Publications by authors named "Sian Harding"

141 Publications

GPER mediates estrogen cardioprotection against epinephrine-induced stress.

J Endocrinol 2021 Apr 1. Epub 2021 Apr 1.

H Sun, physiology department, Xuzhou Medical University, Xuzhou, China.

Currently, there are no conventional treatments for stress-induced cardiomyopathy (SCM, also known as Takotsubo syndrome), and the existing therapies are not effective. The recently discovered G protein- coupled estrogen receptor (GPER) executes the rapid effects of estrogen (E2). In this study, we investigated the effects and mechanism of GPER on epinephrine (Epi)-induced cardiac stress. SCM was developed with a high dose of Epi in adult rats and human-induced pluripotent stem cells-derived cardiomyocytes(hiPSC-CMs). (1) GPER activation with agonist G1/ E2 prevented an increase in left ventricular internal diameter at end-systole, the decrease both in ejection fraction and cardiomyocyte shortening amplitude elicited by Epi. (2) G1/ E2 mitigated heart injury induced by Epi, as revealed by reduced plasma brain natriuretic peptide and lactate dehydrogenase release into culture supernatant. (3) G1/E2 prevented the raised phosphorylation and internalization of β2-adrenergic receptors(β2AR). (4) Blocking Gαi abolished the cardiomyocyte contractile inhibition by Epi. G1/E2 downregulated Gαi activity of cardiomyocytes and further upregulated cyclic adenosine monophosphate concentration in culture supernatant treated with Epi. (5) G1/E2 rescued decreased Ca2+ amplitude and Ca2+ channel current (ICa-L) in rat cardiomyocytes. Notably, the above effects of E2 were blocked by the GPER antagonist, G15. In hiPSC-CM (which expressed GPER, β1AR and β2ARs), knockdown of GPER by siRNA abolished E2 effects on increasing ICa-L and action potential duration in the stress state. In conclusion, GPER played a protective role against SCM. Mechanistically, this effect was mediated by balancing the coupling of β2AR to the Gαs and Gαi signalling pathways.
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http://dx.doi.org/10.1530/JOE-20-0451DOI Listing
April 2021

Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.

Cardiovasc Res 2021 Mar 16. Epub 2021 Mar 16.

National Heart and Lung Institute, Imperial College London, UK.

Aims: Cardiac remodelling is the process by which the heart adapts to its environment. Mechanical load is a major driver of remodelling. Cardiac tissue culture has been frequently employed for in vitro studies of load-induced remodelling; however, current in vitro protocols (e.g. cyclic stretch, isometric load, auxotonic load) are oversimplified and do not accurately capture the dynamic sequence of mechanical conformational changes experienced by the heart in vivo. This limits translational scope and relevance of findings.

Methods And Results: We developed a novel methodology to study chronic load in vitro. We first developed a bioreactor that can recreate the electromechanical events of in vivo pressure-volume loops as in vitro force-length loops. We then used the bioreactor to culture rat living myocardial slices (LMS) for 3 days. The bioreactor operated on the basis of a 3-Element Windkessel circulatory model enabling tissue mechanical loading based on physiologically relevant parameters of afterload and preload. LMS were continuously stretched/relaxed during culture simulating conditions of physiological load (normal preload & afterload), pressure-overload (normal preload & high afterload), or volume-overload (high preload & normal afterload). At the end of culture, functional, structural, and molecular assays were performed to determine load-induced remodelling.Both pressure- and volume-overloaded LMS showed significantly decreased contractility that was more pronounced in the latter compared with physiological load p < 0.0001). Overloaded groups also showed cardiomyocyte hypertrophy; RNAseq identified shared and unique genes expressed in each overload group. The PI3K-Akt pathway was dysregulated in volume-overload while inflammatory pathways were mostly associated with remodelling in pressure-overloaded LMS.

Conclusion: We have developed a proof-of-concept platform and methodology to recreate remodelling under pathophysiological load in vitro. We show that LMS cultured in our bioreactor remodel as a function of the type of mechanical load applied to them.
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http://dx.doi.org/10.1093/cvr/cvab084DOI Listing
March 2021

Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation.

Biofabrication 2021 Mar 12;13(2):025004. Epub 2021 Mar 12.

Department of Materials, Imperial College London, London, United Kingdom. Department of Bioengineering, Imperial College London, London, United Kingdom. Institute of Biomedical Engineering, Imperial College London, London, United Kingdom. National Heart and Lung Institute, Imperial College London, London, United Kingdom.

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 3D shape and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate inverse patterns of the desired 3D shapes in bas relief, which then were used to mold the designed microwell arrays into a hydrogel. Polyacrylamide (PAAm) was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without compromising capacity to modulate stiffness. In this manner, two individual parameters can be finely tuned independently within the hydrogel: the shape of the 3D microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in the absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness that mimics muscle stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2-MYL7 proteins; and higher anisotropism than cells in pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiologic or pathologic microenvironment to an isolated single hiPSC-CM in the absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for-purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision that this system could be easily applied to other tissues and cell types where the influence of cellular shape and stiffness of the surrounding environment is hypothesized to play an important role in physiology.
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http://dx.doi.org/10.1088/1758-5090/abce0aDOI Listing
March 2021

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of βAR in isogenic hPSC-derived cardiomyocytes.

Mol Ther Methods Clin Dev 2021 Mar 27;20:39-53. Epub 2020 Oct 27.

Division of Cancer and Stem Cells, University of Nottingham Biodiscovery Institute, University Park, Nottingham NG7 2RD, UK.

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (βAR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, 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 βAR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level βAR signaling dominated over βAR . 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 βAR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.
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http://dx.doi.org/10.1016/j.omtm.2020.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733025PMC
March 2021

Cross-Priming Dendritic Cells Exacerbate Immunopathology After Ischemic Tissue Damage in the Heart.

Circulation 2021 Feb 10;143(8):821-836. Epub 2020 Dec 10.

National Heart and Lung Institute, Imperial College London, UK (A.S., H.S.K., A.P., C.J., M.A., R.A.C., M.B., M.D.S., S.E.H., F.S.N., N.R., S.S.).

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 heart failure. 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 helper and 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-myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8 T cells.

Methods: We induced type 2 myocardial infarction-like ischemic injury in the heart by treatment with a single high dose of the β-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 -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. mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8 T cells.

Conclusion: Activation of cytotoxic CD8 T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.044581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899721PMC
February 2021

Type 2 MI induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart.

J Cell Mol Med 2021 Jan 29;25(1):229-243. Epub 2020 Nov 29.

National Heart and Lung Institute, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1111/jcmm.15937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810962PMC
January 2021

Natural Biomaterials for Cardiac Tissue Engineering: A Highly Biocompatible Solution.

Front Cardiovasc Med 2020 23;7:554597. Epub 2020 Oct 23.

Faculty of Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom.

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 hence can lead to a considerable reduction in mortality rates due to CVD.
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http://dx.doi.org/10.3389/fcvm.2020.554597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644890PMC
October 2020

Non-invasive detection of exercise-induced cardiac conduction abnormalities in sudden cardiac death survivors in the inherited cardiac conditions.

Europace 2021 Feb;23(2):305-312

Institute of Cardiovascular Science, University College London & Bart's Heart Centre, Bart's Health NHS Trust, London, UK.

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.
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http://dx.doi.org/10.1093/europace/euaa248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868885PMC
February 2021

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of βAR-cAMP Signalling.

Cells 2020 10 12;9(10). Epub 2020 Oct 12.

Cardiac Section, National Heart and Lung Institute (NHLI), Faculty of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

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 βAR-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 βAR. 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 βAR (but not βAR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the βAR-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 βAR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the βAR 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.
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http://dx.doi.org/10.3390/cells9102275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601768PMC
October 2020

Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells.

Biochem Biophys Res Commun 2020 12 19;533(3):376-382. Epub 2020 Sep 19.

Heart Institute (InCor), University of São Paulo Medical School, São Paulo, SP, Brazil. Electronic address:

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and β-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.
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http://dx.doi.org/10.1016/j.bbrc.2020.09.021DOI Listing
December 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 2020 10 9;76(4):1208-1218. Epub 2020 Sep 9.

School of Biological Sciences, University of Reading, United Kingdom (D.N.M., J.J.C., T.M., S.J.F., P.H.S., A.C.), St. George's Healthcare NHS Trust, London, United Kingdom.

Systemic hypertension increases cardiac workload causing cardiomyocyte hypertrophy and increased cardiac fibrosis. An underlying feature is increased production of reactive oxygen species. Redox-sensitive ASK1 (apoptosis signal-regulating kinase 1) activates stress-regulated protein kinases (p38-MAPK [mitogen-activated protein kinases] and JNKs [c-Jun N-terminal kinases]) and promotes fibrosis in various tissues. Here, we determined the specificity of ASK1 signaling in the heart, with the hypothesis that ASK1 inhibitors may be used to manage fibrosis in hypertensive heart disease. Using immunoblotting, we established that moderate levels of HO activate ASK1 in neonatal rat cardiomyocytes and perfused rat hearts. ASK1 was activated during ischemia in adult rat hearts, but not on reperfusion, consistent with activation by moderate (not high) reactive oxygen species levels. In contrast, IL (interleukin)-1β activated an alternative kinase, TAK1 (transforming growth factor-activated kinase 1). ASK1 was not activated by IL1β in cardiomyocytes and activation in perfused hearts was due to increased reactive oxygen species. Selonsertib (ASK1 inhibitor) prevented activation of p38-MAPKs (but not JNKs) by oxidative stresses in cultured cardiomyocytes and perfused hearts. In vivo (C57Bl/6J mice with osmotic minipumps for drug delivery), selonsertib (4 mg/[kg·d]) alone did not affect cardiac function/dimensions (assessed by echocardiography). However, it suppressed hypertension-induced cardiac hypertrophy resulting from angiotensin II (0.8 mg/[kg·d], 7d), with inhibition of mRNA upregulation, reduced cardiomyocyte hypertrophy and, notably, significant reductions in interstitial and perivascular fibrosis. Our data identify a specific reactive oxygen species→ASK1→p38-MAPK pathway in the heart and establish that ASK1 inhibitors protect the heart from hypertension-induced cardiac remodeling. Thus, targeting the ASK1→p38-MAPK nexus has potential therapeutic viability as a treatment for hypertensive heart disease.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480944PMC
October 2020

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

Pflugers Arch 2020 10 1;472(10):1435-1446. Epub 2020 Sep 1.

Faculty of Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK.

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. A total of 1 mM of carbenoxolone was administered at 5 ml/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) increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and 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) - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.
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http://dx.doi.org/10.1007/s00424-020-02446-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476990PMC
October 2020

Mediastinal Lymphadenopathy, Class-Switched Auto-Antibodies and Myocardial Immune-Complexes During Heart Failure in Rodents and Humans.

Front Cell Dev Biol 2020 7;8:695. Epub 2020 Aug 7.

National Heart and Lung Institute, Imperial College London, London, United Kingdom.

Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemic heart failure pointing to an autoimmune response against the heart. T and B cells have been convincingly demonstrated to be activated after myocardial infarction, a prerequisite for the generation of mature auto-antibodies. Yet, little is known about the immunoglobulin isotype repertoire thus pathological potential of anti-heart auto-antibodies during heart failure. We obtained human myocardial tissue from ischemic heart failure patients and induced experimental 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 mature isotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens are present in rat heart failure serum, and IgG and complement C3 deposits are evident in heart failure tissue of both rats and human patients. Previously established myocardial inflammation, and the herein provided proof of B cell maturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide all the hallmark signs of an established autoimmune response in chronic heart failure.
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http://dx.doi.org/10.3389/fcell.2020.00695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426467PMC
August 2020

Human pluripotent stem cell-derived cardiomyocytes as a target platform for paracrine protection by cardiac mesenchymal stromal cells.

Sci Rep 2020 08 3;10(1):13016. Epub 2020 Aug 3.

National Heart and Lung Institute, Imperial College London, Du Cane Road, London, W12 0NN, UK.

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 paracrine signals. 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 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.
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http://dx.doi.org/10.1038/s41598-020-69495-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400574PMC
August 2020

Selective protection of human cardiomyocytes from anthracycline cardiotoxicity by small molecule inhibitors of MAP4K4.

Sci Rep 2020 07 21;10(1):12060. Epub 2020 Jul 21.

National Heart and Lung Institute, Imperial College London, London, W12 0NN, UK.

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.
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http://dx.doi.org/10.1038/s41598-020-68907-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374628PMC
July 2020

β-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes.

Elife 2020 03 31;9. Epub 2020 Mar 31.

Myocardial Function, National Heart and Lung Institute, Imperial College London, ICTEM, Hammersmith Hospital, London, United Kingdom.

Cardiomyocyte β-adrenoceptors (β-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 β-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 β-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 β-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.
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http://dx.doi.org/10.7554/eLife.52221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138611PMC
March 2020

Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics.

J Mol Cell Cardiol 2020 04 19;141:11-16. Epub 2020 Mar 19.

National Heart and Lung Institute, Imperial College London, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yjmcc.2020.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246333PMC
April 2020

Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure.

J Biophotonics 2020 06 12;13(6):e201960239. Epub 2020 Mar 12.

Photonics Group, Department of Physics, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1002/jbio.201960239DOI Listing
June 2020

Future potential of engineered heart tissue patches for repairing the damage caused by heart attacks.

Expert Rev Med Devices 2020 01 6;17(1):1-3. Epub 2019 Dec 6.

Department of translational medicine, Imperial College London, London, UK.

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http://dx.doi.org/10.1080/17434440.2020.1700793DOI Listing
January 2020

A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET.

Cells 2019 11 29;8(12). Epub 2019 Nov 29.

Imperial College London, National Heart and Lung Institute, Du Cane Road, London W12 0NN, UK.

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-S 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.
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http://dx.doi.org/10.3390/cells8121541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952787PMC
November 2019

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Preclinical Cancer Drug Cardiotoxicity Testing: A Scientific Statement From the American Heart Association.

Circ Res 2019 10 19;125(10):e75-e92. Epub 2019 Sep 19.

It is now well recognized that many lifesaving oncology drugs may adversely affect the heart and cardiovascular system, including causing irreversible cardiac injury that can result in reduced quality of life. These effects, which may manifest in the short term or long term, are mechanistically not well understood. Research is hampered by the reliance on whole-animal models of cardiotoxicity that may fail to reflect the fundamental biology or cardiotoxic responses of the human myocardium. The emergence of human induced pluripotent stem cell-derived cardiomyocytes as an in vitro research tool holds great promise for understanding drug-induced cardiotoxicity of oncological drugs that may manifest as contractile and electrophysiological dysfunction, as well as structural abnormalities, making it possible to deliver novel drugs free from cardiac liabilities and guide personalized therapy. This article briefly reviews the challenges of cardio-oncology, the strengths and limitations of using human induced pluripotent stem cell-derived cardiomyocytes to represent clinical findings in the nonclinical research space, and future directions for their further use.
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http://dx.doi.org/10.1161/RES.0000000000000291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398423PMC
October 2019

Understanding dynamic tissue organization by studying the human body one cell at a time: the human cell atlas (HCA) project.

Cardiovasc Res 2018 10;114(12):e93-e95

British Heart Foundation Centre of Research Excellence and British Heart Foundation Centre for Regenerative Medicine, National Heart and Lung Institute, Imperial College London, London, UK.

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http://dx.doi.org/10.1093/cvr/cvy223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148330PMC
October 2018

Characterization of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by multiparametric quantitative cardiac magnetic resonance imaging.

Dis Model Mech 2019 08 16;12(8). Epub 2019 Aug 16.

National Heart and Lung Institute, Imperial College London, London W12 0NN, UK

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 Toll-like receptor 7 (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 overactivation 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 detection of diffuse infiltration, fibrosis and hemorrhages using a combination of T, T and T relaxation times, and compared results with histopathology of cardiac sections corresponding to CMR tissue maps. This allowed detailed correlation between CMR parameters and histopathology, and confirmed the need to include T measurements to detect tissue iron for accurate interpretation of pathology associated with CMR parameter changes. In summary, we provide detailed histological and imaging-based characterization of acute hemorrhagic myocarditis as an acute cardiac complication in the mouse model of Resiquimod-induced SLE, and a refined CMR protocol to allow non-invasive longitudinal studies of heart involvement in acute inflammation. We propose that adding T mapping to CMR protocols for myocarditis diagnosis improves diagnostic sensitivity and interpretation of disease mechanisms.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.040725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737951PMC
August 2019

Multi-cellularity in cardiac tissue engineering, how close are we to native heart tissue?

J Muscle Res Cell Motil 2019 06 20;40(2):151-157. Epub 2019 Jun 20.

National Heart and Lung Institute, Imperial College London Hammersmith Campus, Imperial Centre for Translational and Experimental Medicine, Du Cane Road, London, W12 0NN, UK.

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.
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http://dx.doi.org/10.1007/s10974-019-09528-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726707PMC
June 2019

Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.

Nat Commun 2019 05 15;10(1):2168. Epub 2019 May 15.

National Heart & Lung Institute, Imperial College London, London, W12 0NN, UK.

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.
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http://dx.doi.org/10.1038/s41467-019-10175-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520377PMC
May 2019

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Cell Stem Cell 2019 04 7;24(4):579-591.e12. Epub 2019 Mar 7.

British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London W12 0NN, UK.

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.
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http://dx.doi.org/10.1016/j.stem.2019.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458995PMC
April 2019

Ventricular conduction stability test: a method to identify and quantify changes in whole heart activation patterns during physiological stress.

Europace 2019 Sep;21(9):1422-1431

National Heart & Lung Institute, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1093/europace/euz015DOI Listing
September 2019

Proteomic Analysis Reveals Temporal Changes in Protein Expression in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes In Vitro.

Stem Cells Dev 2019 05 20;28(9):565-578. Epub 2019 Mar 20.

1 Myocardial Function, National Heart and Lung Institute, Imperial College, London, United Kingdom.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold great promise for regenerative medicine and in vitro screening. Despite displaying key cardiomyocyte phenotypic characteristics, they more closely resemble fetal/neonatal cardiomyocytes, and further characterization is necessary. By 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 hiPSC-CM protein expression. We found that hiPSC-CM 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 toward 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 hiPSC-CM. In several cases, a predicted outcome was not clear because of the concerted changes in both stimulatory and inhibitory pathways. Nevertheless, clear development of hiPSC-CM over this time period was evident.
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http://dx.doi.org/10.1089/scd.2018.0210DOI Listing
May 2019

Takotsubo Syndrome: Latest Addition to the Expanding Family of Immune-Mediated Diseases?

JACC Basic Transl Sci 2018 Dec 31;3(6):779-781. Epub 2018 Dec 31.

National Heart and Lung Institute, Imperial College, London, United Kingdom.

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http://dx.doi.org/10.1016/j.jacbts.2018.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314959PMC
December 2018