314 results on '"Godfrey L. Smith"'
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2. Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies
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Alice Main, Andri Boguslavskyi, Jacqueline Howie, Chien-Wen Kuo, Aileen Rankin, Francis L. Burton, Godfrey L. Smith, Roger Hajjar, George S. Baillie, Kenneth S. Campbell, Michael J. Shattock, and William Fuller
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palmitoylation ,hypertrophy ,heart failure ,ZDHHC5 ,cardiac muscle ,depalmitoylation ,Physiology ,QP1-981 - Abstract
S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.
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- 2022
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3. Prediction of Ventricular Arrhythmias by QRS/QTc - Ratio in Citalopram or Escitalopram Intoxication
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Erik Sveberg Dietrichs and Godfrey L. Smith
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citalopram ,escitalopram ,long QT ,ECG ,QRS/QTc ,arrhythmia ,Medicine (General) ,R5-920 - Abstract
BackgroundThe U.S. Food and Drug Administration (FDA) has stated that citalopram and escitalopram should not be used at daily doses above 40 mg/20 mg due to risk for development of fatal ventricular arrhythmias like torsade de pointes (TdP). Yet, supratherapeutic serum concentrations of citalopram are common and predicting patients at risk for TdP is of high clinical value. Accordingly, we investigated whether QRS/QTc; developed for predicting TdP in hypothermic patients could be used in citalopram intoxication.MethodsA total of 16 publications describing patients suffering from complications due to citalopram or escitalopram treatment, or intoxication with the same substances, were included after a systematic search. The main criterion for inclusion was admission ECG, either with given QRS and QTc values or with attached ECG-files that enabled calculation.ResultsQRS/QTc rather that QTc alone emerged as a marker of ventricular arrhythmia in the 16 included case reports, with highly significant (p < 0.0005) lower values in patients displaying ventricular arrhythmias.ConclusionCitalopram and escitalopram are extensively used in treatment of depressive disorders, and a large proportion of patients have supratherapeutic serum concentrations. Calculation of QRS/QTc in available case reports show that this novel ECG-marker has potential to predict patients at risk for developing ventricular arrhythmias.
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- 2022
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4. Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review
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M. Caroline Müllenbroich, Allen Kelly, Corey Acker, Gil Bub, Tobias Bruegmann, Anna Di Bona, Emilia Entcheva, Cecilia Ferrantini, Peter Kohl, Stephan E. Lehnart, Marco Mongillo, Camilla Parmeggiani, Claudia Richter, Philipp Sasse, Tania Zaglia, Leonardo Sacconi, and Godfrey L. Smith
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electrophysiology ,optogenetics ,heart ,arrhythmia ,fluorescence ,Physiology ,QP1-981 - Abstract
Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.
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- 2021
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5. In vivo grafting of large engineered heart tissue patches for cardiac repair
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Richard J. Jabbour, Thomas J. Owen, Pragati Pandey, Marina Reinsch, Brian Wang, Oisín King, Liam Steven Couch, Dafni Pantou, David S. Pitcher, Rasheda A. Chowdhury, Fotios G. Pitoulis, Balvinder S. Handa, Worrapong Kit-Anan, Filippo Perbellini, Rachel C. Myles, Daniel J. Stuckey, Michael Dunne, Mayooran Shanmuganathan, Nicholas S. Peters, Fu Siong Ng, Florian Weinberger, Cesare M. Terracciano, Godfrey L. Smith, Thomas Eschenhagen, and Sian E. Harding
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Cardiology ,Stem cells ,Medicine - Abstract
Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.
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- 2021
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6. Run for your life: can exercise be used to effectively target GLUT4 in diabetic cardiac disease?
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Peter R.T. Bowman, Godfrey L. Smith, and Gwyn W. Gould
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GLUT4 ,Diabetic cardiomyopathy ,Cardiomyocyte ,Exercise ,Glucose ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
The global incidence, associated mortality rates and economic burden of diabetes are now such that it is considered one of the most pressing worldwide public health challenges. Considerable research is now devoted to better understanding the mechanisms underlying the onset and progression of this disease, with an ultimate aim of improving the array of available preventive and therapeutic interventions. One area of particular unmet clinical need is the significantly elevated rate of cardiomyopathy in diabetic patients, which in part contributes to cardiovascular disease being the primary cause of premature death in this population. This review will first consider the role of metabolism and more specifically the insulin sensitive glucose transporter GLUT4 in diabetic cardiac disease, before addressing how we may use exercise to intervene in order to beneficially impact key functional clinical outcomes.
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- 2021
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7. Metformin Reduces Potassium Currents and Prolongs Repolarization in Non-Diabetic Heart
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Layse Malagueta-Vieira, Julieta Fernández-Ruocco, María P. Hortigón-Vinagre, Víctor Zamora, Julián Zayas-Arrabal, Leyre Echeazarra, Godfrey L. Smith, Martín Vila Petroff, Emiliano Medei, Óscar Casis, and Mónica Gallego
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cardiac electrophysiology ,repolarization ,cardiomyocyte ,ion channels ,ventricular arrhythmia ,cardiac action potential ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results in reducing hyperglycaemia and insulin resistance. However, diabetic patients have higher risk of ventricular arrhythmia and sudden cardiac death, and metformin failed to reduce ventricular arrhythmia in clinical trials. In order to explore the mechanisms responsible for the lack of protective effect, we investigated in vivo the effect of metformin on cardiac electrical activity in non-diabetic rats; and in vitro in isolated ventricular myocytes, HEK293 cells expressing the hERG channel and human induced pluripotent stem cells derived cardiomyocytes (hIPS-CMs). Surface electrocardiograms showed that long-term metformin treatment (7 weeks) at therapeutic doses prolonged cardiac repolarization, reflected as QT and QTc interval duration, and increased ventricular arrhythmia during the caffeine/dobutamine challenge. Patch-clamp recordings in ventricular myocytes isolated from treated animals showed that the cellular mechanism is a reduction in the cardiac transient outward potassium current (Ito). In vitro, incubation with metformin for 24 h also reduced Ito, prolonged action potential duration, and increased spontaneous contractions in ventricular myocytes isolated from control rats. Metformin incubation also reduced IhERG in HEK293 cells. Finally, metformin incubation prolonged action potential duration at 30% and 90% of repolarization in hIPS-CMs, which is compatible with the reduction of Ito and IhERG. Our results show that metformin directly modifies the electrical behavior of the normal heart. The mechanism consists in the inhibition of repolarizing currents and the subsequent decrease in repolarization capacity, which prolongs AP and QTc duration.
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- 2022
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8. Regulation of connexin 43 by interleukin 1β in adult rat cardiac fibroblasts and effects in an adult rat cardiac myocyte: fibroblast co-culture model
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Lisa McArthur, Alexandra Riddell, Lisa Chilton, Godfrey L. Smith, and Stuart A. Nicklin
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Cell biology ,Molecular biology ,Cell culture ,Membrane ,Gene expression ,Cardiovascular system ,Science (General) ,Q1-390 ,Social sciences (General) ,H1-99 - Abstract
Connexin 43 expression (Cx43) is increased in cardiac fibroblasts (CFs) following myocardial infarction. Here, potential mediators responsible for increasing Cx43 expression and effects of differential CF phenotype on cardiac myocyte (CM) function were investigated. Stimulating adult rat CFs with proinflammatory mediators revealed that interleukin 1β (IL-1β) significantly enhanced Cx43 levels through the IL-1β pathway. Additionally, IL-1β reduced mRNA levels of the myofibroblast (MF) markers: (i) connective tissue growth factor (CTGF) and (ii) α smooth muscle actin (αSMA), compared to control CFs. A co-culture adult rat CM:CF model was utilised to examine cell-to-cell interactions. Transfer of calcein from CMs to underlying CFs suggested functional gap junction formation. Functional analysis revealed contraction duration (CD) of CMs was shortened in co-culture with CFs, while treatment of CFs with IL-1β reduced this mechanical effect of co-culture. No effect on action potential rise time or duration of CMs cultured with control or IL-1β-treated CFs was observed. These data demonstrate that stimulating CFs with IL-1β increases Cx43 and reduces MF marker expression, suggesting altered cell phenotype. These changes may underlie the reduced mechanical effects of IL-1β treated CFs on CD of co-cultured CMs and therefore have an implication for our understanding of heterocellular interactions in cardiac disease.
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- 2020
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9. A Bioprinted Heart-on-a-Chip with Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Evaluation
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Alan Faulkner-Jones, Victor Zamora, Maria P. Hortigon-Vinagre, Wenxing Wang, Marcus Ardron, Godfrey L. Smith, and Wenmiao Shu
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cell-printing ,organs-on-a-chips ,bio-ink ,hydrogels ,hiPSC-CMs ,in vitro testing ,Technology ,Biology (General) ,QH301-705.5 - Abstract
In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.
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- 2022
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10. Cardiac SNARE Expression in Health and Disease
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Peter R. T. Bowman, Godfrey L. Smith, and Gwyn W. Gould
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diabetes ,cardiomyopathy ,SNARE proteins ,insulin resistance ,GLUT4 ,Diseases of the endocrine glands. Clinical endocrinology ,RC648-665 - Abstract
SNARE proteins are integral to intracellular vesicular trafficking, which in turn is the process underlying the regulated expression of substrate transporters such as the glucose transporter GLUT4 at the cell surface of insulin target tissues. Impaired insulin stimulated GLUT4 trafficking is associated with reduced cardiac function in many disease states, most notably diabetes. Despite this, our understanding of the expression and regulation of SNARE proteins in cardiac tissue and how these may change in diabetes is limited. Here we characterize the array of SNARE proteins expressed in cardiac tissue, and quantify the levels of expression of VAMP2, SNAP23, and Syntaxin4—key proteins involved in insulin-stimulated GLUT4 translocation. We examined SNARE protein levels in cardiac tissue from two rodent models of insulin resistance, db/db mice and high-fat fed mice, and show alterations in patterns of expression are evident. Such changes may have implications for cardiac function.
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- 2019
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11. Electrophysiology of hiPSC-Cardiomyocytes Co-Cultured with HEK Cells Expressing the Inward Rectifier Channel
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Ana Da Silva Costa, Peter Mortensen, Maria P. Hortigon-Vinagre, Marcel A. G. van der Heyden, Francis L. Burton, Hao Gao, Radostin D. Simitev, and Godfrey L. Smith
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hiCMs ,HEK ,IK1 ,electrophysiology ,co-culture ,maturation ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (IK1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-IK1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-IK1:hiCM ratios was compared with co-cultures using wildtype (HEK–WT:hiCM) or hiCM alone on days 3–8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that IK1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK–WT. The addition of the IK1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-IK1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of IK1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient IK1 was integrated into the syncytium.
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- 2021
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12. Corrigendum: Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy
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Iffath A. Ghouri, Allen Kelly, Simona Salerno, Karin Garten, Tomas Stølen, Ole-Johan Kemi, and Godfrey L. Smith
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myocardial infarction ,optical mapping ,two-photon microscopy ,intracellular calcium ,border zone ,Physiology ,QP1-981 - Published
- 2019
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13. Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a β-Adrenoreceptor-Dependent Mechanism
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Przemysław Błyszczuk, Christian Zuppinger, Ana Costa, Daria Nurzynska, Franca Di Meglio, Mara Stellato, Irina Agarkova, Godfrey L. Smith, Oliver Distler, and Gabriela Kania
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cardiac microtissues ,iPSC-derived cardiomyocytes ,cardiac fibroblasts ,cardiac fibrosis ,cardiac rhythm ,TGF-β signalling ,Cytology ,QH573-671 - Abstract
Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.
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- 2020
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14. Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy
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Iffath A. Ghouri, Allen Kelly, Simona Salerno, Karin Garten, Tomas Stølen, Ole-Johan Kemi, and Godfrey L. Smith
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myocardial infarction ,optical mapping ,two-photon microscopy ,intracellular calcium ,border zone ,Physiology ,QP1-981 - Abstract
Background: The origin of electrical behavior in post-myocardial infarction scar tissue is still under debate. This study aims to examine the extent and nature of the residual electrical activity within a stabilized ventricular infarct scar.Methods and Results: An apical infarct was induced in the left ventricle of Wistar rats by coronary artery occlusion. Five weeks post-procedure, hearts were Langendorff-perfused, and optically mapped using di-4-ANEPPS. Widefield imaging of optical action potentials (APs) on the left ventricular epicardial surface revealed uniform areas of electrical activity in both normal zone (NZ) and infarct border zone (BZ), but only limited areas of low-amplitude signals in the infarct zone (IZ). 2-photon (2P) excitation of di-4-ANEPPS and Fura-2/AM at discrete layers in the NZ revealed APs and Ca2+ transients (CaTs) to 500–600 μm below the epicardial surface. 2P imaging in the BZ revealed superficial connective tissue structures lacking APs or CaTs. At depths greater than approximately 300 μm, myocardial structures were evident that supported normal APs and CaTs. In the IZ, although 2P imaging did not reveal clear myocardial structures, low-amplitude AP signals were recorded at discrete layers. No discernible Ca2+ signals could be detected in the IZ. AP rise times in BZ were slower than NZ (3.50 ± 0.50 ms vs. 2.23 ± 0.28 ms) and further slowed in IZ (9.13 ± 0.56 ms). Widefield measurements of activation delay between NZ and BZ showed negligible difference (3.37 ± 1.55 ms), while delay values in IZ showed large variation (11.88 ± 9.43 ms).Conclusion: These AP measurements indicate that BZ consists of an electrically inert scar above relatively normal myocardium. Discrete areas/layers of IZ displayed entrained APs with altered electrophysiology, but the structure of this tissue remains to be elucidated.
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- 2018
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15. Tailoring Mathematical Models to Stem-Cell Derived Cardiomyocyte Lines Can Improve Predictions of Drug-Induced Changes to Their Electrophysiology
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Chon Lok Lei, Ken Wang, Michael Clerx, Ross H. Johnstone, Maria P. Hortigon-Vinagre, Victor Zamora, Andrew Allan, Godfrey L. Smith, David J. Gavaghan, Gary R. Mirams, and Liudmila Polonchuk
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cardiomyocytes ,stem cell derived ,electrophysiology ,mathematical model ,pharmacology ,variability ,Physiology ,QP1-981 - Abstract
Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.
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- 2017
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16. Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes
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Chien-Wen S. Kuo, Sara Dobi, Caglar Gök, Ana Da Silva Costa, Alice Main, Olivia Robertson-Gray, Daniel Baptista-Hon, Krzysztof J. Wypijewski, Hannah Costello, Tim G. Hales, Niall MacQuaide, Godfrey L. Smith, and William Fuller
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Multidisciplinary - Abstract
Mammalian voltage-activated L-type Ca 2+ channels, such as Ca(v)1.2, control transmembrane Ca 2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current–voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca 2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca 2+ channels in excitable cells.
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- 2023
17. Electrophysiological heterogeneity in large populations of rabbit ventricular cardiomyocytes
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Quentin Lachaud, Muhamad Hifzhudin Noor Aziz, Francis L Burton, Niall Macquaide, Rachel C Myles, Radostin D Simitev, and Godfrey L Smith
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Physiology ,Physiology (medical) ,Animals ,Rabbits ,Cardiology and Cardiovascular Medicine ,Ion Channels - Abstract
Aims Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. Methods and results APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD90 value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD90 differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD90 distribution was generated from a larger population of models created by random variation of the maximum conductances (Gmax) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of Gmax values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Conclusion Modelling the measured range of inter-cell APDs required a larger range of key Gmax values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block.
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- 2022
18. Phenomenological analysis of simple ion channel block in large populations of uncoupled cardiomyocytes
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Radostin D Simitev, Antesar Al Dawoud, Muhamad H N Aziz, Rachel Myles, and Godfrey L Smith
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Pharmacology ,General Immunology and Microbiology ,Applied Mathematics ,General Neuroscience ,FOS: Physical sciences ,Quantitative Biology - Tissues and Organs ,General Medicine ,Physics - Medical Physics ,Quantitative Biology - Quantitative Methods ,General Biochemistry, Genetics and Molecular Biology ,Modeling and Simulation ,FOS: Biological sciences ,Cell Behavior (q-bio.CB) ,Quantitative Biology - Cell Behavior ,Medical Physics (physics.med-ph) ,Tissues and Organs (q-bio.TO) ,Quantitative Methods (q-bio.QM) ,General Environmental Science - Abstract
Current understanding of arrhythmia mechanisms and design of anti-arrhythmic drug therapies hinges on the assumption that myocytes from the same region of a single heart have similar, if not identical, action potential waveforms and drug responses. On the contrary, recent experiments reveal significant heterogeneity in uncoupled healthy myocytes both from different hearts as well as from identical regions within a single heart. In this work, a methodology is developed for quantifying the individual electrophysiological properties of large numbers of uncoupled cardiomyocytes under ion channel block in terms of the parameters values of a conceptual fast-slow model of electrical excitability. The approach is applied to a population of nearly 500 rabbit ventricular myocytes for which action potential duration (APD) before and after the application of the drug nifedipine was experimentally measured (Lachaud et al., 2022, Cardiovasc. Res.). To this end, drug action is represented by a multiplicative factor to an effective ion conductance, a closed form asymptotic expression for APD is derived and inverted to determine model parameters as functions of APD and dAPD (drug-induced change in APD) for each myocyte. Two free protocol-related quantities are calibrated to experiment using an adaptive-domain procedure based on an original assumption of optimal excitability. The explicit APD expression and the resulting set of model parameter values allow (a) direct evaluation of conditions necessary to maintain fixed APD or dAPD, (b) predictions of the proportion of cells remaining excitable after drug application, (c) predictions of stimulus period dependency and (d) predictions of dose-response curves, the latter being in agreement with additional experimental data., Accepted for publication in Mathematical Medicine and Biology: A Journal of the IMA (ISSN (Online):1477-8602) on 2023-01-15
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- 2023
19. zDHHC5 expression is increased in cardiac hypertrophy and reduced in heart failure but this does not correlate with changes in substrate palmitoylation
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Alice Main, Andri Bogusalvskyii, Jacqueline Howie, Chien-wen Kuo, Aileen Rankin, Francis L. Burton, Godfrey L. Smith, Roger Hajjar, George S. Baillie, Kenneth S. Campbell, Michael J. Shattock, and William Fuller
- Abstract
S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodiumcalcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Information is lacking about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression was rapidly elevated during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes was not sufficient to drive changes in palmitoylation of zDHHC5 substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its Cterminal tail, and we found the palmitoylation of zDHHC5 may be increased in heart failure in the same manner as NCX1, suggesting additional regulatory mechanisms such as acyl-CoA availability may be involved. Importantly, this study provides the first evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.
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- 2022
20. ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research
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Dierk Thomas, Milan Stengl, Dobromir Dobrev, Matteo E. Mangoni, Jordi Heijman, Carol Ann Remme, Larissa Fabritz, Katja E. Odening, Godfrey L. Smith, Cristina E. Molina, Leonardo Sacconi, A.M. Gomez, Antonio Zaza, Frank R. Heinzel, Cardiologie, RS: Carim - H01 Clinical atrial fibrillation, RS: Carim - H04 Arrhythmogenesis and cardiogenetics, Cardiology, ACS - Heart failure & arrhythmias, APH - Methodology, University of Bern, Odening, K, Gomez, A, Dobrev, D, Fabritz, L, Heinzel, F, Mangoni, M, Molina, C, Sacconi, L, Smith, G, Stengl, M, Thomas, D, Zaza, A, Remme, C, and Heijman, J
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0301 basic medicine ,TORSADE-DE-POINTES ,Cardiac electrophysiology ,Medizin ,Cardiomyopathy ,Arrhythmias ,030204 cardiovascular system & hematology ,0302 clinical medicine ,BIO/09 - FISIOLOGIA ,Mechanisms ,Position paper ,Induced pluripotent stem cell ,LEFT-VENTRICULAR WALL ,SINOATRIAL NODE ,Atrial fibrillation ,Animal models ,3. Good health ,PRESERVED EJECTION FRACTION ,Ion channels ,cardiovascular system ,HEART-FAILURE ,Mechanism ,Ion channel ,Electrophysiologic Techniques, Cardiac ,Cardiology and Cardiovascular Medicine ,Experimental models ,PLURIPOTENT STEM-CELLS ,Arrhythmia ,Myocarditis ,Cellular electrophysiology ,LONG-QT SYNDROME ,03 medical and health sciences ,[SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system ,Physiology (medical) ,SINUS NODE DYSFUNCTION ,medicine ,Animals ,Humans ,Animal model ,Experimental model ,business.industry ,TRANSGENIC RABBIT MODEL ,Cardiac arrhythmia ,Models, Theoretical ,medicine.disease ,Electrophysiological Phenomena ,030104 developmental biology ,Heart failure ,ATRIAL-FIBRILLATION ,business ,Neuroscience - Abstract
Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.
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- 2021
21. Atrial resting membrane potential confers sodium current sensitivity to propafenone, flecainide and dronedarone
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S. Nashitha Kabir, Jasmeet S. Reyat, Antony J. Workman, Priyanka Saxena, Dannie Fobian, Davor Pavlovic, Larissa Fabritz, Clara Apicella, Stefan M. Kuhlmann, Godfrey L. Smith, Paulus Kirchhof, Molly O’Reilly, Andrew P. Holmes, Fahima Syeda, Suranjana Gupta, and Christopher O’Shea
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Male ,medicine.medical_specialty ,Atrial action potential ,Refractory period ,Action Potentials ,Propafenone ,030204 cardiovascular system & hematology ,Membrane Potentials ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Physiology (medical) ,Internal medicine ,Atrial Fibrillation ,medicine ,Animals ,Myocyte ,Heart Atria ,030212 general & internal medicine ,Dronedarone ,Flecainide ,Voltage-Gated Sodium Channel Blockers ,business.industry ,Sodium channel ,Sodium ,Atrial fibrillation ,medicine.disease ,Disease Models, Animal ,cardiovascular system ,Cardiology ,Female ,Cardiology and Cardiovascular Medicine ,business ,Anti-Arrhythmia Agents ,medicine.drug - Abstract
Background Although atrial fibrillation ablation is increasingly used for rhythm control therapy, antiarrhythmic drugs (AADs) are commonly used, either alone or in combination with ablation. The effectiveness of AADs is highly variable. Previous work from our group suggests that alterations in atrial resting membrane potential (RMP) induced by low Pitx2 expression could explain the variable effect of flecainide. Objective The purpose of this study was to assess whether alterations in atrial/cardiac RMP modify the effectiveness of multiple clinically used AADs. Methods The sodium channel blocking effects of propafenone (300 nM, 1 μM), flecainide (1 μM), and dronedarone (5 μM, 10 μM) were measured in human stem cell–derived cardiac myocytes, HEK293 expressing human NaV1.5, primary murine atrial cardiac myocytes, and murine hearts with reduced Pitx2c. Results A more positive atrial RMP delayed INa recovery, slowed channel inactivation, and decreased peak action potential (AP) upstroke velocity. All 3 AADs displayed enhanced sodium channel block at more positive atrial RMPs. Dronedarone was the most sensitive to changes in atrial RMP. Dronedarone caused greater reductions in AP amplitude and peak AP upstroke velocity at more positive RMPs. Dronedarone evoked greater prolongation of the atrial effective refractory period and postrepolarization refractoriness in murine Langendorff-perfused Pitx2c+/– hearts, which have a more positive RMP compared to wild type. Conclusion Atrial RMP modifies the effectiveness of several clinically used AADs. Dronedarone is more sensitive to changes in atrial RMP than flecainide or propafenone. Identifying and modifying atrial RMP may offer a novel approach to enhancing the effectiveness of AADs or personalizing AAD selection.
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- 2021
22. Inhibition of myocardial cathepsin-L release during reperfusion following myocardial infarction improves cardiac function and reduces infarct size
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Elspeth B. Elliott, Aadil Shaukat, Weihong He, Kristopher Ford, Lisa McArthur, Katrin Nather, Mitchell Lindsay, Colin Berry, Stuart Hood, Ali Abdullah I. Zaeri, Catherine Hawksby, Tamara P. Martin, John D. McClure, Keith G. Oldroyd, David Carrick, Paul Rocchiccioli, Hany Eteiba, Keith M. Channon, Andrew Davie, Dylan O'Toole, Margaret McEntegart, Mark C. Petrie, Kenneth Mangion, Charlotte S. McCarroll, Stuart Watkins, Mathew M Y Lee, Stuart A. Nicklin, David Corcoran, Christopher M. Loughrey, Richard Good, Alexandra Riddell, Godfrey L. Smith, and Study, Oxford Acute Myocardial Infarction (OxAMI)
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0301 basic medicine ,Cardiac function curve ,medicine.medical_specialty ,Physiology ,medicine.medical_treatment ,Myocardial Infarction ,chemistry.chemical_element ,Myocardial Reperfusion ,Myocardial Reperfusion Injury ,030204 cardiovascular system & hematology ,Calcium ,Cathepsin L ,03 medical and health sciences ,Percutaneous Coronary Intervention ,0302 clinical medicine ,Physiology (medical) ,Internal medicine ,medicine ,Humans ,Myocardial infarction ,biology ,business.industry ,Percutaneous coronary intervention ,medicine.disease ,Cathepsins ,Treatment Outcome ,030104 developmental biology ,chemistry ,Apoptosis ,Heart failure ,Reperfusion ,Cardiology ,biology.protein ,ST Elevation Myocardial Infarction ,Cardiology and Cardiovascular Medicine ,business ,Reperfusion injury - Abstract
Aims: \ud Identifying novel mediators of lethal myocardial reperfusion injury that can be targeted during primary percutaneous coronary intervention (PPCI) is key to limiting the progression of patients with ST-elevated myocardial infarction (STEMI) to heart failure. Here we show through parallel clinical and integrative preclinical studies the significance of the protease cathepsin-L on cardiac function during reperfusion injury.\ud \ud Methods and Results: \ud We found that direct cardiac release of cathepsin-L in STEMI patients (n = 76) immediately post-PPCI leads to elevated serum cathepsin-L levels and that serum levels of cathepsin-L in the first 24 hour post-reperfusion are associated with reduced cardiac contractile function and increased infarct size. Preclinical studies, demonstrate that inhibition of cathepsin-L release following reperfusion injury with CAA0225 reduces infarct size and improves cardiac contractile function by limiting abnormal cardiomyocyte calcium handling and apoptosis.\ud \ud Conclusion: \ud Our findings suggest that cathepsin-L is a novel therapeutic target that could be exploited clinically to counteract the deleterious effects of acute reperfusion injury after an acute STEMI.\ud \ud Translational perspective: \ud New therapeutic targets are urgently required to limit myocardial damage after reperfusion injury. We identified cardiac release of the protease cathepsin-L among patients following primary percutaneous coronary intervention (PPCI). Elevated serum levels of cathepsin-L were associated with reduced contractile function and increased infarct size at 24 hour and 6 months post-PPCI. Work conducted using animal models indicated that cardiac release of cathepsin-L mediated cardiac dysfunction following reperfusion injury. Specific inhibition of cathepsin-L prevented abnormal calcium handling, reduced infarct size and improved contractile function. These novel findings offer the prospect of targeting cathepsin-L-mediated cardiac dysfunction after PPCI.
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- 2021
23. Cardiac myosin binding protein-C palmitoylation is associated with increased myofilament affinity, reduced myofilament Ca2+ sensitivity and is increased in ischaemic heart failure
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Alice Main, Gregory N. Milburn, Rajvi M. N. Balesar, Aileen C. Rankin, Godfrey L. Smith, Kenneth S. Campbell, George S. Baillie, Jolanda van der Velden, and William Fuller
- Abstract
Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of cardiac contractility through its interactions with the thick and thin filament. cMyBP-C is heavily influenced by post-translational modifications, including phosphorylation which improves cardiac inotropy and lusitropy, and S-glutathionylation, which impairs phosphorylation and is increased in heart failure. Palmitoylation is an essential cysteine modification that regulates the activity of cardiac ion channels and soluble proteins, however, its relevance to myofilament proteins has not been investigated. In the present study, we purified palmitoylated proteins from ventricular cardiomyocytes and identified that cardiac actin, myosin and cMyBP-C are palmitoylated. The palmitoylated form of cMyBP-C was more resistant to salt extraction from the myofilament lattice than the non-palmitoylated form. Isometric tension measurements suggest c-MyBP-C palmitoylation reduces myofilament Ca2+ sensitivity, with no change to maximum force or passive tension. Importantly, cMyBP-C palmitoylation levels are reduced at the site of injury in a rabbit model of heart failure but increased in samples from patients with ischaemic heart failure. Identification of cMyBP-C palmitoylation site revealed S-glutathionylated cysteines C635 and C651 are required for cMyBP-C palmitoylation, suggesting an interplay between the modifications at these sites. We conclude that structural and contractile proteins within the myofilament lattice are palmitoylated, with important functional consequences for cardiac contractile performance.
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- 2022
24. Adrenoceptor sub-type involvement in Ca
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Priyanka, Saxena, Rachel C, Myles, Godfrey L, Smith, and Antony J, Workman
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Calcium Channels, L-Type ,Adrenergic beta-Antagonists ,Prazosin ,Receptors, Adrenergic, alpha ,Norepinephrine ,Receptors, Adrenergic, alpha-2 ,Atrial Fibrillation ,Receptors, Adrenergic, beta ,Animals ,Humans ,Myocytes, Cardiac ,Rabbits ,Heart Atria ,Adrenergic alpha-Antagonists - Abstract
Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca
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- 2022
25. The Use of Voltage Sensitive Dye di-4-ANEPPS and Video-Based Contractility Measurements to Assess Drug Effects on Excitation–Contraction Coupling in Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes
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Maria P. Hortigon-Vinagre, Victor Zamora, Francis L. Burton, and Godfrey L. Smith
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0301 basic medicine ,Drug ,Inotrope ,Time Factors ,Calcium Channels, L-Type ,media_common.quotation_subject ,Induced Pluripotent Stem Cells ,Action Potentials ,Pyridinium Compounds ,030204 cardiovascular system & hematology ,Pharmacology ,Risk Assessment ,Amrinone ,Contractility ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Myofibrils ,Toxicity Tests ,medicine ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,Cells, Cultured ,Excitation Contraction Coupling ,Fluorescent Dyes ,media_common ,Cardiotoxicity ,Microscopy, Video ,Chemistry ,Arrhythmias, Cardiac ,Cell Differentiation ,Myocardial Contraction ,Bay K8644 ,030104 developmental biology ,Microscopy, Fluorescence ,Pimobendan ,Cardiology and Cardiovascular Medicine ,medicine.drug - Abstract
Because cardiotoxicity is one of the leading causes of drug failure and attrition, the design of new protocols and technologies to assess proarrhythmic risks on cardiac cells is in continuous development by different laboratories. Current methodologies use electrical, intracellular Ca2+, or contractility assays to evaluate cardiotoxicity. Increasingly, the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are the in vitro tissue model used in commercial assays because it is believed to recapitulate many aspects of human cardiac physiology. In this work, we demonstrate that the combination of a contractility and voltage measurements, using video-based imaging and fluorescence microscopy, on hiPSC-CMs allows the investigation of mechanistic links between electrical and mechanical effects in an assay design that can address medium throughput scales necessary for drug screening, offering a view of the mechanisms underlying drug toxicity. To assess the accuracy of this novel technique, 10 commercially available inotropic drugs were tested (5 positive and 5 negative). Included were drugs with simple and specific mechanisms, such as nifedipine, Bay K8644, and blebbistatin, and others with a more complex action such as isoproterenol, pimobendan, digoxin, and amrinone, among others. In addition, the results provide a mechanism for the toxicity of itraconazole in a human model, a drug with reported side effects on the heart. The data demonstrate a strong negative inotropic effect because of the blockade of L-type Ca2+ channels and additional action on the cardiac myofilaments. We can conclude that the combination of contractility and action potential measurements can provide wider mechanistic knowledge of drug cardiotoxicity for preclinical assays.
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- 2021
26. Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction
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Anne Berit Johnsen, Karin Garten, Eirik Skogvoll, Nathan R. Scrimgeour, Godfrey L. Smith, Øyvind Ellingsen, Kari Jørgensen, Bjarne M. Nes, Tomas Stølen, Julie R. McMullen, José Bianco Nascimento Moreira, Muhammad Shakil Ahmed, Ulrik Wisløff, Maria P. Hortigon-Vinagre, Håvard Attramadal, Anne Marie Ormbostad Berre, Morten A. Høydal, and Victor Zamora
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0301 basic medicine ,Cardiac function curve ,medicine.medical_specialty ,Myocardial Infarction ,Infarction ,Cardiomegaly ,030204 cardiovascular system & hematology ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Endurance training ,Physical Conditioning, Animal ,Internal medicine ,medicine ,Animals ,Aerobic exercise ,Myocytes, Cardiac ,Myocardial infarction ,Molecular Biology ,computer.programming_language ,Heart Failure ,sed ,business.industry ,Arrhythmias, Cardiac ,medicine.disease ,Myocardial Contraction ,Aerobiosis ,Electrophysiological Phenomena ,MicroRNAs ,Electrophysiology ,030104 developmental biology ,Gene Expression Regulation ,Heart failure ,Ventricular Fibrillation ,Cardiology ,Female ,Cardiology and Cardiovascular Medicine ,business ,computer ,Biomarkers - Abstract
Aims Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. Methods and results Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these “exercise-miRNAs” individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the “exercise-miRNAs”, miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. Conclusion Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.
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- 2020
27. SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca
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Bracy, Fertig, Jiayue, Ling, Edgar E, Nollet, Sara, Dobi, Tara, Busiau, Kiyotake, Ishikawa, Kelly, Yamada, Ahyoung, Lee, Changwon, Kho, Lauren, Wills, Amy J, Tibbo, Mark, Scott, Kirsten, Grant, Kenneth S, Campbell, Emma J, Birks, Niall, MacQuaide, Roger, Hajjar, Godfrey L, Smith, Jolanda, van der Velden, and George S, Baillie
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Myofibrils ,Lysine ,Troponin I ,Humans ,Sumoylation ,Calcium ,Phosphorylation - Abstract
Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.
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- 2022
28. Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart
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Allen Kelly, Karin Garten, Godfrey L. Smith, Simona Salerno, and Tomas Stølen
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Optical Phenomena ,030303 biophysics ,Biophysics ,Action Potentials ,Voltage sensitive dyes ,Article ,law.invention ,Mice ,03 medical and health sciences ,Two-photon excitation microscopy ,Confocal microscopy ,law ,Microscopy ,Animals ,Ventricular Function ,Repolarization ,Molecular Biology ,Photons ,0303 health sciences ,Chemistry ,Heart ,Depolarization ,Photobleaching ,Fluorescence ,Electrophysiology ,Isolated perfused heart ,Two photon excitation microscopy ,Biomedical engineering - Abstract
Background and aims:\ud Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging.\ud \ud Methods:\ud Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy.\ud \ud Results:\ud FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation.\ud \ud Conclusion:\ud Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties.
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- 2020
29. Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes
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Nurul A N Mohd Yusof, Christine L. Mummery, Leon G.J. Tertoolen, Jessica Nebel, Peter Clements, Eric I. Rossman, Umber Saleem, Ana Krotenberg Garcia, Kate Harris, Maria L. H. Vlaming, Karen McGlynn, Godfrey L. Smith, Xiaoping Xu, Puspita A Katili, Tessa de Korte, Francis L. Burton, Berend J. van Meer, Ingra Mannhardt, Arne Hansen, Chris Denning, Anthony Bahinski, Thomas Eschenhagen, and Applied Stem Cell Technologies
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0301 basic medicine ,Drug ,Inotrope ,CRACK-IT project ,medicine.medical_specialty ,Contraction (grammar) ,media_common.quotation_subject ,Induced Pluripotent Stem Cells ,Alternatives to animal testing ,cardiomyocytes ,predictive toxicology ,030204 cardiovascular system & hematology ,Toxicology ,contractility ,Contractility ,03 medical and health sciences ,alternatives to animal testing ,0302 clinical medicine ,Internal medicine ,Emerging Technologies, Methods, and Models ,safety pharmacology ,medicine ,human-induced pluripotent stem cells ,Animals ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,media_common ,Dose-Response Relationship, Drug ,business.industry ,Safety pharmacology ,inotropy ,electrophysiology ,Electrophysiology ,030104 developmental biology ,Pharmaceutical Preparations ,Cardiology ,business - Abstract
Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these “secondary” parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation.
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- 2020
30. General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy
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Adam P. Hill, Zhen Song, Haibo Ni, Norman Stockbridge, David G. Strauss, Jamie I. Vandenberg, Todd Wisialowski, Mikiko Nakamura, Takashi Yoshinaga, Godfrey L. Smith, Jill Steidl‐Nichols, Mark Holbrook, Stefano Severi, Pierre Morissette, Derek J. Leishman, Colleen E. Clancy, Gary R. Mirams, Andrea Greiter-Wilke, Andrew G. Edwards, Xiaomei Han, Zhihua Li, Flora Musuamba Tshinanu, Stefano Morotti, Stephane Nave, Aaron Fullerton, Bradley J. Ridder, Zhilin Qu, Michelangelo Paci, Alfonso Bueno-Orovio, Janell E. Chen, Thomas Singer, Peter R. Kowey, Liudmila Polonchuk, Eric A. Sobie, Blanca Rodriguez, Randall L. Rasmusson, Elisa Passini, Eleonora Grandi, Ken Wang, Michael Liu, Bruce P. Damiano, Xin Zhou, Li Z., Mirams G.R., Yoshinaga T., Ridder B.J., Han X., Chen J.E., Stockbridge N.L., Wisialowski T.A., Damiano B., Severi S., Morissette P., Kowey P.R., Holbrook M., Smith G., Rasmusson R.L., Liu M., Song Z., Qu Z., Leishman D.J., Steidl-Nichols J., Rodriguez B., Bueno-Orovio A., Zhou X., Passini E., Edwards A.G., Morotti S., Ni H., Grandi E., Clancy C.E., Vandenberg J., Hill A., Nakamura M., Singer T., Polonchuk L., Greiter-Wilke A., Wang K., Nave S., Fullerton A., Sobie E.A., Paci M., Musuamba Tshinanu F., Strauss D.G., Tampere University, BioMediTech, and Research group: Computational Biophysics and Imaging Group
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Drug-Related Side Effects and Adverse Reactions ,none ,In silico ,White Paper ,Harmonization ,Context (language use) ,Arrhythmias ,Validation Studies as Topic ,Risk prediction models ,Risk Assessment ,030226 pharmacology & pharmacy ,Session (web analytics) ,03 medical and health sciences ,0302 clinical medicine ,Consistency (negotiation) ,White paper ,Theoretical ,Models ,medicine ,Humans ,Computer Simulation ,Pharmacology (medical) ,Pharmacology & Pharmacy ,Pharmacology ,Proarrhythmia ,Management science ,Arrhythmias, Cardiac ,217 Medical engineering ,Pharmacology and Pharmaceutical Sciences ,Models, Theoretical ,medicine.disease ,030220 oncology & carcinogenesis ,Cardiac - Abstract
This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration–sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model‐based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm.
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- 2019
31. Stimulation of isolated ventricular myocytes within an open architecture microarray.
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Norbert Klauke, Godfrey L. Smith, and Jonathan M. Cooper
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- 2005
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32. Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes
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Nikolaj Gadegaard, Francis L. Burton, Godfrey L. Smith, Hao Gao, Radostin D. Simitev, Virpi Talman, Peter Mortensen, Lotta Pohjolainen, Heikki Ruskoaho, Eline Huethorst, Regenerative cardiac pharmacology, Division of Pharmacology and Pharmacotherapy, Drug Research Program, Divisions of Faculty of Pharmacy, and Regenerative pharmacology group
- Subjects
Chronotropic ,DYNAMICS ,Contraction (grammar) ,Physiology ,TENSION ,recombinant collagen polymer ,Induced Pluripotent Stem Cells ,Action Potentials ,030204 cardiovascular system & hematology ,ALPHA-CATENIN ,FORCE ,Contractility ,03 medical and health sciences ,0302 clinical medicine ,RELEVANCE ,Isoprenaline ,Monolayer ,medicine ,CARDIAC-MUSCLE ,Cell Adhesion ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,030304 developmental biology ,pharmaceutical assay ,0303 health sciences ,cardiac physiology ,Chemistry ,substrate rigidity ,STIFFNESS ,Cell Differentiation ,Adhesion ,IN-VITRO ,Myocardial Contraction ,COLLAGEN ,317 Pharmacy ,Self-healing hydrogels ,Biophysics ,MATRIX ,mathematical model ,medicine.drug - Abstract
Key points Spatiotemporal contractility analysis of human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only 'twitch'-like transients are observed. HiPSC-CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress-activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell-substrate adhesion underly multiphasic contractile behaviour of hiPSC-CMs. Abstract Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers 1) attached to glass or plastic (Young's modulus (E) >1 GPa), 2) detached (substrate-free) and 3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multi-phasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared to glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multi-phasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. This article is protected by copyright. All rights reserved.
- Published
- 2021
33. In vivo grafting of large engineered heart tissue patches for cardiac repair
- Author
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Rasheda A. Chowdhury, Marina Reinsch, Godfrey L. Smith, Dafni Pantou, Mayooran Shanmuganathan, Florian Weinberger, Fu Siong Ng, Thomas Eschenhagen, Liam Couch, Thomas J. Owen, Oisín King, Cesare M. Terracciano, David S. Pitcher, Daniel J. Stuckey, Nicholas S. Peters, Balvinder S. Handa, Richard J. Jabbour, Brian Wang, Pragati Pandey, Michael Dunne, Rachel C. Myles, Worrapong Kit-Anan, Filippo Perbellini, Fotios G. Pitoulis, Sian E. Harding, and British Heart Foundation
- Subjects
Cardiac function curve ,medicine.medical_specialty ,Contraction (grammar) ,Induced Pluripotent Stem Cells ,Myocardial Infarction ,Cardiology ,Human stem cells ,Stem cells ,Arrhythmias ,Fibrin ,In vivo ,Internal medicine ,Animals ,Humans ,Medicine ,Myocardial infarction ,Cardiac Surgical Procedures ,Heart Failure ,Tissue Engineering ,biology ,Guided Tissue Regeneration ,business.industry ,Myocardium ,Arrhythmias, Cardiac ,Hydrogels ,General Medicine ,medicine.disease ,Cardiovascular disease ,Myocardial Contraction ,Heart failure ,biology.protein ,Rabbits ,Stem cell ,business ,Ex vivo ,Research Article - Abstract
Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.
- Published
- 2021
34. Dynamic clamping human and rabbit atrial calcium current: narrowing I CaL window abolishes early afterdepolarizations
- Author
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Godfrey L. Smith, Sarah Kettlewell, Priyanka Saxena, Antony J. Workman, John Dempster, Michael A. Colman, and Rachel C. Myles
- Subjects
0301 basic medicine ,Fibrillation ,medicine.medical_specialty ,Atrial action potential ,Physiology ,Chemistry ,Atrial fibrillation ,medicine.disease ,Afterdepolarization ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Nifedipine ,Internal medicine ,medicine ,Cardiology ,Repolarization ,Myocyte ,Patch clamp ,medicine.symptom ,030217 neurology & neurosurgery ,medicine.drug - Abstract
KEY POINTS Early-afterdepolarizations (EADs) are abnormal action potential oscillations and a known cause of cardiac arrhythmias. Ventricular EADs involve reactivation of a Ca2+ current (ICaL ) in its 'window region' voltage range. However, electrical mechanisms of atrial EADs, a potential cause of atrial fibrillation, are poorly understood. Atrial cells were obtained from consenting patients undergoing heart surgery, as well as from rabbits. ICaL was blocked with nifedipine and then a hybrid patch clamp/mathematical-modelling technique, 'dynamic clamping', was used to record action potentials at the same time as injecting an artificial, modifiable, ICaL (ICaL,D-C ). Progressively widening the ICaL,D-C window region produced EADs of various types, dependent on window width. EAD production was strongest upon moving the activation (vs. inactivation) side of the window. EADs were then induced by a different method: increasing ICaL,D-C amplitude and/or K+ channel-blockade (4-aminopyridine). Narrowing of the ICaL,D-C window by ∼10 mV abolished these EADs. Atrial ICaL window narrowing is worthy of further testing as a potential anti-atrial fibrillation drug mechanism. ABSTRACT Atrial early-afterdepolarizations (EADs) may contribute to atrial fibrillation (AF), perhaps involving reactivation of L-type Ca2+ current (ICaL ) in its window region voltage range. The present study aimed (i) to validate the dynamic clamp technique for modifying the ICaL contribution to atrial action potential (AP) waveform; (ii) to investigate the effects of widening the window ICaL on EAD-propensity; and (iii) to test whether EADs from increased ICaL and AP duration are supressed by narrowing the window ICaL . ICaL and APs were recorded from rabbit and human atrial myocytes by whole-cell-patch clamp. During AP recording, ICaL was inhibited (3 µm nifedipine) and replaced by a dynamic clamp model current, ICaL,D-C (tuned to native ICaL characteristics), computed in real-time (every 50 µs) based on myocyte membrane potential. ICaL,D-C -injection restored the nifedipine-suppressed AP plateau. Widening the window ICaL,D-C , symmetrically by stepwise simultaneous equal shifts of half-voltages (V0.5 ) of ICaL,D-C activation (negatively) and inactivation (positively), generated EADs (single, multiple or preceding repolarization failure) in a window width-dependent manner, as well as AP alternans. A stronger EAD-generating effect resulted from independently shifting activation V0.5 (asymmetrical widening) than inactivation V0.5 ; for example, a 15 mV activation shift produced EADs in nine of 17 (53%) human atrial myocytes vs. 0 of 18 from inactivation shift (P
- Published
- 2019
35. Electrophysiology of hiPSC-Cardiomyocytes Co-Cultured with HEK Cells Expressing the Inward Rectifier Channel
- Author
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Radostin D. Simitev, Marcel A.G. van der Heyden, Hao Gao, Godfrey L. Smith, Maria P. Hortigon-Vinagre, Ana Costa, Peter Mortensen, and Francis L. Burton
- Subjects
0301 basic medicine ,IK1 ,QH301-705.5 ,Induced Pluripotent Stem Cells ,030204 cardiovascular system & hematology ,Giant Cells ,Catalysis ,Article ,Inorganic Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Myocytes, Cardiac ,Biology (General) ,Physical and Theoretical Chemistry ,Potassium Channels, Inwardly Rectifying ,QD1-999 ,Molecular Biology ,Spectroscopy ,Syncytium ,HEK ,Inward-rectifier potassium ion channel ,Cardiac electrophysiology ,Chemistry ,maturation ,Organic Chemistry ,HEK 293 cells ,Wild type ,hiCMs ,General Medicine ,electrophysiology ,Embryonic stem cell ,co-culture ,Myocardial Contraction ,Coculture Techniques ,Computer Science Applications ,Cell biology ,Electrophysiology ,030104 developmental biology ,HEK293 Cells ,Intracellular - Abstract
The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (IK1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-IK1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-IK1:hiCM ratios was compared with co-cultures using wildtype (HEK–WT:hiCM) or hiCM alone on days 3–8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that IK1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK–WT. The addition of the IK1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-IK1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of IK1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient IK1 was integrated into the syncytium.
- Published
- 2021
36. Is hypothermia more neuroprotective than avoiding fever after cardiac arrest?
- Author
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Godfrey L. Smith, Erik Sveberg Dietrichs, and Rachel C. Myles
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Physiology ,business.industry ,medicine.medical_treatment ,Clinical Commentaries ,Cardiovascular Research Onlife ,Hypothermia ,Targeted temperature management ,Cardiac arrest ,Neuroprotection ,Heart Arrest ,Hypothermia, Induced ,Physiology (medical) ,Anesthesia ,medicine ,Humans ,AcademicSubjects/MED00200 ,Therapeutic hypothermia ,QRS/QTc ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business - Abstract
Hypothermia was increasingly proposed as a neuroprotective therapy in the 1990s, culminating in the publication of two randomized trials in 2002, which showed beneficial effects of therapeutic hypothermia in comatose survivors of cardiac arrest.1,2 Therapeutic hypothermia was subsequently included in guidelines for the treatment of this patient group. However, doubt as to whether hypothermia provided benefit over targeted normothermia (i.e. preventing fever) was raised in 2013 when a large randomized trial showed no difference in survival or neurological function between cardiac arrest survivors cooled to 33 or 36°C.3 Recently the results from a large (1900 patients) randomized open-label trial, comparing patients cooled to 33°C or maintained normothermic (≤37.5°C), was published by Dankiewicz et al.4 in the New England Journal of Medicine. In agreement with the 2013 publication,3 no difference in disability or death was detected between the treatment groups after 6 months. The only significant difference in adverse events was arrhythmias; those resulting in haemodynamic compromise were more common in the hypothermia group than in the normothermia group (24% vs. 17%).4
- Published
- 2021
37. Electrical activity in exercise trained infarcted rat hearts
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Tomas O. Stolen, Anne Berit Johnsen, Mathias Nyman, Karin Garten, Allen Kelly, Francis L. Burton, Godfrey L. Smith, and Jan Pål Loennechen
- Subjects
Biophysics - Published
- 2022
38. Resistance to ventricular fibrillation predicted by the QRS/QTc - Ratio in an intact rat model of hypothermia/rewarming
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Torkjel Tveita, Karen McGlynn, Timofei V. Kondratiev, Erik Sveberg Dietrichs, Godfrey L. Smith, and Anders Lund Selli
- Subjects
medicine.medical_specialty ,Accidental hypothermia ,Rat model ,Hypothermia ,QT interval ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,QRS complex ,0302 clinical medicine ,Hypothermia, Induced ,Internal medicine ,medicine ,Animals ,Humans ,cardiovascular diseases ,Rewarming ,Cryopreservation ,030219 obstetrics & reproductive medicine ,business.industry ,0402 animal and dairy science ,VDP::Medisinske Fag: 700::Basale medisinske, odontologiske og veterinærmedisinske fag: 710 ,Arrhythmias, Cardiac ,04 agricultural and veterinary sciences ,General Medicine ,medicine.disease ,040201 dairy & animal science ,VDP::Medical disciplines: 700::Basic medical, dental and veterinary science disciplines: 710 ,Rats ,Moderate hypothermia ,Ventricular fibrillation ,Ventricular Fibrillation ,Cardiology ,cardiovascular system ,High incidence ,medicine.symptom ,General Agricultural and Biological Sciences ,business ,circulatory and respiratory physiology - Abstract
Accidental hypothermia is associated with increased risk for arrhythmias. QRS/QTc is proposed as an ECG-marker, where decreasing values predict hypothermia-induced ventricular arrhythmias. If reliable it should also predict nonappearance of arrhythmias, observed in species like rat that regularly tolerate prolonged hypothermia. A rat model designed for studying cardiovascular function during cooling, hypothermia and subsequent rewarming was chosen due to species-dependent resistance to ventricular arrhythmias. ECG was recorded throughout the protocol. No ventricular arrhythmias occurred during experiments. QRS/QTc increased throughout the cooling period and remained above normothermic baseline until rewarmed. Different from the high incidence of hypothermia-induced ventricular arrhythmias in accidental hypothermia patients, where QRS/QTc ratio is decreased in moderate hypothermia; hypothermia and rewarming of rats is not associated with increased risk for ventricular fibrillation. This resistance to lethal hypothermia-induced arrhythmias was predicted by QRS/QTc.
- Published
- 2020
39. Phosphodiesterase Type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins
- Author
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Brian O. Smith, Amy J. Tibbo, William Fuller, George S. Baillie, Caglar Gök, Aisling McFall, Niall MacQuaide, Gonzalo S. Tejeda, Ruth MacLeod, Godfrey L. Smith, Connor M. Blair, Thomas Brand, and Sara Dobi
- Subjects
chemistry.chemical_classification ,Chemistry ,CAMP signaling ,Effector ,Second messenger system ,Phosphodiesterase ,Myocyte ,Peptide ,Receptor ,Intracellular ,Cell biology - Abstract
Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that Popeye proteins exist in complexes with members of the PDE4 family in cardiac myocytes thus restricting cAMP signaling. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 is essential to maintain action potential duration in beating cardiac myocytes.
- Published
- 2020
40. C In-vivo grafting of large engineered heart tissue patches for cardiac repair
- Author
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Richard J. Jabbour, Florian Weinberger, Thomas J. Owen, Godfrey L. Smith, Thomas Eschenhagen, Sian E. Harding, Marina Reinsch, and Pragati Pandey
- Subjects
medicine.medical_specialty ,biology ,business.industry ,Provocation test ,medicine.disease ,Fibrin ,Fractional area change ,In vivo ,Internal medicine ,Cardiac repair ,medicine ,Myocardial cell ,Cardiology ,biology.protein ,Myocardial infarction ,business ,Ex vivo - Abstract
Introduction Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix may overcome the limitations of intracoronary/myocardial cell delivery routes. EHTs regenerate heart muscle in small animal models but data regarding clinically relevant engineered heart tissue (EHT) patches large enough for first-in-human studies are lacking. Methods An upscaled EHT patch (approx. 3 cm × 2 cm × 1.5 mm) consisting of 15–20 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) embedded in a fibrin based hydrogel was developed. A rabbit myocardial infarction model was then developed to test for feasibility and efficacy of EHT grafting. Results The patches began to beat spontaneously within 3 days of fabrication and after 28 days of dynamic culture (late EHTs) showed the development of several mature characteristics when compared to early patches ( We then tested the EHT patch in-vivo using a rabbit model. Patches were applied to infarcted hearts (n=14 [n=7 EHT vs n=7 sham]). Sham operations used non-cellular fibrin patches. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that underwent EHT patch grafting (n=7; absolute difference of 10.04 ± 3.1% over sham group; fractional area change, P In-vivo telemetry recordings (n=5 MI/sham vs n=7 MI/EHT) indicated that no clinically relevant arrhythmia was seen in the MI/EHT group and arrhythmia provocation protocols (ex vivo n=5 MI/sham vs n=6 MI/EHT) confirmed that the patch was not pro-arrhythmic (arrhythmia inducibility score 5.6 ± 1.0 [MI/patch] vs 5.0 ± 0.6 [MI/sham]; p=ns). Conclusion An upscaled clinically relevant EHT patch was developed and improved function in infarcted hearts without causing arrhythmia. Therefore EHT may have specific advantages over the direct intramyocardial injection of cells.
- Published
- 2020
41. Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations
- Author
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Tomoharu Osada, Yama A. Abassi, Hong Shi, Jennifer B. Pierson, Sonja Stoelzle-Feix, Gary Gintant, Qianyu Dang, T.K. Feaster, Emily Pfeiffer Kaushik, Hua Rong Lu, Todd J. Herron, Li Pang, Ksenia Blinova, Godfrey L. Smith, Martin Traebert, Beibei Cai, Yasunari Kanda, Ralf Kettenhofen, Francis L. Burton, Katherine Czysz, and Publica
- Subjects
Ventricular Repolarization ,Future studies ,Computer science ,Safety pharmacology ,Best practice ,Nonclinical safety ,Arrhythmias, Cardiac ,General Medicine ,010501 environmental sciences ,Validation Studies as Topic ,Toxicology ,030226 pharmacology & pharmacy ,01 natural sciences ,Cardiotoxins ,Membrane Potentials ,03 medical and health sciences ,Adult Stem Cells ,0302 clinical medicine ,Practice Guidelines as Topic ,Repolarization ,Humans ,Myocytes, Cardiac ,Stem cell ,Neuroscience ,0105 earth and related environmental sciences - Abstract
Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and ""cellrhythmias"" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define ""fit for purpose"" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable ""fit for purpose"" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.
- Published
- 2020
42. A novel ECG-biomarker for cardiac arrest during hypothermia
- Author
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Torkjel Tveita, Rachel C. Myles, Erik Sveberg Dietrichs, and Godfrey L. Smith
- Subjects
Male ,medicine.medical_specialty ,Accidental hypothermia ,Hypothermia ,Critical Care and Intensive Care Medicine ,QT interval ,Electrocardiography ,03 medical and health sciences ,QRS complex ,Ventricular arrhythmias ,0302 clinical medicine ,Hypothermia, Induced ,Internal medicine ,medicine ,Animals ,Humans ,Therapeutic hypothermia ,VDP::Medisinske Fag: 700 ,cardiovascular diseases ,030212 general & internal medicine ,New zealand white ,Rewarming ,business.industry ,lcsh:Medical emergencies. Critical care. Intensive care. First aid ,030208 emergency & critical care medicine ,lcsh:RC86-88.9 ,Cardiac arrest ,medicine.disease ,Heart Arrest ,Electrophysiology ,VDP::Medical disciplines: 700 ,Disease Models, Animal ,Ventricular Fibrillation ,Ventricular fibrillation ,Commentary ,cardiovascular system ,Emergency Medicine ,Cardiology ,Biomarker (medicine) ,Female ,Rabbits ,medicine.symptom ,business ,Biomarkers ,circulatory and respiratory physiology - Abstract
Background Treatment of arrhythmias evoked by accidental or therapeutic hypothermia and rewarming remains challenging. We aim to find an ECG-biomarker that can predict ventricular arrhythmias at temperatures occurring in therapeutic and accidental hypothermia. Main body Evaluation of ECG-data from accidental and therapeutic hypothermia patients and experimental data on ECG and ventricular fibrillation (VF) threshold in hypothermic New Zealand White Rabbits. VF threshold was measured in rabbit hearts cooled to moderate (31 °C) and severe (17 °C) hypothermia. QRS-interval divided by corrected QT-interval (QTc) was calculated at same temperatures. Clinical QRS/QTc data were obtained after a systematic literature review. Rabbit QRS/QTc values correlated with risk for VF (correlation coefficient: 0.97). Human QRS/QTc values from hypothermic patients, showed similar correlation with risk for ventricular fibrillation in the experimental data (correlation coefficient: 1.00). Conclusions These calculations indicate that QRS/QTc has potential as novel biomarker for predicting risk of hypothermia-induced cardiac arrest. Our findings apply both to victims of accidental hypothermia and to patients undergoing therapeutic hypothermia during surgery or after e.g. cardiac arrest.
- Published
- 2020
43. OP6 Investigating spatio-temporal dynamics of GLUT4 dispersal in cardiomyocytes
- Author
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Gwyn W. Gould, Anna Magdalena Koester, Godfrey L. Smith, and Nikolaj Gadegaard
- Subjects
endocrine system diseases ,biology ,business.industry ,Insulin ,medicine.medical_treatment ,Glucose uptake ,Cardiac muscle ,nutritional and metabolic diseases ,Skeletal muscle ,medicine.disease ,Exocytosis ,Cell biology ,medicine.anatomical_structure ,Diabetic cardiomyopathy ,medicine ,biology.protein ,Myocyte ,business ,hormones, hormone substitutes, and hormone antagonists ,GLUT4 - Abstract
Individuals with Type 2 diabetes mellitus (T2DM) are at significantly increased risk of cardiovascular disease and the development of diabetic cardiomyopathy. One of the major hallmarks of T2DM and diabetic cardiomyopathy is a significant decrease in myocardial glucose uptake. Understanding the mechanisms underpinning this effect is an important research goal with the potential to have far-reaching health and economic benefits. Regulated trafficking of the facilitative insulin-regulated GLUT4 mediates glucose transport in adipose tissue, skeletal and cardiac muscle. In the absence of insulin GLUT4 is sequestered in intracellular GLUT4 storage vesicles (GSV). Insulin stimulates translocation of GSVs to the plasma membrane (PM). In addition to stimulating GSV exocytosis, insulin regulates the spatial distribution of GLUT4 in the PM. In adipocytes GLUT4 is localized to relatively stationary clusters or freely diffusible monomers in the PM. Insulin stimulation increases GLUT4 dispersal from its fusion site to the surrounding PM by more than 60-fold. GLUT4 trafficking in muscle tissue is more complex because contraction is also a major stimulus of GLUT4 translocation. In skeletal muscle insulin and contraction recruit GLUT4 from different pools by distinct mechanisms. However, it is unknown whether contraction in cardiomyocytes involves GLUT4 dispersal and how this is influenced by T2DM. The emergence of super resolution microscopy techniques has allowed cell biologists to examine characteristics of molecules such as GLUT4 organization in the PM at the single-molecule level beyond the diffraction limit of conventional light microscopy. Stochastic reconstruction microscopy (STORM) investigated GLUT4 dispersal in basal and insulin-stimulated adipocytes and confirmed that dispersal of GLUT4 within the plasma membrane was a key action of insulin. Most studies employing STORM to investigate GLUT4 trafficking have been carried out in adipocytes; our approach is to use this technique in cardiac myocytes and test the hypothesis that such dispersed mechanisms operate and may be defective in diabetic cardiomyopathy using induced pluripotent stem cell derived cardiomyocytes and primary cardiomyocytes as model systems.
- Published
- 2020
44. Novel optics-based approaches for cardiac electrophysiology
- Author
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Leonardo Sacconi, Godfrey L. Smith, Gil Bub, and Marco Mongillo
- Subjects
Electrophysiology ,Optical Phenomena ,Cardiac electrophysiology ,Computer science ,cardiac electrophysiolog ,Biophysics ,Animals ,Humans ,Heart ,Molecular Biology ,Neuroscience - Published
- 2020
45. Moderate but not severe hypothermia causes pro-arrhythmic changes in cardiac electrophysiology
- Author
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Erik Sveberg Dietrichs, Karen McGlynn, Rachel C. Myles, Andrew Allan, Sarah Kettlewell, Godfrey L. Smith, Martin J. Bishop, Adam Connolly, Torkjel Tveita, and Francis L. Burton
- Subjects
medicine.medical_specialty ,Physiology ,030204 cardiovascular system & hematology ,QT interval ,03 medical and health sciences ,QRS complex ,0302 clinical medicine ,Physiology (medical) ,Internal medicine ,medicine ,Repolarization ,VDP::Medisinske Fag: 700 ,cardiovascular diseases ,Heptanol ,Cardiac electrophysiology ,business.industry ,030208 emergency & critical care medicine ,Hypothermia ,medicine.disease ,Electrophysiology ,Ventricular fibrillation ,Cardiology ,cardiovascular system ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business - Abstract
Aims Treatment of arrhythmias evoked by hypothermia/rewarming remains challenging, and the underlying mechanisms are unclear. This in vitro experimental study assessed cardiac electrophysiology in isolated rabbit hearts at temperatures occurring in therapeutic and accidental hypothermia. Methods and results Detailed ECG, surface electrogram, and panoramic optical mapping were performed in isolated rabbit hearts cooled to moderate (31°C) and severe (17°C) hypothermia. Ventricular activation was unchanged at 31°C while action potential duration (APD) was significantly prolonged (176.9 ± 4.2 ms vs. 241.0 ± 2.9 ms, P Conclusions Moderate hypothermia does not significantly change ventricular conduction time but prolongs repolarization and is pro-arrhythmic. Further cooling to severe hypothermia causes parallel changes in ventricular activation and repolarization, changes which are anti-arrhythmic. Therefore, relative changes in QRS and QT intervals (QR/QTc) emerge as an ECG-biomarker of pro-arrhythmic activity. Risk for ventricular fibrillation appears to be linked to the relatively low temperature sensitivity of ventricular transmural conduction, a conclusion supported by the anti-arrhythmic effect of heptanol at 31°C.
- Published
- 2020
46. Cardiac SNARE expression in health and disease
- Author
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Gwyn W. Gould, Godfrey L. Smith, and Peter R T Bowman
- Subjects
0301 basic medicine ,Cardiac function curve ,SNARE proteins ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,030209 endocrinology & metabolism ,lcsh:Diseases of the endocrine glands. Clinical endocrinology ,RS ,03 medical and health sciences ,Endocrinology ,0302 clinical medicine ,Insulin resistance ,insulin resistance ,SNAP23 ,medicine ,Original Research ,lcsh:RC648-665 ,biology ,diabetes ,Insulin ,Glucose transporter ,Transporter ,medicine.disease ,Cell biology ,030104 developmental biology ,biology.protein ,cardiomyopathy ,GLUT4 ,Intracellular - Abstract
SNARE proteins are integral to intracellular vesicular trafficking, which in turn is the process underlying the regulated expression of substrate transporters such as the glucose transporter GLUT4 at the cell surface of insulin target tissues. Impaired insulin stimulated GLUT4 trafficking is associated with reduced cardiac function in many disease states, most notably diabetes. Despite this, our understanding of the expression and regulation of SNARE proteins in cardiac tissue and how these may change in diabetes is limited. Here we characterize the array of SNARE proteins expressed in cardiac tissue, and quantify the levels of expression of VAMP2, SNAP23, and Syntaxin4—key proteins involved in insulin-stimulated GLUT4 translocation. We examined SNARE protein levels in cardiac tissue from two rodent models of insulin resistance, db/db mice and high-fat fed mice, and show alterations in patterns of expression are evident. Such changes may have implications for cardiac function.
- Published
- 2019
47. Ca2+ leak—What is it? Why should we care? Can it be managed?
- Author
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Penelope A. Boyden and Godfrey L. Smith
- Subjects
0301 basic medicine ,Leak ,business.industry ,Dysfunctional family ,030204 cardiovascular system & hematology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Physiology (medical) ,Medicine ,Cardiology and Cardiovascular Medicine ,business ,Neuroscience ,Intracellular - Abstract
For arrhythmia triggers that are secondary to dysfunctional intracellular Ca2+ cycling, there are few, if any, agents that specifically target the Ca2+ handling machinery. However, several candidates have been proposed in the literature. Here we review the idea that these agents or their derivatives will prove invaluable in clinical applications in the future.
- Published
- 2018
48. Human cardiomyocyte calcium handling and transverse tubules in mid-stage of post-myocardial-infarction heart failure
- Author
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Øyvind Ellingsen, Idar Kirkeby-Garstad, Gianluigi Condorelli, Godfrey L. Smith, Rune Wiseth, Ole J. Kemi, Riccardo Contu, Asbjørn Karevold, Rune Haaverstad, Ulrik Wisløff, Tomas L. Stølen, Morten A. Høydal, and Alexander Wahba
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Contraction (grammar) ,business.industry ,Diastole ,Stimulation ,030204 cardiovascular system & hematology ,medicine.disease ,Phospholamban ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Ventricle ,Internal medicine ,Heart failure ,medicine ,Cardiology ,Myocardial infarction ,Cardiology and Cardiovascular Medicine ,business ,Homeostasis - Abstract
Aims: Cellular processes in the heart rely mainly on studies from experimental animal models or explanted hearts from patients with terminal end-stage heart failure (HF). To address this limitation, we provide data on excitation contraction coupling, cardiomyocyte contraction and relaxation, and Ca2+ handling in post-myocardial-infarction (MI) patients at mid-stage of HF. Methods and results: Nine MI patients and eight control patients without MI (non-MI) were included. Biopsies were taken from the left ventricular myocardium and processed for further measurements with epifluorescence and confocal microscopy. Cardiomyocyte function was progressively impaired in MI cardiomyocytes compared with non-MI cardiomyocytes when increasing electrical stimulation towards frequencies that simulate heart rates during physical activity (2 Hz); at 3 Hz, we observed almost total breakdown of function in MI. Concurrently, we observed impaired Ca2+ handling with more spontaneous Ca2+ release events, increased diastolic Ca2+, lower Ca2+ amplitude, and prolonged time to diastolic Ca2+ removal in MI (P < 0.01). Significantly reduced transverse-tubule density (−35%, P < 0.01) and sarcoplasmic reticulum Ca2+ adenosine triphosphatase 2a (SERCA2a) function (−26%, P < 0.01) in MI cardiomyocytes may explain the findings. Reduced protein phosphorylation of phospholamban (PLB) serine-16 and threonine-17 in MI provides further mechanisms to the reduced function. Conclusions: Depressed cardiomyocyte contraction and relaxation were associated with impaired intracellular Ca2+ handling due to impaired SERCA2a activity caused by a combination of alteration in the PLB/SERCA2a ratio and chronic dephosphorylation of PLB as well as loss of transverse tubules, which disrupts normal intracellular Ca2+ homeostasis and handling. This is the first study that presents these mechanisms from viable and intact cardiomyocytes isolated from the left ventricle of human hearts at mid-stage of post-MI HF.
- Published
- 2018
49. Deconvoluting Kinase Inhibitor Induced Cardiotoxicity
- Author
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Sarah D. Lamore, Godfrey L. Smith, Scott Boyer, Maria P. Hortigon-Vinagre, Victor Zamora Rodriguez, Michelle Lamb, Clay W Scott, Johanna Sagemark, Allison Laura Choy, Ernst Ahlberg, Matthew F. Peters, Stephanie M. Bates, Jonna Stålring, and Lars Carlsson
- Subjects
0301 basic medicine ,kinase ,kinase inhibitor ,Induced Pluripotent Stem Cells ,cardiotoxicity ,030204 cardiovascular system & hematology ,Pharmacology ,Biology ,Toxicology ,03 medical and health sciences ,0302 clinical medicine ,Calcium flux ,IKBKE ,Humans ,Kinome ,Myocytes, Cardiac ,Protein Kinase Inhibitors ,Cardiotoxicity ,Kinase ,Drug discovery ,Reverse Transcriptase Polymerase Chain Reaction ,Heart ,RPS6KA3 ,030104 developmental biology ,Calcium ,Signal transduction ,cellular impedance ,Kinase Inhibitors and Cardiotoxicity ,Protein Kinases - Abstract
Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation–contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways.
- Published
- 2017
50. Flexible Substrate is Key to Appropriate Contractile Behaviour of hiPSC Derived Cardiomyocytes
- Author
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Francis L. Burton, Godfrey L. Smith, Nikolaj Gadegaard, and Eline Huethorst
- Subjects
Chemistry ,Biophysics ,Substrate (printing) - Published
- 2020
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