MyoLoop: Design, development and validation of a standalone bioreactor for pathophysiological electromechanical in vitro cardiac studies.

Mechanical load is one of the main determinants of cardiac structure and function. Mechanical load is studied in vitro using cardiac preparations together with loading protocols (e.g., auxotonic, isometric). However, such studies are often limited by reductionist models and poorly simulated mechanical load profiles. This hinders the physiological relevance of findings. Living myocardial slices have been used to study load in vitro. Living myocardial slices (LMS) are 300-µm-thick intact organotypic preparations obtained from explanted animal or human hearts. They have preserved cellular populations and the functional, structural, metabolic and molecular profile of the tissue from which they are prepared. Using a three-element Windkessel (3EWK) model we previously showed that LMSs can be cultured while performing cardiac work loops with different preload and afterload. Under such conditions, LMSs remodel as a function of the mechanical load applied to them (physiological load, pressure or volume overload). These studies were conducted in commercially available length actuators that had to be extensively modified for culture experiments. In this paper, we demonstrate the design, development and validation of a novel device, MyoLoop. MyoLoop is a bioreactor that can pace, thermoregulate, acquire and process data, and chronically load LMSs and other cardiac tissues in vitro. In MyoLoop, load is parametrised using a 3EWK model, which can be used to recreate physiological and pathological work loops and the remodelling response to these. We believe MyoLoop is the next frontier in basic cardiovascular research enabling reductionist but physiologically relevant in vitro mechanical studies.

Functional microvascularization of human myocardium in vitro.

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.

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Polysaccharide-Polyplex Nanofilm Coatings Enhance Nanoneedle-Based Gene Delivery and Transfection Efficiency.

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.

Extracellular Vesicles from Human Cardiac Fibroblasts Modulate Calcium Cycling in Human Stem Cell-Derived Cardiomyocytes.

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.

Mechanosensitive molecular mechanisms of myocardial fibrosis in living myocardial slices.

AIMS: Altered mechanical load in response to injury is a main driver of myocardial interstitial fibrosis. No current in vitro model can precisely modulate mechanical load in a multicellular environment while maintaining physiological behaviour. Living myocardial slices (LMS) are a 300 µm-thick cardiac preparation with preserved physiological structure and function. Here we apply varying degrees of mechanical preload to rat and human LMS to evaluate early cellular, molecular, and functionality changes related to myocardial fibrosis. METHODS AND RESULTS: Left ventricular LMS were obtained from Sprague Dawley rat hearts and human cardiac samples from healthy and failing (dilated cardiomyopathy) hearts. LMS were mounted on custom stretchers and two degrees of diastolic load were applied: physiological sarcomere length (SL) (SL = 2.2 µm) and overload (SL = 2.4 µm). LMS were maintained for 48 h under electrical stimulation in circulating, oxygenated media at 37°C. In overloaded conditions, LMS displayed an increase in nucleus translocation of Yes-associated protein (YAP) and an up-regulation of mechanotransduction markers without loss in cell viability. Expression of fibrotic and inflammatory markers, as well as Collagen I deposition were also observed. Functionally, overloaded LMS displayed lower contractility (7.48 ± 3.07 mN mm-2 at 2.2 SL vs. 3.53 ± 1.80 mN mm-2 at 2.4 SL). The addition of the profibrotic protein interleukin-11 (IL-11) showed similar results to the application of overload with enhanced fibrosis (8% more of collagen surface coverage) and reduced LMS contractility at physiological load. Conversely, treatment with the Transforming growth factor ß receptor (TGF-ßR) blocker SB-431542, showed down-regulation of genes associated with mechanical stress, prevention of fibrotic response and improvement in cardiac function despite overload (from 2.40 ± 0.8 mN mm-2 to 4.60 ± 1.08 mN mm-2 ). CONCLUSIONS: The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-ßR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.

Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure.

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 in the absence of culprit 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 Cavß subunit), RGS4 (regulator of G-protein signalling 4), and G-protein subunit Gß (GNB1) as underlying these effects. CONCLUSION: miR-16 and miR-26a sensitize 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.

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

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 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.

In vivo grafting of large engineered heart tissue patches for cardiac repair.

Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.

CaMKII inhibition reduces arrhythmogenic Ca2+ events in subendocardial cryoinjured rat living myocardial slices.

Spontaneous Ca2+ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca2+ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca2+ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca2+ transients to peak after injury. In ENDO LMS, the Ca2+ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation-contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.

Vascularisation of pluripotent stem cell-derived myocardium: biomechanical insights for physiological relevance in cardiac tissue engineering.

The myocardium is a diverse environment, requiring coordination between a variety of specialised cell types. Biochemical crosstalk between cardiomyocytes (CM) and microvascular endothelial cells (MVEC) is essential to maintain contractility and healthy tissue homeostasis. Yet, as myocytes beat, heterocellular communication occurs also through constantly fluctuating biomechanical stimuli, namely (1) compressive and tensile forces generated directly by the beating myocardium, and (2) pulsatile shear stress caused by intra-microvascular flow. Despite endothelial cells (EC) being highly mechanosensitive, the role of biomechanical stimuli from beating CM as a regulatory mode of myocardial-microvascular crosstalk is relatively unexplored. Given that cardiac biomechanics are dramatically altered during disease, and disruption of myocardial-microvascular communication is a known driver of pathological remodelling, understanding the biomechanical context necessary for healthy myocardial-microvascular interaction is of high importance. The current gap in understanding can largely be attributed to technical limitations associated with reproducing dynamic physiological biomechanics in multicellular in vitro platforms, coupled with limited in vitro viability of primary cardiac tissue. However, differentiation of CM from human pluripotent stem cells (hPSC) has provided an unlimited source of human myocytes suitable for designing in vitro models. This technology is now converging with the diverse field of tissue engineering, which utilises in vitro techniques designed to enhance physiological relevance, such as biomimetic extracellular matrix (ECM) as 3D scaffolds, microfluidic perfusion of vascularised networks, and complex multicellular architectures generated via 3D bioprinting. These strategies are now allowing researchers to design in vitro platforms which emulate the cell composition, architectures, and biomechanics specific to the myocardial-microvascular microenvironment. Inclusion of physiological multicellularity and biomechanics may also induce a more mature phenotype in stem cell-derived CM, further enhancing their value. This review aims to highlight the importance of biomechanical stimuli as determinants of CM-EC crosstalk in cardiac health and disease, and to explore emerging tissue engineering and hPSC technologies which can recapitulate physiological dynamics to enhance the value of in vitro cardiac experimentation.

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

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.

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells.

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.

Integrative Bioinformatic Analyses of Global Transcriptome Data Decipher Novel Molecular Insights into Cardiac Anti-Fibrotic Therapies.

Integrative bioinformatics is an emerging field in the big data era, offering a steadily increasing number of algorithms and analysis tools. However, for researchers in experimental life sciences it is often difficult to follow and properly apply the bioinformatical methods in order to unravel the complexity and systemic effects of omics data. Here, we present an integrative bioinformatics pipeline to decipher crucial biological insights from global transcriptome profiling data to validate innovative therapeutics. It is available as a web application for an interactive and simplified analysis without the need for programming skills or deep bioinformatics background. The approach was applied to an ex vivo cardiac model treated with natural anti-fibrotic compounds and we obtained new mechanistic insights into their anti-fibrotic action and molecular interplay with miRNAs in cardiac fibrosis. Several gene pathways associated with proliferation, extracellular matrix processes and wound healing were altered, and we could identify micro (mi) RNA-21-5p and miRNA-223-3p as key molecular components related to the anti-fibrotic treatment. Importantly, our pipeline is not restricted to a specific cell type or disease and can be broadly applied to better understand the unprecedented level of complexity in big data research.

Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics.

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.

Heart Plasticity in Response to Pressure- and Volume-Overload: A Review of Findings in Compensated and Decompensated Phenotypes.

The adult human heart has an exceptional ability to alter its phenotype to adapt to changes in environmental demand. This response involves metabolic, mechanical, electrical, and structural alterations, and is known as cardiac plasticity. Understanding the drivers of cardiac plasticity is essential for development of therapeutic agents. This is particularly important in contemporary cardiology, which uses treatments with peripheral effects (e.g., on kidneys, adrenal glands). This review focuses on the effects of different hemodynamic loads on myocardial phenotype. We examine mechanical scenarios of pressure- and volume overload, from the initial insult, to compensated, and ultimately decompensated stage. We discuss how different hemodynamic conditions occur and are underlined by distinct phenotypic and molecular changes. We complete the review by exploring how current basic cardiac research should leverage available cardiac models to study mechanical load in its different presentations.

Remote Magnetic Nanoparticle Manipulation Enables the Dynamic Patterning of Cardiac Tissues.

The ability to manipulate cellular organization within soft materials has important potential in biomedicine and regenerative medicine; however, it often requires complex fabrication procedures. Here, a simple, cost-effective, and one-step approach that enables the control of cell orientation within 3D collagen hydrogels is developed to dynamically create various tailored microstructures of cardiac tissues. This is achieved by incorporating iron oxide nanoparticles into human cardiomyocytes and applying a short-term external magnetic field to orient the cells along the applied field to impart different shapes without any mechanical support. The patterned constructs are viable and functional, can be detected by T2 *-weighted magnetic resonance imaging, and induce no alteration to normal cardiac function after grafting onto rat hearts. This strategy paves the way to creating customized, macroscale, 3D tissue constructs with various cell-types for therapeutic and bioengineering applications, as well as providing powerful models for investigating tissue behavior.

Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.

Although past decades have witnessed significant reductions in mortality of heart failure together with advances in our understanding of its cellular, molecular, and whole-heart features, a lot of basic cardiac research still fails to translate into clinical practice. In this review we examine myocardial slices, a novel model in the translational arena. Myocardial slices are living ultra-thin sections of heart tissue. Slices maintain the myocardium's native function (contractility, electrophysiology) and structure (multicellularity, extracellular matrix) and can be prepared from animal and human tissue. The discussion begins with the history and current advances in the model, the different interlaboratory methods of preparation and their potential impact on results. We then contextualize slices' advantages and limitations by comparing it with other cardiac models. Recently, sophisticated methods have enabled slices to be cultured chronically in vitro while preserving the functional and structural phenotype. This is more timely now than ever where chronic physiologically relevant in vitro platforms for assessment of therapeutic strategies are urgently needed. We interrogate the technological developments that have permitted this, their limitations, and future directions. Finally, we look into the general obstacles faced by the translational field, and how implementation of research systems utilizing slices could help in resolving these.

Physiological Biomimetic Culture System for Pig and Human Heart Slices.

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to ß-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3-dimensional heart model.