Myocardial Tissue Slices: Organotypic Pseudo-2D Models for Cardiac Research & Development
2009; Future Medicine; Volume: 5; Issue: 5 Linguagem: Inglês
10.2217/fca.09.32
ISSN1744-8298
AutoresTeun P. de Boer, Patrizia Camelliti, Ursula Ravens, Peter Köhl,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoFuture CardiologyVol. 5, No. 5 EditorialFree AccessMyocardial tissue slices: organotypic pseudo-2D models for cardiac research & developmentTeun P de Boer*, Patrizia Camelliti*, Ursula Ravens & Peter KohlTeun P de Boer*† Author for correspondenceDepartment of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands and University of Oxford, Department of Physiology, Anatomy & Genetics, MEF Lab, Parks Road, Oxford OX1 3TP, UK. ; , Patrizia Camelliti*University of Oxford, Department of Physiology, Anatomy & Genetics, MEF Lab, Parks Road, Oxford OX1 3TP, UK. , Ursula RavensDepartment of Pharmacology & Toxicology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden 01307, Germany. & Peter KohlUniversity of Oxford, Department of Physiology, Anatomy & Genetics, MEF Lab, Parks Road, Oxford OX1 3TP, UK. Published Online:28 Aug 2009https://doi.org/10.2217/fca.09.32AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Cardiac function is studied using experimental and theoretical models that range from sub-cellular structures to the individual patient. Each level of structural complexity offers unique insights, shaped by a model-specific mix of utility and limitations. As a rule of thumb, the 'more reduced' models tend to benefit from better control over interventions and more direct observation of responses, aiding identification of causal chains of events. This is offset by the increasingly artificial environment in which mechanisms are studied, which may render observed chains of events nonrepresentative of behavior at more integrated levels of structural complexity. This has given (and will continue to give) rise to both false-positive and/or false-negative observations, which must be weeded out in the process of translation from reduced mechanisms to integrated responses.Vital cardiac tissue slices represent an interesting balance between structural complexity and ease of data interpretation. After more than three decades of intermittent efforts by a relatively small number of research groups to develop and validate this organotypic pseudo-2D model of myocardium, and more recently aided by significant improvements in sectioning techniques, cardiac slices appear ready now for more widespread use in cardiac research and development. This editorial will illustrate how living cardiac slices may help the study of integrated myocardial functions, such as impulse propagation, the relation between cardiac mechanics and electrophysiology, and responses to pharmacological interventions.Cardiac research modelsBasic research into cardiac function is largely reliant on nonhuman model systems, including intact organisms, isolated whole hearts, tissue segments such as coronary-perfused wedges or excised trabeculae, and cardiomyocytes studied either in isolation, after reconnection with other cells to form multicellular cultures, or subsequent to breaking them down into even smaller components such as ion channels or myofibrils. These experimental models – by definition simplified representations of reality – all have their strengths and weaknesses in the extent to which they are representative of myocardial function, and hence in their relevance for specific research questions [1].Given the important role of disturbances in cardiac electrophysiological behavior by producing cardiac arrest, the leading cause of morbidity and mortality in the developed world, it is important to point out that clinically relevant changes in heart rhythm are multicellular phenomena. Thus, in terms of model applicability, it is helpful to distinguish between single cells (for simplicity often treated as zero-dimensional [0D] model systems) and multicellular constructs (whether one-dimensional [1D] strands of cells (see [2]), 2D tissue-culture systems or 3D tissue). Computational modeling studies suggest that 2D models offer an excellent compromise between simplification and representative complexity for studies into cardiac electromechanical function [3].In particular, this is the case for thin sections of vital cardiac tissue. These could be referred to as 'pseudo-2D' because their thickness is one to two orders of magnitude smaller than the in-plane extent. In these slices, cells are exposed to a more realistic distribution of external electrophysiological source-sink relations than in culture models, and they experience a more physiological mechanical microenvironment. At the same time, the pseudo-2D nature of the preparation allows for optical tracking of key structural and functional properties of the tissue (e.g., using fluorescent reporter dyes or contact mapping). This is a key feature, absent from thicker 3D model systems, as even voltage-sensitive dye recordings from the epicardial surface suffer from significant depth-contributions to observed effects [4], making clear structure–function interrelation difficult.In principle, one can distinguish three kinds of pseudo-2D myocardial tissue models: cell cultures, cardiac tissue slices and thin layers of (usually epicardial) myocardium, generated in whole heart studies by chemical or physical destruction of deeper ventricular free-wall tissue [5] (note that atrial myocardium, often portrayed as a simple thin muscle membrane, tends to have a highly complex 3D structure in mammals). In contrast to cardiac cell cultures, 'reconstituted' from cells after isolation to assemble a pseudo-2D (and potentially structured [6]) representation of myocardial tissue, native myocardial tissue slices represent an organotypic model of the heart. Other organotypic multicellular models include ventricular wedges, papillary muscle, trabeculae and Purkinje fibers, but these are not pseudo-2D.Compared with the more established myocardial model systems, tissue slices have received relatively little attention in cardiac research, which is in stark contrast to their widespread use in neuroscience. This is probably caused by difficulties in preparing and preserving cardiac tissue sections, in particular ensuring proper oxygenation of the densely packed, metabolically active myocardium. By comparison, ventricular wedge preparations must be perfused via associated arterial vasculature, while the maintenance of papillary muscles, trabeculae and Purkinje fibers critically depends on their diameter, which at levels exceeding 400 µm usually restricts oxygen diffusion [7]. Such considerations are particularly important when attempting to maintain cardiac tissue for prolonged periods of time, for instance to allow gene transfection or cell biology studies.If implemented correctly, cardiac slices benefit from user-defined tissue thickness (not usually achieved in studies involving intracardiac tissue ablation). Typically vibratome-cut at a thickness of 250–350 µm, they can benefit from oxygen diffusion through both the bottom and top surfaces of the tissue. Even if attached to a rigid surface, whether a culture dish or contact electrode array for electrophysiological recordings, their thickness is well below the above mentioned effective limit for oxygen diffusion. If maintained in appropriate culture-like conditions, adult slices can be electromechanically active for up to 1 week, although a thorough quantitative assessment of functionality over time is still largely missing.A further advantage of the technique is that multiple slices (often ten or more) can be obtained from one heart, depending on cutting angle, species and developmental stage. Given the delicacy of the slice preparation, injury due to cutting must be minimized, as otherwise a relatively large portion of the tissue may become unsuitable for experimentation [8]. With the availability of vibratomes that combine very low blade advancement speeds (in the order of mm × min-1) with submicrometer out-of-plane blade deviations, this has become more feasible. Another essential 'trick' is to minimize tissue contraction and energy consumption of the myocardium while slices are being prepared. This can be achieved by cutting in the presence of excitation–contraction uncouplers (such as 2,3-butanedione monoxime or blebbistatin) and/or high potassium concentrations to depolarize cells.Short history of cardiac slice workEarly studies using cardiac slices focused on biochemical aspects of tissue function, demonstrating the usefulness of thin myocardial sections, initially over 24-h time periods (for review see [9]). The initial application of this technique in 1976 was the study by Claycomb et al., who demonstrated that DNA synthesis in neonatal rat-heart slices and cardiomyocyte differentiation may be controlled by adrenergic innervation, acting through cAMP [10]. Pharmacological research was among the 'early adopters' of cardiac slice work in the 1980s. Thus, the toxicity of substances such as epirubicin, doxorubicin and mitoxantrone was assessed by measuring oxygen uptake and ATP production in slices [11]. At the same time, initial studies relevant for cardiac electrophysiology included the assessment, by radioactive Rb+ uptake assay, of Na+/K+-ATPase activity, which was found to be higher in adult than in young rat hearts [12].Living cardiac tissue slices were also employed successfully as model systems to investigate ischemic preconditioning [13]. Application of the cardiac slice model to metabolism research was pioneered by Suga, Takaki and colleagues. In an extensive series of experimental studies, they used mechanically unloaded tissue slices to identify key contributors to O2 consumption of normal and pathologically disturbed myocardium [14–19], and highlighted that tissue sectioned in parallel to the epicardial surface yields particularly useful samples if one is interested in extended areas of in-plane myocyte populations [14].In terms of direct cell–electrophysiological investigations, Burnashev et al. were first to demonstrate, in 1990, that cardiac slices can be used – analogous to brain slices – in patch-clamp experiments [20]. In this seminal paper, cells within neonatal rat slices were demonstrated to be excitable (although with reduced resting membrane potentials) and recorded ion currents were compatible with those known from other cardiac tissue preparations at the time.In spite of these encouraging observations, cardiac slices were scarcely employed in subsequent (electro-)physiological studies for another decade. In 2003, interest in the cardiac slice model system resurfaced, when it was demonstrated that neonatal rat heart slices possess more representative electrophysiological characteristics than cultured monolayers or tissue-engineered myocardial constructs [21]. Subsequently, building on prior work by Takaki and colleagues [18], a substantial body of work from Hescheler, Pillekamp and colleagues, demonstrated that slices can be prepared not only from rat, but also from murine embryonic, neonatal and adult heart tissue, allowing research into cardiac phenotypes of transgenic mice [22–26]. These investigators also developed techniques to maintain slices in culture conditions for more extended periods of time (generally 3–4 days, but in certain cases even up to 1 week [25]). Recordings with sharp microelectrodes confirmed preserved viability and electrophysiological phenotype of cardiomyocytes in the slices, and demonstrated that the model can be used to test the effects of drugs on the cardiac action potential, for example, in the context of adrenergic and muscarinic stimulation, as well as during Na+- and K+-channel block. Furthermore, using slices cut in a plane orthogonal to the heart's long axis, ventricular tissue rings were obtained, whose contractile behavior could be monitored using a pair of small hooks and a force transducer [24].As cardiac slices partially preserve electrotonic and paracrine cell–cell interactions, they constitute an attractive model for cardiac cell biology research. This was elegantly demonstrated by Stuckmann et al., who used cultured cardiac tissue slices to illustrate that the epicardium of embryonic chick heart secretes erythropoietin and retinoic acid, which are necessary for proliferation of cardiomyocytes [27]. A special case of cell–cell interaction is that between native myocardium and cells transplanted into the heart, for example as part of stem cell research. In whole animals or hearts, it is difficult to directly observe these interactions, while they can be tracked visually in tissue slices. A number of studies have used human, murine or rat cardiac slices in this context, investigating interactions between myocardium and either human embryonic stem cells or bone marrow cells [24,28,29].Future potentialUp to now, much of the cardiac tissue slice work has focused on validation of the technique. This is of course necessary for any new experimental model system, but as the slice preparation is used increasingly now for research and development, the focus is shifting from establishing 'gold standards' to their broader application in pathophysiological, pharmacological and cell-biological research. This benefits from the combined potential of noninvasive imaging and computational reconstruction of structure and function for extended cardiac tissue regions [30,31]. The pseudo-2D nature of slices makes them a particularly suitable target for this approach, where computational models can provide more or less specific reconstructions of individual sections, which may be used to reproduce observations, interpret data, aid hypothesis formation and plan further interventions [32].In terms of integrated myocardial function, tissue slices have the potential of serving as a uniquely controlled electromechanical system, in which the bidirectional cross-talk between electrical excitation and mechanical activity can be quantitatively controlled and/or assessed [33]. A key technological development required for this is the establishment of approaches to precisely control the mechanical environment of tissue sections, whether cut across the short axis [25] or in the plane of the ventricular myocardium [14]. Obvious targets for this work include basic research into regional, developmental, species- or disease-related differences in tissue function, as well as pharmacological compound screening [34].Another interesting direction is the experimental assessment of specific contributions by nonmyocytes to cardiac function. Illustrative examples range from the experimental simulation of higher-level functionality, such as direct observation of responses to epicardial ganglia stimulation [35], to quantitative elucidation of contributions to cardiac function by epithelial or connective tissue cells [36]. As demonstrated by the effects of sphingosine-1-phosphate, effects on cardiomyocyte electrophysiology that are mediated via nonmyocytes may be significant [37]. Cardiac slices can effectively be transfected with viruses [38]. Using cell-type specific promoters, it would be possible to affect specific cardiac cell populations and assess their contribution to cardiac tissue function, for example via heterotypic cell–cell interactions [39], whether biophysical (e.g., through gap junctional or mechanical coupling) or biochemical in nature (such as paracrine signaling).In the context of development of biological pacemakers or myocardial repair using stem cell-derived cardiomyocytes [28,40–42], myocardial slices offer an exciting model system as they allow the observation of tissue integration and electrotonic interactions between grafted cells and native myocardium. This is particularly difficult to study in other organotypic model systems that are not pseudo-2D. Finally, it will be interesting to see whether putative 'resident' cardiac progenitor cells can be directly identified in cardiac slices [43,44]. If this is possible, their in situ phenotype could be studied for the first time, and compared or contrasted to myocardial repair mechanisms involving intrinsic cardiomyocyte proliferation [45], which would help to tap into the potential of regenerative cardiology.Financial & competing interests disclosureTeun de Boer is supported by the Ter Meulen Fund of the Royal Netherlands Academy of Arts and Sciences and the Interuniversity Cardiology Institute of the Netherlands (20082355). Patrizia Camelliti is a Junior Research Fellow of Christ Church College Oxford, UK. Peter Kohl is a Senior Research Fellow of the British Heart Foundation. Work in the Oxford and Dresden laboratories is supported by the BHF, the EU NormaCOR project and the Oxford EP Abraham Cephalosporin fund. 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Patrizia Camelliti is a Junior Research Fellow of Christ Church College Oxford, UK. Peter Kohl is a Senior Research Fellow of the British Heart Foundation. Work in the Oxford and Dresden laboratories is supported by the BHF, the EU NormaCOR project and the Oxford EP Abraham Cephalosporin fund. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download
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