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

383 results found

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

Journal article

Owen TJ, Harding SE, 2019, Multi-cellularity in cardiac tissue engineering, how close are we to native heart tissue?, J Muscle Res Cell Motil

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

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

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, 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, 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

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, 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

Brito L, Mylonaki I, Moroz E, Grigsby C, Smart N, Rosenthal N, Harding SE, Stevens MMet al., 2019, Epicardial cell transfection with cationic polymeric nanocomplexes, British-Society-for-Gene-and-Cell-Therapy Autumn Conference, Publisher: MARY ANN LIEBERT, INC, Pages: A9-A9, ISSN: 1043-0342

Conference paper

Owen TJ, Smith JS, Bhagwan JR, Mosqueira D, Scott E, Mannhardt I, Barriales-Villa R, Monserrat L, Hansen A, Eschenhagen T, Harding SE, Marston SB, Denning Cet al., 2019, Isogenic pairs of patient-derived hiPSC-CMs with an apical hypertrophic cardiomyopathy/left ventricular non-compaction-associated ACTC1 E99K mutation unveil differential functional deficits, British-Society-for-Gene-and-Cell-Therapy Autumn Conference, Publisher: MARY ANN LIEBERT, INC, Pages: A7-A8, ISSN: 1043-0342

Conference paper

Dubey P, Humphrey E, Majid Q, Grigsby C, Stevens MM, Terracciano C, Harding SE, Roy Iet al., 2019, Polyhydroxyalkanoates, ideal materials for cardiac regeneration, ISSN: 1526-7547

© 2019 Omnipress - All rights reserved. Statement of purpose: Polyhydroxyalkanoates (PHAs), a family of biodegradable and biocompatible polymers with a range of material properties and degradation rates are known to be particularly cardio-regenerative in nature 1,2,3 . Myocardial infarction results in the generation of scar tissue with limited or no regeneration due to the modest nature of intrinsic myocardial regenerative capability. The concept of a cardiac patch is tailored to meet the unmet medical need of cardiac regeneration where a biomaterial-based patch with/without cells would be used to induce efficient cardiac regeneration. Medium chain length PHAs (MCL-PHAs) with monomer chain length between C 6 -C 16 are highly elastomeric in nature 1,2 and have been shown to be excellent substrates for the growth and function of neonatal cardiomyocytes 3 . This work describes an in-depth study of the potential of MCL-PHAs for the development of functional cardiac patches laden with human pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), providing mechanical support and cell based therapy.

Conference paper

Sattler S, Couch LS, Harding SE, 2018, Takotsubo Syndrome: Latest addition to the expanding family of immune-mediated diseases?, JACC: Basic to Translational Science, Vol: 3, Pages: 779-781, ISSN: 2452-302X

Journal article

Smith JGW, Owen T, Bhagwan JR, Mosqueira D, Scott E, Mannhardt I, Patel A, Barriales-Villa R, Monserrat L, Hansen A, Eschenhagen T, Harding SE, Marston S, Denning Cet al., 2018, Isogenic pairs of hiPSC-CMs with hypertrophic cardiomyopathy/LVNC-associated ACTC1 E99K mutation unveil differential functional deficits, Stem Cell Reports, Vol: 11, Pages: 1226-1243, ISSN: 2213-6711

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.

Journal article

Noseda M, Harding SE, 2018, Understanding dynamic tissue organization by studying the human body one cell at a time: the human cell atlas (HCA) project, CARDIOVASCULAR RESEARCH, Vol: 114, Pages: E93-E95, ISSN: 0008-6363

Journal article

Watson SA, Duff JD, Bardi IB, Zabielska M, Atanur SS, Jabbour RJ, Smolenski RT, Harding SE, Perbellini F, Terracciano CMet al., 2018, A novel platform to maintain adult cardiac tissue in vitro: myocardial slices and electromechanical stimulation with physiological preload, European-Society-of-Cardiology Congress, Publisher: OXFORD UNIV PRESS, Pages: 1090-1090, ISSN: 0195-668X

Conference paper

Panahi M, Papanikolaou A, Torabi A, Zhang JG, Khan H, Vazir A, Hasham MG, Cleland J, Rosenthal N, Harding S, Sattler Set al., 2018, Immunomodulatory interventions in myocardial infarction and heart failure: a systematic review of clinical trials and meta-analysis of IL-1 inhibition., Cardiovascular Research, ISSN: 1755-3245

Following a myocardial infarction (MI), the immune system helps to repair ischaemic damage and restore tissue integrity, but excessive inflammation has been implicated in adverse cardiac remodelling and development towards heart failure (HF). Pre-clinical studies suggest that timely resolution of inflammation may help prevent HF development and progression. Therapeutic attempts to prevent excessive post-MI inflammation in patients have included pharmacological interventions ranging from broad immunosuppression to immunomodulatory approaches targeting specific cell types or factors with the aim to maintain beneficial aspects of the early post-MI immune response. These include the blockade of early initiators of inflammation including reactive oxygen species and complement, inhibition of mast cell degranulation and leucocyte infiltration, blockade of inflammatory cytokines, and inhibition of adaptive B and T-lymphocytes. Herein, we provide a systematic review on post-MI immunomodulation trials and a meta-analysis of studies targeting the inflammatory cytokine Interleukin-1. Despite an enormous effort into a significant number of clinical trials on a variety of targets, a striking heterogeneity in study population, timing and type of treatment, and highly variable endpoints limits the possibility for meaningful meta-analyses. To conclude, we highlight critical considerations for future studies including (i) the therapeutic window of opportunity, (ii) immunological effects of routine post-MI medication, (iii) stratification of the highly diverse post-MI patient population, (iv) the potential benefits of combining immunomodulatory with regenerative therapies, and at last (v) the potential side effects of immunotherapies.

Journal article

Kapnisi M, Mansfield C, Marijon C, Guex AG, Perbellini F, Bardi I, Humphrey EJ, Puetzer J, Mawad D, Koutsogeorgis DC, Stuckey DJ, Terracciano CM, Harding SE, Stevens MMet al., 2018, Auxetic cardiac patches with tunable mechanical and conductive properties toward treating myocardial infarction, Advanced Functional Materials, Vol: 28, ISSN: 1616-301X

An auxetic conductive cardiac patch (AuxCP) for the treatment of myocardial infarction (MI) is introduced. The auxetic design gives the patch a negative Poisson's ratio, providing it with the ability to conform to the demanding mechanics of the heart. The conductivity allows the patch to interface with electroresponsive tissues such as the heart. Excimer laser microablation is used to micropattern a re‐entrant honeycomb (bow‐tie) design into a chitosan‐polyaniline composite. It is shown that the bow‐tie design can produce patches with a wide range in mechanical strength and anisotropy, which can be tuned to match native heart tissue. Further, the auxetic patches are conductive and cytocompatible with murine neonatal cardiomyocytes in vitro. Ex vivo studies demonstrate that the auxetic patches have no detrimental effect on the electrophysiology of both healthy and MI rat hearts and conform better to native heart movements than unpatterned patches of the same material. Finally, the AuxCP applied in a rat MI model results in no detrimental effect on cardiac function and negligible fibrotic response after two weeks in vivo. This approach represents a versatile and robust platform for cardiac biomaterial design and could therefore lead to a promising treatment for MI.

Journal article

Couch LS, Harding SE, 2018, Takotsubo Syndrome: Stress or NO Stress?, JACC: Basic to Translational Science, Vol: 3, Pages: 227-229, ISSN: 2452-302X

Journal article

Foldes G, Lawlor K, Harding SE, Randi AMet al., 2018, STAT3 mediates differentiation and maintenance of human pluripotent stem-derived endothelial cells, 5th Congress of the ESC-Council-on-Basic-Cardiovascular-Science on Frontiers in Cardio Vascular Biology, Publisher: OXFORD UNIV PRESS, Pages: S39-S39, ISSN: 0008-6363

Background: High plasticity derivatives of human pluripotent stem cells (hPSC) such as embryonic stem cells (hESC) are being intensively developed for their use in endothelial replacement.Methods/Results: In this study, we found that transient addition of Activin A, followed by culture with VEGF165, BMP4 and FGF2 was an effective mechanism to induce differentiation of hESC toward the endothelial lineage. Indeed, human GeneChip microarray analysis revealed that endothelial gene regulatory networks were gradually increased during 12 days of differentiation. Isolated hESC-derived endothelial cells (hESC-EC) expressed mature endothelial-associated genes, including CD31, NRP1, VE-cadherin, Tie2, VWF and ICAM2, reaching levels comparable with human umbilical cord vascular endothelial cells by day 19. We found that a network of CD31+ tubes comprising endothelial precursor cells had formed in culture from 10 days after start of differentiation of hESC. As assessed by automated high content microscopy, the alignment and tube formation of the newly formed CD31+ vascular network were markedly decreased in response to C188-9, a novel small molecule inhibitor of STAT3 transcription factor (tube length: 57%, connected tube area: 22% of those in control, both p<0.001). Human ESC-EC were capable of transdifferentiating into mesenchymal cells in long-term cultures. Endothelial-mesenchymal transition was characterised by gradual loss of endothelial marker expression and increased mesenchymal marker FSP1 expression. We found that inhibition of STAT3 tyr705 phosphorylation by C188-9 resulted in a decreased proliferation of FSP1+ mesenchymal cells (2-fold decrease in Ki67%-positive population, p<0.001), and subsequently reduced number of FSP+ cells (36% reduction, p<0.05). At the same time, C188-9 increased the number of CD31+ hESC-EC by 30% (p=0.05, n=6). Viability remained unchanged in C188-9-treated cells (Topro3 necrosis marker, p=0.32, n=3).Conclusions: These results sugge

Conference paper

Husveth-Toth M, Gara E, Nemes A, Molnar AA, Csepi K, Radovits T, Harding SE, Merkely B, Foldes Get al., 2018, Human pluripotent stem cell-derived endothelial cells are vasoactive in vitro and capable of engineering 3D vascular grafts, 5th Congress of the ESC-Council-on-Basic-Cardiovascular-Science on Frontiers in Cardio Vascular Biology, Publisher: Oxford University Press (OUP), Pages: S111-S111, ISSN: 1755-3245

Conference paper

Wright PT, Bhogal N, Diakonov I, Pannell L, Perera R, Bork N, Schobesberger S, Lucarelli C, Faggian G, Alvarez-Laviada A, Zaccolo M, Kamp TJ, Balijepalli R, Lyon A, Harding SE, Nikolaev V, Gorelik Jet al., 2018, Cardiomyocyte membrane structure and cAMP compartmentation produce anatomical variation in β2AR-cAMP responsiveness in murine hearts, Cell Reports, Vol: 23, Pages: 459-469, ISSN: 2211-1247

Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to β2-adrenoceptor (β2AR) stimulation, which may underlie the development of Takotsubo cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived β2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to β2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.

Journal article

Hou H, Zhao Z, Machuki JO, Zhang L, Zhang Y, Fu L, Wu J, Liu Y, Harding SE, Sun Het al., 2018, Estrogen deficiency compromised the beta(2)AR-Gs/Gi coupling: implications for arrhythmia and cardiac injury, PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, Vol: 470, Pages: 559-570, ISSN: 0031-6768

Journal article

Ricardo CP, Hellen N, Foldes G, Kodagoda T, Terracciano CM, Duchen MR, Harding SEet al., 2018, Low levels of the A3243G MTDNA mutation in human induced pluripotent stem cellcardiomyocytes do not cause functional or metabolic disturbances but increase with further passaging, Autumn Meeting of the British-Society-for-Cardiovascular-Research on Cardiac Metabolic Disorders and Mitochondrial Dysfunction, Publisher: BMJ Publishing Group, Pages: A8-A9, ISSN: 1355-6037

Conference paper

Machuki JO, Zhang HY, Harding SE, Sun Het al., 2018, Molecular pathways of oestrogen receptors and -adrenergic receptors in cardiac cells: Recognition of their similarities, interactions and therapeutic value, ACTA PHYSIOLOGICA, Vol: 222, ISSN: 1748-1708

Oestrogen receptors (ERs) and β‐adrenergic receptors (βARs) play important roles in the cardiovascular system. Moreover, these receptors are expressed in cardiac myocytes and vascular tissues. Numerous experimental observations support the hypothesis that similarities and interactions exist between the signalling pathways of ERs (ERα, ERβ and GPR30) and βARs (β1AR, β2AR and β3AR). The recently discovered oestrogen receptor GPR30 shares structural features with the βARs, and this forms the basis for the interactions and functional overlap. GPR30 possesses protein kinase A (PKA) phosphorylation sites and PDZ binding motifs and interacts with A‐kinase anchoring protein 5 (AKAP5), all of which enable its interaction with the βAR pathways. The interactions between ERs and βARs occur downstream of the G‐protein‐coupled receptor, through the Gαs and Gαi proteins. This review presents an up‐to‐date description of ERs and βARs and demonstrates functional synergism and interactions among these receptors in cardiac cells. We explore their signalling cascades and the mechanisms that orchestrate their interactions and propose new perspectives on the signalling patterns for the GPR30 based on its structural resemblance to the βARs. In addition, we explore the relevance of these interactions to cell physiology, drugs (especially β‐blockers and calcium channel blockers) and cardioprotection. Furthermore, a receptor‐independent mechanism for oestrogen and its influence on the expression of βARs and calcium‐handling proteins are discussed. Finally, we highlight promising therapeutic avenues that can be derived from the shared pathways, especially the phosphatidylinositol‐3‐OH kinase (PI3K/Akt) pathway.

Journal article

Adamowicz M, Morgan CC, Haubner BJ, Noseda M, Collins MJ, Paiva MA, Srivastava PK, Gellert P, Razzaghi B, O'Gara P, Raina P, Game L, Bottolo L, Schneider MD, Harding SE, Penninger J, Aitman TJet al., 2018, Functionally conserved noncoding regulators of cardiomyocyte proliferation and regeneration in mouse and human, Circulation: Cardiovascular Genetics, Vol: 11, ISSN: 1942-325X

Background: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI.Methods and Results: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR-451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes.Conclusions: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.

Journal article

Leong KMW, Ng FS, Roney C, Cantwell C, Shun-Shin M, Linton N, Whinnett Z, Lefroy D, Davies DW, Harding S, Lim PB, Francis D, Peters N, Varnava A, Kanagaratnam Pet al., 2017, Repolarization abnormalities unmasked with exercise in sudden cardiac death survivors with structurally normal hearts, Journal of Cardiovascular Electrophysiology, Vol: 29, Pages: 115-126, ISSN: 1045-3873

BACKGROUND: Models of cardiac arrhythmogenesis predict that non-uniformity in repolarization and/or depolarization promotes ventricular fibrillation and is modulated by autonomic tone, but this is difficult to evaluate in patients. We hypothesize that such spatial heterogeneities would be detected by non-invasive ECG imaging (ECGi) in sudden cardiac death (SCD) survivors with structurally normal hearts under physiological stress. METHODS: ECGi was applied to 11 SCD survivors, 10 low-risk Brugada Syndrome patients (BrS) and 10 controls undergoing exercise treadmill testing. ECGi provides whole heart activation maps and > 1200 unipolar electrograms over the ventricular surface from which global dispersion of activation recovery interval (ARI) and regional delay in conduction were determined. These were used as surrogates for spatial heterogeneities in repolarization and depolarization. Surface ECG markers of dispersion (QT and Tpeak-end intervals) were also calculated for all patients for comparison. RESULTS: Following exertion, the SCD group demonstrated the largest increase in ARI dispersion compared to BrS and control groups (13±8 ms vs 4±7 ms vs 4±5 ms; p = 0.009), with baseline dispersion being similar in all groups. In comparison, surface ECG markers of dispersion of repolarisation were unable to discriminate between the groups at baseline or following exertion. Spatial heterogeneities in conduction were also present following exercise but were not significantly different between SCD survivors and the other groups. CONCLUSION: Increased dispersion of repolarization is apparent during physiological stress in SCD survivors and is detectable with ECGi but not with standard ECG parameters. The electrophysiological substrate revealed by ECGi could be the basis of alternative risk-stratification techniques. This article is protected by copyright. All rights reserved.

Journal article

Leong KMW, Ng FS, yao C, Roney C, Linton N, Whinnett Z, lefroy D, Davies DW, Lim PB, Harding S, Peters N, Kanagaratnam P, Varnava Aet al., 2017, ST-Elevation Magnitude Correlates With Right Ventricular Outflow Tract Conduction Delay in Type I Brugada ECG, Circulation: Arrhythmia and Electrophysiology, Vol: 10, ISSN: 1941-3084

Background: The substrate location and underlying electrophysiological mechanisms that contribute to the characteristic ECG pattern of Brugada syndrome (BrS) are still debated. Using noninvasive electrocardiographical imaging, we studied whole heart conduction and repolarization patterns during ajmaline challenge in BrS individuals.Methods and Results: A total of 13 participants (mean age, 44±12 years; 8 men), 11 concealed patients with type I BrS and 2 healthy controls, underwent an ajmaline infusion with electrocardiographical imaging and ECG recordings. Electrocardiographical imaging activation recovery intervals and activation timings across the right ventricle (RV) body, outflow tract (RVOT), and left ventricle were calculated and analyzed at baseline and when type I BrS pattern manifested after ajmaline infusion. Peak J-ST point elevation was calculated from the surface ECG and compared with the electrocardiographical imaging–derived parameters at the same time point. After ajmaline infusion, the RVOT had the greatest increase in conduction delay (5.4±2.8 versus 2.0±2.8 versus 1.1±1.6 ms; P=0.007) and activation recovery intervals prolongation (69±32 versus 39±29 versus 21±12 ms; P=0.0005) compared with RV or left ventricle. In controls, there was minimal change in J-ST point elevation, conduction delay, or activation recovery intervals at all sites with ajmaline. In patients with BrS, conduction delay in RVOT, but not RV or left ventricle, correlated to the degree of J-ST point elevation (Pearson R, 0.81; P<0.001). No correlation was found between J-ST point elevation and activation recovery intervals prolongation in the RVOT, RV, or left ventricle.Conclusions: Magnitude of ST (J point) elevation in the type I BrS pattern is attributed to degree of conduction delay in the RVOT and not prolongation in repolarization time.

Journal article

Chow A, Stuckey DJ, Kidher E, Rocco M, Jabbour RJ, Mansfield CA, Darzi A, Harding SE, Stevens MM, Athanasiou Tet al., 2017, Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction., Stem Cell Reports, Vol: 9, Pages: 1415-1422, ISSN: 2213-6711

Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment.

Journal article

Martinez VG, Ontoria-Oviedo I, Ricardo CP, Harding SE, Sacedon R, Varas A, Zapata A, Sepulveda P, Vicente Aet al., 2017, Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells, Stem Cell Research and Therapy, Vol: 8, ISSN: 1757-6512

BackgroundHuman dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression.MethodsHuman dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA).ResultsWhile HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription.ConclusionsImmunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases the

Journal article

Sikkel MB, Francis DP, Howard J, Gordon F, Rowlands C, Peters NS, Lyon AR, Harding SE, MacLeod KTet al., 2017, Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies, Cardiovascular Research, Vol: 113, Pages: 1743-1752, ISSN: 1755-3245

AimsIt is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests).Methods and resultsCa2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF.Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF.ConclusionCa2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for relia

Journal article

Perbellini F, Watson SA, Scigliano M, Alayoubi S, Tkach S, Bardi I, Quaife N, Kane C, Dufton NP, Simon A, Sikkel MB, Faggian G, Randi AM, Gorelik J, Harding S, Terracciano CMNet al., 2017, Investigation of cardiac fibroblasts using myocardial slices, Cardiovascular Research, Vol: 114, Pages: 77-89, ISSN: 1755-3245

AimsCardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro, CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices.Methods and resultsUnloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture. CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(−) despite transforming growth factor-β (20 ng/ml) or angiotensin II (200 µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 0.001).ConclusionsMyocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.

Journal article

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