: 61±9%, 50µM: 3±2%, p < 0.001).In CF, VF activity was disorganised and the RA-sustained VF time was the lowest (CF: 27±7% versus PF: 75±5%, p < 0.001). Global fibrillatory organisation measured by FDI was highest in PF (PF: 0.67±0.05 versus CF: 0.33±0.03, p < 0.001). PF harboured the longest duration and most spatially stable RAs (patchy: 1411&plusm
Handa BS, Roney CH, Houston C, et al., 2018, Analytical approaches for myocardial fibrillation signals, Computers in Biology and Medicine, Vol: 102, Pages: 315-326, ISSN: 0010-4825
Atrial and ventricular fibrillation are complex arrhythmias, and their underlying mechanisms remain widely debated and incompletely understood. This is partly because the electrical signals recorded during myocardial fibrillation are themselves complex and difficult to interpret with simple analytical tools. There are currently a number of analytical approaches to handle fibrillation data. Some of these techniques focus on mapping putative drivers of myocardial fibrillation, such as dominant frequency, organizational index, Shannon entropy and phase mapping. Other techniques focus on mapping the underlying myocardial substrate sustaining fibrillation, such as voltage mapping and complex fractionated electrogram mapping. In this review, we discuss these techniques, their application and their limitations, with reference to our experimental and clinical data. We also describe novel tools including a new algorithm to map microreentrant circuits sustaining fibrillation.
Rotolo A, Caputo VS, Holubova M, et al., 2018, Enhanced anti-lymphoma activity of CAR19-iNKT cells underpinned by dual CD19 and CD1d targeting, Cancer Cell, Vol: 34, Pages: 596-610, ISSN: 1535-6108
Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19+ B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19+ chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers.
Houston CPJ, Tzortzis KN, Roney C, et al., 2018, Characterisation of re-entrant circuit (or rotational activity) in vitro using the HL1-6 myocyte cell line, Journal of Molecular and Cellular Cardiology, Vol: 119, Pages: 155-164, ISSN: 0022-2828
Fibrillation is the most common arrhythmia observed in clinical practice. Understanding of the mechanisms underlying its initiation and maintenance remains incomplete. Functional re-entries are potential drivers of the arrhythmia. Two main concepts are still debated, the “leading circle” and the “spiral wave or rotor” theories. The homogeneous subclone of the HL1 atrial-derived cardiomyocyte cell line, HL1-6, spontaneously exhibits re-entry on a microscopic scale due to its slow conduction velocity and the presence of triggers, making it possible to examine re-entry at the cellular level.We therefore investigated the re-entry cores in cell monolayers through the use of fluorescence optical mapping at high spatiotemporal resolution in order to obtain insights into the mechanisms of re-entry.Re-entries in HL1-6 myocytes required at least two triggers and a minimum colony area to initiate (3.5 to 6.4 mm2). After electrical activity was completely stopped and re-started by varying the extracellular K+ concentration, re-entries never returned to the same location while 35% of triggers re-appeared at the same position. A conduction delay algorithm also allows visualisation of the core of the re-entries. This work has revealed that the core of re-entries is conduction blocks constituted by lines and/or groups of cells rather than the round area assumed by the other concepts of functional re-entry. This highlights the importance of experimentation at the microscopic level in the study of re-entry mechanisms.
Pitcher DS, de Mattos-Shipley K, Tzortzis K, et al., 2015, Bortezomib Amplifies Effect on Intracellular Proteasomes by Changing Proteasome Structure, EBioMedicine, Vol: 2, Pages: 642-648
The proteasome inhibitor Bortezomib is used to treat multiple myeloma (MM). Bortezomib inhibits protein degradation by inactivating proteasomes’ active-sites. MM cells are exquisitely sensitive to Bortezomib - exhibiting a low-nanomolar IC50 - suggesting that minimal inhibition of degradation suffices to kill MM cells. Instead, we report, a low Bortezomib concentration, contrary to expectation, achieves severe inhibition of proteasome activity in MM cells: the degree of inhibition exceeds what one would expect from the small proportion of active-sites that Bortezomib inhibits. Our data indicate that Bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity. Comparing MM cells to other, Bortezomib-resistant, cancer cells shows that the degree of proteasome inhibition is the greatest in MM cells and only there leads to proteasome stress, providing an explanation for why Bortezomib is effective against MM but not other cancers.
Pitcher DS, De Mattos-Shipley K, Wang Z, et al., 2014, Nuclear proteasomes carry a constitutive posttranslational modification which derails SDS-PAGE (but not CTAB-PAGE), Biochimica et Biophysica Acta - Proteins and Proteomics, Vol: 1844, Pages: 2222-2228
We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.
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