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Cardiac tissue slices: preparation, handling, and successful optical mapping.

ABSTRACT: Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained well-aligned in-slice myocardial cell strands ("fibers") in subepicardial and midmyocardial sections. Cut with a high-precision slow-advancing microtome at a thickness of 350 to 400 ?m, tissue slices preserved essential action potential (AP) properties of the precutting Langendorff-perfused heart. We identified the need for a postcutting recovery period of 36 min (guinea pig) and 63 min (rabbit) to reach 97.5% of final steady-state values for AP duration (APD) (identified by exponential fitting). There was no significant difference between the postcutting recovery dynamics in slices obtained using 2,3-butanedione 2-monoxime or blebistatin as electromechanical uncouplers during the cutting process. A rapid increase in APD, seen after cutting, was caused by exposure to ice-cold solution during the slicing procedure, not by tissue injury, differences in uncouplers, or pH-buffers (bicarbonate; HEPES). To characterize intrinsic patterns of CaT, AP, and conduction, a combination of multipoint and field stimulation should be used to avoid misinterpretation based on source-sink effects. In summary, we describe in detail the preparation, mapping, and data analysis approaches for reproducible cardiac tissue slice-based investigations into AP and CaT dynamics.

SUBMITTER: Wang K

PROVIDER: S-EPMC4551126 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Cardiac tissue slices: preparation, handling, and successful optical mapping.

Wang Ken K Lee Peter P Mirams Gary R GR Sarathchandra Padmini P Borg Thomas K TK Gavaghan David J DJ Kohl Peter P Bollensdorff Christian C

American journal of physiology. Heart and circulatory physiology 20150116 9

Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained well-aligned in-slice myocardial cell strands ("fibers") in subepicardial ...[more]

PMID: 25595366

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Project description:BackgroundSimultaneous optical mapping of multiple electrophysiologically relevant parameters in living myocardium is desirable for integrative exploration of mechanisms underlying heart rhythm generation under normal and pathophysiologic conditions. Current multiparametric methods are technically challenging, usually involving multiple sensors and moving parts, which contributes to high logistic and economic thresholds that prevent easy application of the technique.ObjectiveThe purpose of this study was to develop a simple, affordable, and effective method for spatially resolved, continuous, simultaneous, and multiparametric optical mapping of the heart, using a single camera.MethodsWe present a new method to simultaneously monitor multiple parameters using inexpensive off-the-shelf electronic components and no moving parts. The system comprises a single camera, commercially available optical filters, and light-emitting diodes (LEDs), integrated via microcontroller-based electronics for frame-accurate illumination of the tissue. For proof of principle, we illustrate measurement of four parameters, suitable for ratiometric mapping of membrane potential (di-4-ANBDQPQ) and intracellular free calcium (fura-2), in an isolated Langendorff-perfused rat heart during sinus rhythm and ectopy, induced by local electrical or mechanical stimulation.ResultsThe pilot application demonstrates suitability of this imaging approach for heart rhythm research in the isolated heart. In addition, locally induced excitation, whether stimulated electrically or mechanically, gives rise to similar ventricular propagation patterns.ConclusionCombining an affordable camera with suitable optical filters and microprocessor-controlled LEDs, single-sensor multiparametric optical mapping can be practically implemented in a simple yet powerful configuration and applied to heart rhythm research. The moderate system complexity and component cost is destined to lower the threshold to broader application of functional imaging and to ease implementation of more complex optical mapping approaches, such as multiparametric panoramic imaging. A proof-of-principle application confirmed that although electrically and mechanically induced excitation occur by different mechanisms, their electrophysiologic consequences downstream from the point of activation are not dissimilar.

Project description:Background:Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol. Methods:Four single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition. Results:On average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC-MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%. Conclusions:In essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research.

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Cardiac tissue slices: preparation, handling, and successful optical mapping.

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Cardiac tissue slices: preparation, handling, and successful optical mapping.

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