Imperial College London

ProfessorSianHarding

Faculty of MedicineNational Heart & Lung Institute

Professor of Cardiac Pharmacology
 
 
 
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Contact

 

+44 (0)20 7594 3009sian.harding Website

 
 
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Location

 

435ICTEM buildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

413 results found

Kondrashov A, Mohd Yusof NAN, Hasan A, Goulding J, Kodagoda T, Hoang DM, Vo NTN, Melarangi T, Dolatshad N, Gorelik J, Hill SJ, Harding SE, Denning Cet al., 2021, CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes, Molecular Therapy - Methods and Clinical Development, Vol: 20, Pages: 39-53, ISSN: 2329-0501

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.

Journal article

Leong KMW, Ng FS, Shun-Shin MJ, Koa-Wing M, Qureshi N, Whinnett ZI, Linton NF, Lefroy D, Francis DP, Harding SE, Davies DW, Peter NS, Lim PB, Behr E, Lambiase PD, Varnava A, Kanagaratnam Pet al., 2021, Non-invasive detection of exercise-induced cardiac conduction abnormalities in sudden cardiac death survivors in the inherited cardiac conditions., Europace, Vol: 23, Pages: 305-312

AIMS : Rate adaptation of the action potential ensures spatial heterogeneities in conduction across the myocardium are minimized at different heart rates providing a protective mechanism against ventricular fibrillation (VF) and sudden cardiac death (SCD), which can be quantified by the ventricular conduction stability (V-CoS) test previously described. We tested the hypothesis that patients with a history of aborted SCD due to an underlying channelopathy or cardiomyopathy have a reduced capacity to maintain uniform activation following exercise. METHODS AND RESULTS : Sixty individuals, with (n = 28) and without (n = 32) previous aborted-SCD event underwent electro-cardiographic imaging recordings following exercise treadmill test. These included 25 Brugada syndrome, 13 hypertrophic cardiomyopathy, 12 idiopathic VF, and 10 healthy controls. Data were inputted into the V-CoS programme to calculate a V-CoS score that indicate the percentage of ventricle that showed no significant change in ventricular activation, with a lower score indicating the development of greater conduction heterogeneity. The SCD group, compared to those without, had a lower median (interquartile range) V-CoS score at peak exertion [92.8% (89.8-96.3%) vs. 97.3% (94.9-99.1%); P < 0.01] and 2 min into recovery [95.2% (91.1-97.2%) vs. 98.9% (96.9-99.5%); P < 0.01]. No significant difference was observable later into recovery at 5 or 10 min. Using the lowest median V-CoS scores obtained during the entire recovery period post-exertion, SCD survivors had a significantly lower score than those without for each of the different underlying aetiologies. CONCLUSION : Data from this pilot study demonstrate the potential use of this technique in risk stratification for the inherited cardiac conditions.

Journal article

Forte E, Perkins B, Sintou A, Kallkat HS, Papanikolaou A, Jenkins C, Alsubaie M, Chowdhury RA, Duffy TM, Skelly DA, Branca J, Bellahcene M, Schneider M, Harding S, Furtado MB, Ng FS, Hasham MG, Rosenthal N, Sattler Set al., 2020, Cross-priming dendritic cells exacerbate immunopathology after ischemic tissue damage in the heart, Circulation, ISSN: 0009-7322

Background: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of HF. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helperand CD8+ cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross priming DC in post-MI myocardial impairment through presentation of self-antigen fromnecrotic cardiomyocytes to cytotoxic CD8+ T cells.Methods: We induced type-2 myocardial infarction (MI)-like ischemic injury in the heart by treatment with a single high dose of the beta-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long term cardiac immunopathology and functional decline in wild type and Clec9a-depleted mice lacking DC cross-priming function.Results: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a -/- mice deficient in DC cross-priming are protected from long-term immune-mediated myocardial damage and decline of cardiac function, likely dueto dampened activation of cytotoxic CD8+ T cells.Conclusion: Activation of cytotoxic CD8+ T cells by cross-priming DC contributes to exacerbation of post-ischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent immune-mediated worsening of post-ischemic HF.

Journal article

Crestani T, Steichen C, Neri E, Rodrigues M, Fonseca-Alaniz MH, Ormrod B, Holt MR, Pandey P, Harding S, Ehler E, Krieger JEet al., 2020, Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 533, Pages: 376-382, ISSN: 0006-291X

Journal article

Forte E, Panahi M, Baxan N, Ng FS, Boyle JJ, Branca J, Bedard O, Hasham MG, Benson L, Harding SE, Rosenthal N, Sattler Set al., 2020, Type 2 MI induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart, Journal of Cellular and Molecular Medicine, ISSN: 1582-1838

Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol hydrochloride is used for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis. To assess whether isoproterenol-induced cardiomyocyte necrosis triggers an adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.

Journal article

Kit-Anan W, Mazo M, Wang BX, Leonardo V, Pence I, Gopal S, Gelmi A, Becce M, Chiappini C, Harding SE, Terracciano C, Stevens Met al., 2020, Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation, Biofabrication, ISSN: 1758-5082

Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely microscopic topography (3D shape resembling that of adult CM) and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate pillars of the desired 3D shapes, which would be used to mould the designed microwell arrays into a hydrogel. Polyacrylamide was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without comprising its capacity to modulate the stiffness. In this manner, individual parameters can be finely tuned independently within the hydrogel: the dimension of 3D shaped microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in an absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell Peer reviewed version of the manuscript published in final form at Biofabrication (2020). membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2- MYL7 proteins; while displaying higher anisotropism in comparison to pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiological or pathological microenvironment to an isolated single hiPSC-CM in absen

Journal article

Hasan A, Mohammadi N, Nawaz A, Kodagoda T, Diakonov I, Harding SE, Gorelik Jet al., 2020, Age-dependent maturation of iPSC-CMs leads to the enhanced compartmentation of beta(2)AR-cAMP signalling, Cells, Vol: 9, ISSN: 2073-4409

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

Journal article

Meijles DN, Cull JJ, Markou T, Cooper STE, Haines ZHR, Fuller SJ, O'Gara P, Sheppard MN, Harding SE, Sugden PH, Clerk Aet al., 2020, Redox Regulation of Cardiac ASK1 (Apoptosis Signal-Regulating Kinase 1) Controls p38-MAPK (Mitogen-Activated Protein Kinase) and Orchestrates Cardiac Remodeling to Hypertension, HYPERTENSION, Vol: 76, Pages: 1208-1218, ISSN: 0194-911X

Journal article

Brook J, Kim M-Y, Koutsoftidis S, Pitcher D, Agha-Jaffar D, Sufi A, Jenkins C, Tzortzis K, Ma S, Jabbour R, Houston C, Handa B, Li X, Chow J-J, Jothidasan A, Bristow P, Perkins J, Harding S, Bharath A, Ng FS, Peters N, Cantwell C, Chowdhury Ret al., 2020, Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling, Pflügers Archiv European Journal of Physiology, Vol: 472, Pages: 1435-1446, ISSN: 0031-6768

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap-junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting Porcine (n=9) and human (n=4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. 1mM of carbenoxolone was administered at 5ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed.We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9±4.1ms to 67.2±2.7ms) indicating conduction slowing. The features with the largest percentage change between baseline to Carbenoxolone were Fractionation +185.3%, Endpoint amplitude -106.9%, S-Endpoint Gradient +54.9%, S Point, -39.4%, RS Ratio +38.6% and (-dV/dt)max -20.9%.The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically-induced proarrhythmic substrates.

Journal article

Majid QA, Fricker ATR, Gregory DA, Davidenko N, Hernandez Cruz O, Jabbour RJ, Owen TJ, Basnett P, Lukasiewicz B, Stevens M, Best S, Cameron R, Sinha S, Harding SE, Roy Iet al., 2020, Natural biomaterials for cardiac tissue engineering: a highly biocompatible solution., Frontiers in Cardiovascular Medicine, Vol: 7, Pages: 1-32, ISSN: 2297-055X

Cardiovascular diseases (CVD) constitute a major fraction of the current major global diseases and lead to about 30% of the deaths, i.e., 17.9 million deaths per year. CVD include coronary artery disease (CAD), myocardial infarction (MI), arrhythmias, heart failure, heart valve diseases, congenital heart disease, and cardiomyopathy. Cardiac Tissue Engineering (CTE) aims to address these conditions, the overall goal being the efficient regeneration of diseased cardiac tissue using an ideal combination of biomaterials and cells. Various cells have thus far been utilized in pre-clinical studies for CTE. These include adult stem cell populations (mesenchymal stem cells) and pluripotent stem cells (including autologous human induced pluripotent stem cells or allogenic human embryonic stem cells) with the latter undergoing differentiation to form functional cardiac cells. The ideal biomaterial for cardiac tissue engineering needs to have suitable material properties with the ability to support efficient attachment, growth, and differentiation of the cardiac cells, leading to the formation of functional cardiac tissue. In this review, we have focused on the use of biomaterials of natural origin for CTE. Natural biomaterials are generally known to be highly biocompatible and in addition are sustainable in nature. We have focused on those that have been widely explored in CTE and describe the original work and the current state of art. These include fibrinogen (in the context of Engineered Heart Tissue, EHT), collagen, alginate, silk, and Polyhydroxyalkanoates (PHAs). Amongst these, fibrinogen, collagen, alginate, and silk are isolated from natural sources whereas PHAs are produced via bacterial fermentation. Overall, these biomaterials have proven to be highly promising, displaying robust biocompatibility and, when combined with cells, an ability to enhance post-MI cardiac function in pre-clinical models. As such, CTE has great potential for future clinical solutions and he

Journal article

Sattler S, Baxan N, Ng FS, Benson L, Boyle J, Harding S, Rosenthal Net al., 2020, Myocardial damage induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart, Journal of Cellular and Molecular Medicine, ISSN: 1582-1838

Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol-hydrochloride isused for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis.To assess if isoproterenol-induced cardiomyocyte necrosistriggersan adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of 160mg/kg isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum, as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.

Journal article

Sintou A, Mansfield C, Iacob A-O, Chowdhury RA, Narodden S, Rothery SM, Podoveo R, Sanchez Alonso JL, Ferraro E, Swiatlowska P, Harding S, Prasad S, Rosenthal N, Gorelik J, Sattler Set al., 2020, Mediastinal lymphadenopathy, class-switched auto-antibodies and myocardial immune-complexes during heart failure in rodents and humans, Frontiers in Cell and Developmental Biology, Vol: 8, Pages: 1-12, ISSN: 2296-634X

Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemicheart failure pointing to an autoimmune response against the heart. T and B cell have beenconvincingly demonstrated to be activated after myocardial infarction, a prerequisite for thegeneration of mature auto-antibodies. Yet, little is known about the immunoglobulin isotyperepertoire thus pathological potential of anti-heart auto-antibodies during heart failure.We obtained human myocardial tissue from ischemic heart failure patients and inducedexperimental MI in rats. We found that anti-heart autoimmunity persists during heart failure.Rat mediastinal lymph nodes are enlarged and contain active secondary follicles with matureisotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens arepresent in rat heart failure serum, and IgG and complement C3 deposits are evident in heartfailure tissue of both rats and human patients.Previously established myocardial inflammation, and the herein provided proof of B cellmaturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide allthe hallmark signs of an established autoimmune response in chronic heart failure.

Journal article

Constantinou C, Miranda Almeida A, Chaves Guerrero P, Bellahcene M, Massaia A, Cheng K, Samari S, Rothery S, Chandler A, Schwarz R, Harding S, Punjabi P, Schneider MD, Noseda Met al., 2020, Human pluripotent stem cell-derived cardiomyocytes as a targetplatform for paracrine protection by cardiac mesenchymal stromalcells, Scientific Reports, Vol: 10, ISSN: 2045-2322

Ischemic heart disease remains the foremost cause of death globally, with survivors at risk for subsequent heart failure. Paradoxically, cell therapies to offset cardiomyocyte loss after ischemic injury improve long-term cardiac function despite a lack of durable engraftment. An evolving consensus, inferred preponderantly from non-human models, is that transplanted cells benefit the heart via early paracrinesignals. Here, we tested the impact of paracrine signals on human cardiomyocytes, using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the target of mouse and human cardiac mesenchymal stromal cells (cMSC) with progenitor-like features. In co-culture and conditioned medium studies, cMSCs markedly inhibited human cardiomyocyte death. Little or no protection was conferred by mouse tail tip or human skin fibroblasts. Consistent with the results of transcriptomic profiling, functional analyses showed that the cMSC secretome suppressed apoptosis and and preserved cardiac mitochondrial transmembrane potential. Protection was independent of exosomes under the conditions tested. In mice, injecting cMSC-conditioned media into the infarct border zone reduced apoptotic cardiomyocytes >70% locally. Thus, hPSC-CMs provide an auspicious, relevant human platform to investigate extracellular signals for cardiac muscle survival, substantiating human cardioprotection by cMSCs, and suggesting the cMSC secretome or its components as potential cell-free therapeutic products.

Journal article

Schneider M, Golforoush P, Narasimhan P, Chaves-Guerrero P, Lawrence E, Newton G, Yan R, Harding S, Perrior T, Chapman Ket al., 2020, Selective protection of human cardiomyocytes from anthracycline cardiotoxicity by small molecule inhibitors of MAP4K4, Scientific Reports, Vol: 10, Pages: 1-12, ISSN: 2045-2322

Given the poor track record to date of animal models for creating cardioprotective drugs, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been proposed as a therapeutically relevant human platform to guide target validation and cardiac drug development. Mitogen-Activated Protein Kinase Kinase Kinase Kinase-4 (MAP4K4) is an “upstream” member of the MAPK superfamily that is implicated in human cardiac muscle cell death from oxidative stress, based on gene silencing and pharmacological inhibition in hPSC-CMs. A further role for MAP4K4 was proposed in heart muscle cell death triggered by cardiotoxic anti-cancer drugs, given its reported activation in failing human hearts with doxorubicin (DOX) cardiomyopathy, and its activation acutely by DOX in cultured cardiomyocytes. Here, we report successful protection from DOX in two independent hPSC-CM lines, using two potent, highly selective MAP4K4 inhibitors. The MAP4K4 inhibitors enhanced viability and reduced apoptosis at otherwise lethal concentrations of DOX, and preserved cardiomyocyte function, as measured by spontaneous calcium transients, at sub-maximal ones. Notably, in contrast, no intereference was seen in tumor cell killing, caspase activation, or mitochondrial membrane dissipation by DOX, in human cancer cell lines. Thus, MAP4K4 is a plausible, tractable, selective therapeutic target in DOX-induced human heart muscle cell death.

Journal article

Lyon A, Babalis D, Morley-Smith AC, Hedger M, Suarez Barrientos A, Foldes G, Couch LS, Chowdhury RA, Tzortzis KN, Peters NS, Rog-Zielinska EA, Yang YH, Welch S, Bowles CT, Rahman Haley S, Bell AR, Rice A, Sasikaran T, Johnson NA, Falaschetti E, Parameshwar J, Lewis C, Tsui S, Simon A, Pepper J, Rudy JJ, Zsebo KM, MacLeod KT, Terracciano CM, Hajjar RJ, Banner N, Harding SEet al., 2020, Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device – the SERCA-LVAD TRIAL, Gene Therapy, Vol: 27, Pages: 579-590, ISSN: 0969-7128

The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. Enrolled subjects were randomised to receive a single intracoronary infusion of 1x1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort, which may help guide future trial design.

Journal article

Jabbour RJ, Owen TJ, Pandey P, Reinsch M, Smith G, Weinberger F, Eschenhagen T, Harding SEet al., 2020, IN-VIVO GRAFTING OF LARGE ENGINEERED HEART TISSUE PATCHES FOR CARDIAC REPAIR, Publisher: BMJ PUBLISHING GROUP, Pages: A111-A112, ISSN: 1355-6037

Conference paper

Sparks H, Dvinskikh L, Firth J, Francis A, Harding S, Paterson C, MacLeod K, Dunsby Cet al., 2020, Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure, Journal of Biophotonics, Vol: 13, ISSN: 1864-063X

We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.

Journal article

Pitoulis FG, Hasan W, Papadaki M, Clavere NG, Perbellini F, Harding SE, Kirk JA, Boateng SY, de Tombe PP, Terracciano CMet al., 2020, Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics, Journal of Molecular and Cellular Cardiology, Vol: 141, Pages: 11-16, ISSN: 0022-2828

Determining transmural mechanical properties in the heart provides a foundation to understand physiological and pathophysiological cardiac mechanics. Although work on mechanical characterisation has begun in isolated cells and permeabilised samples, the mechanical profile of living individual cardiac layers has not been examined. Myocardial slices are 300 μm-thin sections of heart tissue with preserved cellular stoichiometry, extracellular matrix, and structural architecture. This allows for cardiac mechanics assays in the context of an intact in vitro organotypic preparation. In slices obtained from the subendocardium, midmyocardium and subepicardium of rats, a distinct pattern in transmural contractility is found that is different from that observed in other models. Slices from the epicardium and midmyocardium had a higher active tension and passive tension than the endocardium upon stretch. Differences in total myocyte area coverage, and aspect ratio between layers underlined the functional readouts, while no differences were found in total sarcomeric protein and phosphoprotein between layers. Such intrinsic heterogeneity may orchestrate the normal pumping of the heart in the presence of transmural strain and sarcomere length gradients in the in vivo heart.

Journal article

Schobesberger S, Wright PT, Poulet C, Mardones JLSA, Mansfield C, Friebe A, Harding SE, Balligand J-L, Nikolaev VO, Gorelik Jet al., 2020, beta(3)-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes, eLife, Vol: 9, Pages: 1-15, ISSN: 2050-084X

Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

Journal article

Fiedler LR, Chapman K, Xie M, Maifoshie E, Jenkins M, Golforoush PA, Bellahcene M, Noseda M, Faust D, Jarvis A, Newton G, Paiva MA, Harada M, Stuckey DJ, Song W, Habib J, Narasimhan P, Aqil R, Sanmugalingam D, Yan R, Pavanello L, Sano M, Wang SC, Sampson RD, Kanayaganam S, Taffet GE, Michael LH, Entman ML, Tan T-H, Harding SE, Low CMR, Tralau-Stewart C, Perrior T, Schneider MDet al., 2020, MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo (vol 24, 579.e1,2019), CELL STEM CELL, Vol: 26, Pages: 458-458, ISSN: 1934-5909

Journal article

Pitoulis FG, Nunez-Toldra R, Kit-Anan WS, Dries E, Bardi I, Perbellini F, Harding SE, de Tombe PP, Terracciano CMet al., 2020, Exploring Mechanical Load-Induced Cardiac Remodelling Using a Novel Organotypic Myocardial Slice Model, 64th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 425A-425A, ISSN: 0006-3495

Conference paper

Jabbour RJ, Owen TJ, Pandey P, Harding SEet al., 2019, Future potential of engineered heart tissue patches for repairing the damage caused by heart attacks, EXPERT REVIEW OF MEDICAL DEVICES, Vol: 17, Pages: 1-3, ISSN: 1743-4440

Journal article

Ramuz M, Hasan A, Gruscheski L, Diakonov I, Pavlaki N, Nikolaev VO, Harding S, Dunsby C, Gorelik Jet al., 2019, A software tool for high-throughput real-time measurement of intensity-based ratio-metric FRET, Cells, Vol: 8, ISSN: 2073-4409

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

Journal article

Shun-Shin MJ, Leong KMW, Ng FS, Linton NWF, Whinnett ZI, Koa-Wing M, Qureshi N, Lefroy DC, Harding SE, Lim PB, Peters NS, Francis DP, Varnava AM, Kanagaratnam Pet al., 2019, Ventricular conduction stability test: a method to identify and quantify changes in whole heart activation patterns during physiological stress, EP-Europace, Vol: 21, Pages: 1422-1431, ISSN: 1099-5129

AIMS: Abnormal rate adaptation of the action potential is proarrhythmic but is difficult to measure with current electro-anatomical mapping techniques. We developed a method to rapidly quantify spatial discordance in whole heart activation in response to rate cycle length changes. We test the hypothesis that patients with underlying channelopathies or history of aborted sudden cardiac death (SCD) have a reduced capacity to maintain uniform activation following exercise. METHODS AND RESULTS: Electrocardiographical imaging (ECGI) reconstructs >1200 electrograms (EGMs) over the ventricles from a single beat, providing epicardial whole heart activation maps. Thirty-one individuals [11 SCD survivors; 10 Brugada syndrome (BrS) without SCD; and 10 controls] with structurally normal hearts underwent ECGI vest recordings following exercise treadmill. For each patient, we calculated the relative change in EGM local activation times (LATs) between a baseline and post-exertion phase using custom written software. A ventricular conduction stability (V-CoS) score calculated to indicate the percentage of ventricle that showed no significant change in relative LAT (<10 ms). A lower score reflected greater conduction heterogeneity. Mean variability (standard deviation) of V-CoS score over 10 consecutive beats was small (0.9 ± 0.5%), with good inter-operator reproducibility of V-CoS scores. Sudden cardiac death survivors, compared to BrS and controls, had the lowest V-CoS scores post-exertion (P = 0.011) but were no different at baseline (P = 0.50). CONCLUSION: We present a method to rapidly quantify changes in global activation which provides a measure of conduction heterogeneity and proof of concept by demonstrating SCD survivors have a reduced capacity to maintain uniform activation following exercise.

Journal article

Sattler S, Baxan N, Chowdhury R, Rosenthal N, Prasad S, Zhao L, Harding Set al., 2019, Characterization of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by multi-parametric quantitative cardiac MRI, Disease Models & Mechanisms, Vol: 12, Pages: 1-10, ISSN: 1754-8403

Hemorrhagic myocarditis is a potentially fatal complication of excessive levels of systemic inflammation. It has been reported in viral infection, but is also possible in systemic autoimmunity. Epicutaneous treatment of mice with the TLR-7 agonist Resiquimod induces auto-antibodies and systemic tissue damage including in the heart, and is used as an inducible mouse model of Systemic Lupus Erythematosus (SLE).Here, we show that over-activation of the TLR-7 pathway of viral recognition by Resiquimod-treatment of CFN mice induces severe thrombocytopenia and internal bleeding which manifests most prominently as hemorrhagic myocarditis. We optimized a cardiac magnetic resonance (CMR) tissue mapping approach for the in vivo detection of diffuse infiltration, fibrosis and hemorrhages using a combination of T1, T2 and T2* relaxation times, and compared results to ex vivo histopathology of cardiac sections corresponding to CMR tissue maps. This allowed a detailed correlation between in vivo CMR parameters and ex vivo histopathology, and confirmed the need to include T2* measurements to detect tissue iron for accurate interpretation of pathology associated with CMR parameter changes.In summary, we provide detailed histological and in vivo imaging-based characterization of acute hemorrhagic myocarditis as acute cardiac complication in the mouse model of Resiquimod-induced SLE, and a refined CMR protocol to allow non-invasive longitudinal in vivo studies of heart involvement in acute inflammation. We propose that adding T2* mapping to CMR protocols for myocarditis diagnosis will improve interpretation of disease mechanisms and diagnostic sensitivity.

Journal article

Owen TJ, Harding SE, 2019, Multi-cellularity in cardiac tissue engineering, how close are we to native heart tissue?, Journal of Muscle Research and Cell Motility, Vol: 40, Pages: 151-157, ISSN: 0142-4319

Tissue engineering is a complex field where the elements of biology and engineering are combined in an attempt to recapitulate the native environment of the body. Tissue engineering has shown one thing categorically; that the human body is extremely complex and it is truly a difficult task to generate this in the lab. There have been varied attempts at trying to generate a model for the heart with numerous cell types and different scaffolds or materials. The common underlying theme in these approaches is to combine together matrix material and different cell types to make something similar to heart tissue. Multi-cellularity is an essential aspect of the heart and therefore critical to any approach which would try to mimic such a complex tissue. The heart is made up of many cell types that combine to form complex structures like: deformable chambers, a tri-layered heart muscle, and vessels. Thus, in this review we will summarise how tissue engineering has progressed in modelling the heart and what gaps still exist in this dynamic field.

Journal article

Watson S, Duff J, Bardi I, Zabielska M, Atanur S, Jabbour R, Simon A, Tomas A, Smolenski R, Harding S, Terracciano Cet al., 2019, Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro, Nature Communications, Vol: 10, ISSN: 2041-1723

Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured outside the body. The in vivo environment, particularly constant electromechanical stimulation, is fundamental to the regulation of cardiac structure and function. We investigated the role of electromechanical stimulation in preventing culture-induced dedifferentiation of adult cardiac tissue using rat, rabbit and human heart failure myocardial slices. Here we report that the application of a preload equivalent to sarcomere length (SL) = 2.2 μm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Gene sets associated with the preservation of structure and function are activated, while gene sets involved in dedifferentiation are suppressed. The maximum contractility of human heart failure myocardial slices at 24 h is also optimally maintained at SL = 2.2 μm. Rabbit myocardial slices cultured at SL = 2.2 μm remain stable for 5 days. This approach substantially prolongs the culture of adult cardiac tissue in vitro.

Journal article

Hellen N, Pinto Ricardo C, Vauchez K, Whiting G, Wheeler J, Harding SEet al., 2019, Proteomic analysis reveals temporal changes in protein expression in human induced pluripotent stem cell-derived cardiomyocytes in vitro, Stem Cells and Development, Vol: 28, ISSN: 1547-3287

Human induced pluripotent stem cell-derived cardiomyocytes hold great promise for regenerative medicine and in vitro screening. Despite displaying key cardiomyocyte phenotypic characteristics, they more closely resemble foetal/neonatal cardiomyocytes and further characterisation is necessary. Combining the use of tandem mass tags to label cell lysates, followed by multiplexing, we have determined the effects of short term (30 day) in vitro culture on human induced pluripotent stem cell derived cardiomyocyte protein expression. We found that human induced pluripotent stem cell derived cardiomyocytes exhibit temporal changes in global protein expression; alterations in protein expression were pronounced during the first 2 weeks following thaw and dominated by reductions in proteins associated with protein synthesis and ubiquitination. Between 2 and 4 weeks proceeding thaw alterations in protein expression were dominated by metabolic pathways, indicating a potential temporal metabolic shift from glycolysis towards oxidative phosphorylation. Time-dependent changes in proteins associated with cardiomyocyte contraction, excitation-contraction coupling and metabolism were detected. While some were associated with expected functional outcomes in terms of morphology or electrophysiology, others such as metabolism did not produce the anticipated maturation of human induced pluripotent stem cell derived cardiomyocytes. In several cases, a predicted outcome was not clear because of the concerted changes in both stimulatory and inhibitory pathways. Nevertheless, clear development of human induced pluripotent stem cell derived cardiomyocytes over this time period was evident.

Journal article

Schneider M, Fiedler L, Chapman K, Xie M, Maifosie E, Jenkins M, Golforoush P, Bellahcene M, Noseda M, Faust D, Jarvis A, Newton G, Paiva MA, Harada M, Stuckey DJ, Song W, Habib J, Narasimham P, Aqil R, Sanmugalingam D, Yan R, Pavanello L, Sano M, Wang SC, Sampson RD, Kanayaganam S, Taffet GE, Michael LH, Entman ML, Tan T, Harding S, Low CMR, Tralau-Stewart C, Perrior T, Schneider MDet al., 2019, MAP4K4 inhibition promotes survival of human stem cell derived cardiomyocyte and reduces infarct size in vivo, Cell Stem Cell, Vol: 24, Pages: 579-591.e12, ISSN: 1875-9777

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Journal article

Wang BX, Kit-Anan W, Whittaker T, Couch L, Nagelkerke A, Deidda G, Mitraki A, Harding SE, Stevens MM, MacLeod KT, Terracciano CMet al., 2019, Human Cardiac Fibroblast-Secreted Exosomes Improve Efficiency of Human Cardiomyocyte Calcium Cycling, Publisher: SPRINGER, Pages: 269-269, ISSN: 0920-3206

Conference paper

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