Publications
302 results found
Shintani Y, Drexler HC, Kioka H, et al., 2024, Author Correction: Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2., EMBO Rep
Shintani Y, Fukushima S, Varela-Carver A, et al., 2024, Corrigendum to "Donor cell-type specific paracrine effects of cell transplantation for post-infarction heart failure" [J Mol Cell Cardiol 47 (2009) 288-295]., J Mol Cell Cardiol, Vol: 187, Pages: 118-119
Pitoulis FG, Smith JJ, Pamias-Lopez B, et al., 2023, MyoLoop: Design, development and validation of a standalone bioreactor for pathophysiological electromechanical in vitro cardiac studies, EXPERIMENTAL PHYSIOLOGY, ISSN: 0958-0670
Nunez-Toldra R, Del Canizo A, Secco I, et al., 2023, Living myocardial slices for the study of nucleic acid-based therapies, Frontiers in Bioengineering and Biotechnology, Vol: 11, ISSN: 2296-4185
Gene therapy based on viral vectors offers great potential for the study and the treatment of cardiac diseases. Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac tissue from failing (dilated cardiomyopathy) hearts was also used to validate the model in a more translational setting. LMS were cultured at physiological sarcomere length for 72-h under electrical stimulation. Two recombinant AAV serotypes (AAV6 and AAV9) at different multiplicity of infection (MOI) expressing enhanced green fluorescent protein (eGFP) were added to the surface of rat LMS. AAV6 at 20,000 MOI proved to be the most suitable serotype without affecting LMS contractility or kinetics and showing high transduction and penetrability efficiency in rat LMS. This serotype exhibited 40% of transduction efficiency in cardiomyocytes and stromal cells while 20% of the endothelial cells were transduced. With great translational relevance, this protocol introduces the use of LMS as a model for nucleic acid-based therapies, allowing the acceleration of preclinical studies for cardiac diseases.
Cler M, Perez Amodio S, Martinez E, et al., 2023, Exogenous lactate improves cardiac function of living myocardial slices, European Chapter of the Tissue-Engineering-and-Regenerative-Medicine-International-Society (TERMIS), Publisher: MARY ANN LIEBERT, INC, ISSN: 1937-3341
Hayman D, Terracciano CM, 2023, Development of an ischemia/reperfusion injury model in the living myocardial slice, Publisher: CELL PRESS, Pages: 165A-165A, ISSN: 0006-3495
van der Velden J, Asselbergs FW, Bakkers J, et al., 2022, Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC working group on myocardial function and the ESC Working Group on Cellular Biology of the Heart, Cardiovascular Research, Vol: 118, Pages: 3016-3051, ISSN: 0008-6363
Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task. In particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and co-morbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models cannot provide a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on a organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and improved current animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction and refinement (3R) as a guiding concept.
Zabielska-Kaczorowska MA, Bogucka AE, Macur K, et al., 2022, Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation, SCIENTIFIC REPORTS, Vol: 12, ISSN: 2045-2322
Nunez-Toldra R, Del Canizo A, Secco I, et al., 2022, Nucleic acid-based therapies using living myocardial slices, Publisher: OXFORD UNIV PRESS, Pages: 2930-2930, ISSN: 0195-668X
King O, Cruz-Moreira D, Sayed A, et al., 2022, Functional microvascularization of human myocardium in vitro, Cell Reports: Methods, Vol: 2, Pages: 1-16, ISSN: 2667-2375
In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research.
Hachim D, Zhao J, Bhankharia J, et al., 2022, Polysaccharide-polyplex nanofilm coatings enhance nanoneedle-based gene delivery and transfection efficiency, Small, Vol: 18, ISSN: 1613-6810
Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.
Wang BX, Kane C, Nicastro L, et al., 2022, Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol: 23
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Nunez-Toldra R, Del Canizo A, Secco I, et al., 2022, Living myocardial slices for the study of nucleic acid-based therapies, Publisher: WILEY, Pages: 276-277, ISSN: 1388-9842
Couch LS, Fiedler J, Chick G, et al., 2022, Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure, Cardiovascular Research, Vol: 118, Pages: 1758-1770, ISSN: 0008-6363
AIMS: Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and absence of coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS. METHODS AND RESULTS: miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal) cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4) and G-protein subunit Gβ (GNB1) as underlying these effects. CONCLUSION: miR-16 and miR-26a sensitise the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future. TRANSLATIONAL PERSPECTIVE: TTS-associated miRs have the potential to be active players predisposing to TTS. Feasibly, their measurement in recovered TTS patients during subsequent peri
Nunez-Toldra R, Kirwin T, Ferraro E, et al., 2022, Mechanosensitive molecular mechanisms of myocardial fibrosis in living myocardial slices, Publisher: WILEY PERIODICALS, INC, Pages: 1400-1412, ISSN: 2055-5822
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- Citations: 7
Roy I, Dubey P, Majid Q, et al., 2022, POLYHYDROXYALKANOATES, NATURAL MATERIALS OF BACTERIAL ORIGIN, IDEAL FOR CARDIAC TISSUE ENGINEERING, Publisher: MARY ANN LIEBERT, INC, Pages: S36-S37, ISSN: 1937-3341
Wang BX, Nicastro L, Couch L, et al., 2022, Extracellular vesicles from human cardiac fibroblasts modulate calcium cycling in human stem cell-derived cardiomyocytes, Cells, Vol: 11, Pages: 1171-1171, ISSN: 2073-4409
Cardiac fibroblasts regulate the development of the adult cardiomyocyte phenotype and cardiac remodeling in disease. We investigate the role that cardiac fibroblasts-secreted extracellular vesicles (EVs) have in the modulation of cardiomyocyte Ca2+ cycling–a fundamental mechanism in cardiomyocyte function universally altered during disease. EVs collected from cultured human cardiac ventricular fibroblasts were purified by centrifugation, ultrafiltration and size-exclusion chromatography. The presence of EVs and EV markers were identified by dot blot analysis and electron microscopy. Fibroblast-conditioned media contains liposomal particles with a characteristic EV phenotype. EV markers CD9, CD63 and CD81 were highly expressed in chromatography fractions that elute earlier (Fractions 1–15), with most soluble contaminating proteins in the later fractions collected (Fractions 16–30). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with fibroblast-secreted EVs and intracellular Ca2+ transients were analyzed. Fibroblast-secreted EVs abbreviate the Ca2+ transient time to peak and time to 50% decay versus serum-free controls. Thus, EVs from human cardiac fibroblasts represent a novel mediator of human fibroblast-cardiomyocyte interaction, increasing the efficiency of hiPSC-CM Ca2+ handling.
Nicastro L, Kyriakou A, Downing B, et al., 2022, Mechanical load affects the regulation of contractility of rat living myocardial slices via extracellular vesicles, Publisher: CELL PRESS, Pages: 304A-304A, ISSN: 0006-3495
Pitoulis FG, Nunez-Toldra R, Xiao K, et al., 2022, Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro, Cardiovascular Research, Vol: 118, Pages: 814-827, ISSN: 0008-6363
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, and 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 based on 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 and afterload), pressure-overload (normal preload and 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 pathophysiol
Nicastro L, Kyriakou A, Emanueli C, et al., 2021, Extracellular vesicles derived from physiologically loaded living myocardial slices improve the contractility of healthy living myocardial slices, Publisher: OXFORD UNIV PRESS, Pages: 3222-3222, ISSN: 0195-668X
Jabbour R, Owen T, Pandey P, et al., 2021, In vivo grafting of large engineered heart tissue patches for cardiac repair, JCI Insight, Vol: 6, Pages: 1-13, ISSN: 2379-3708
Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be anovel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more dataare needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5mm) consisting of up to 20 million human induced pluripotent stem cell–derived cardiomyocytes(hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarctionmodel was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs inEHTs became more aligned over 28 days and had improved contraction kinetics and faster calciumtransients. Blinded echocardiographic analysis revealed a significant improvement in function ininfarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularizationfrom the host to the patch was observed at week 1 and stable to week 4, but electrical couplingbetween patch and host heart was not observed. In vivo telemetry recordings and ex vivoarrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTsimproved function and reduced scar size without causing arrhythmia, which may be due to the lackof electrical coupling between patch and host heart.
Basnett P, Matharu RK, Taylor CS, et al., 2021, Harnessing Polyhydroxyalkanoates and Pressurized Gyration for Hard and Soft Tissue Engineering, ACS APPLIED MATERIALS & INTERFACES, Vol: 13, Pages: 32624-32639, ISSN: 1944-8244
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- Citations: 23
King O, Sunyovszki I, Terracciano CM, 2021, Vascularisation of pluripotent stem cell-derived myocardium: biomechanical insights for physiological relevance in cardiac tissue engineering, PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, Vol: 473, Pages: 1117-1136, ISSN: 0031-6768
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- Citations: 4
Dries E, Bardi I, Nunez-Toldra R, et al., 2021, CaMKII inhibition reduces arrhythmogenic Ca<SUP>2+</SUP> events in subendocardial cryoinjured rat living myocardial slices, JOURNAL OF GENERAL PHYSIOLOGY, Vol: 153, ISSN: 0022-1295
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- Citations: 4
Kit-Anan W, Mazo M, Wang BX, et al., 2021, Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation, Biofabrication, Vol: 13, Pages: 1-16, ISSN: 1758-5082
Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely microscopic topography (3D shape resembling that of adult CM) and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate pillars of the desired 3D shapes, which would be used to mould the designed microwell arrays into a hydrogel. Polyacrylamide was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without comprising its capacity to modulate the stiffness. In this manner, individual parameters can be finely tuned independently within the hydrogel: the dimension of 3D shaped microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in an absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell Peer reviewed version of the manuscript published in final form at Biofabrication (2020). membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2- MYL7 proteins; while displaying higher anisotropism in comparison to pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiological or pathological microenvironment to an isolated single hiPSC-CM in absen
Poulet C, Sanchez-Alonso J, Swiatlowska P, et al., 2021, Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes, Cardiovascular Research, Vol: 117, Pages: 149-161, ISSN: 0008-6363
Aim: In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodeling in cardiac diseases is associated with down-regulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signaling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes.Methods and results: Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodeling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with Methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions: JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol an
Nicastro L, Calapano F, Nunez Toldra R, et al., 2020, Mechanical load modulates the cargo of secreted extracellular vesicles (EVs) from living myocardial slices, European-Society-of-Cardiology (ESC) Congress, Publisher: OXFORD UNIV PRESS, Pages: 3614-3614, ISSN: 0195-668X
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King O, Cruz-Moreira D, Kit-Anan W, et al., 2020, Influence of perfusable microvasculature on excitation-contraction coupling in IPSC-derived myocardium, European-Society-of-Cardiology (ESC) Congress, Publisher: OXFORD UNIV PRESS, Pages: 3590-3590, ISSN: 0195-668X
Zwi Dantsis L, Winter CW, Kauscher U, et al., 2020, Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells, Nanoscale, Vol: 12, Pages: 19844-19854, ISSN: 2040-3364
Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration.
Lyon A, Babalis D, Morley-Smith AC, et al., 2020, Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device – the SERCA-LVAD TRIAL, Gene Therapy, Vol: 27, Pages: 579-590, ISSN: 0969-7128
The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. Enrolled subjects were randomised to receive a single intracoronary infusion of 1x1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort, which may help guide future trial design.
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