Preparation of viable adult ventricular myocardial slices from large and small mammals

Abstract

This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-μm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation–contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in 4 h.

Introduction

Myocardial slices are 100- to 400-μm-thick slices of living adult ventricular myocardium, prepared using a high-precision vibratome1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 (Fig. 1). They retain the multicellularity, complex architecture and physiology of adult cardiac tissue, and their thinness allows the diffusion of oxygen and other metabolic substrates into their innermost cells, maintaining viability in the absence of coronary perfusion in vitro2. Slices have been produced from a number of small and large mammals, including mice3,15,16,21, rats4,8,9,10,11,12,13,14,17,18,19,20,22,23,24, guinea pigs5,13,14, rabbits5, dogs3,6,25, pigs and human tissue biopsies2,3,6,7,25. Multiple slices can be produced from each heart or biopsy, producing multiple tissue samples for experimentation and thus reducing the number of animals required for studies.

Figure 1: Representative images of a rat myocardial slice.
figure1

(a) Original myocardial slice. Photograph taken directly after slicing. (b) Aligned area. Photograph taken after the aligned area of the slice had been visualized under a macroscope and cut out using a scalpel (for further information, see Fig. 4).

Development and applications

Myocardial slices were first used as far back as the 1970s; however, in recent years, they have been increasingly used. Early studies investigated a number of aspects of cardiac function, including biochemistry8, metabolism9,18,19,20,21, electrophysiology10,11 and pharmacological cardiotoxicity4. These studies demonstrated that myocardial slices had wide-ranging applications, but it was not until the early 2000s that their uses became more widely recognized. During this period, myocardial slices were found to have more representative electrophysiological properties than other cardiac models12, and other functional parameters, including myocardial slice contractility15, were measured for the first time. Importantly, techniques to culture slices for up to 1 week were developed15, and more recent use of an air–liquid interface has demonstrated that slices can be maintained for up to 1 month in vitro2.

As myocardial slices provide a clinically relevant and representative human model system in vitro, they are particularly useful for translational studies7. A substantial amount of work has demonstrated that myocardial slices can be used for pharmacological testing and in vitro drug safety screening6,7,13,14,22. They have also been used to study the integrative capacity of a number of cell types as part of the development of cell-replacement strategies16,26,27. More recently, slices have been used to test the clinical applicability of conductive polymer patches17.

Despite the number of studies published, the protocols used for their preparation have varied greatly. There are a number of critical steps that must be carefully controlled to produce myocardial slices with high viability, and by optimizing the method used, their contractility can be significantly improved (Supplementary Fig. 1), in addition to improving other structural and functional parameters. A critical step, that is often overlooked, is the identification of myocardial fiber direction and fiber alignment within the tissue block. In a large number of papers published, hearts are sliced along the short axis. As myocardial fibers run parallel to the epicardium, this results in large numbers of fibers being transected and substantial tissue damage. Other critical steps include carefully maintaining the correct temperature, the use of an excitation–contraction uncoupler and optimal vibratome settings, and correctly handling the myocardial slices. These factors and the techniques used to control them are discussed in depth in this protocol.

Comparison with other methods

Cardiovascular research has been conducted using a variety of model systems. Isolated adult cardiomyocytes. Isolated adult cardiomyocytes have been used widely. During isolation, cardiomyocytes lose connections with other cardiac cells and the extracellular matrix (ECM), both of which are known to modulate myocardial function28,29, and the outcomes of different isolation protocols are known to vary noticeably30. Isolated cardiomyocytes also undergo substantial remodeling within hours31,32, with substantial implications for the relevance of chronic studies32.

Whole hearts. Whole-heart studies avoid the issues associated with cell isolation. However, it is extremely difficult to use isolated whole hearts for more than a few hours, and investigations therefore require large numbers of animals. Their complexity also makes it difficult to study events at the cellular level, and such studies require specific and expensive setups. Other intermediate preparations exist, including cardiac wedges and papillary muscles, but these often become ischemic ex vivo33, particularly when prepared from large mammals.

Stem cell-derived cardiomyocytes. In recent years, human pluripotent stem cell-derived cardiomyocytes have been used. Although these cells have the advantage of re-creating pathological phenotypes in vitro34, they also have a relatively naive phenotype35, with many aspects, including morphology, metabolism36, electrophysiology and Ca2+ handling37, regarded as particularly immature.

Myocardial slices. Myocardial slices bridge the gap between isolated cardiomyocytes and whole-heart studies. The multicellular composition and noncellular architecture of the myocardium are almost fully maintained during slicing, resulting in a model with the correct cell ratios and cell–cell/cell–ECM interactions. This allows all cardiac cell populations, including fibroblasts and endothelial cells, to be studied alongside one another, which is required to improve our understanding of cardiac physiology and pathology38. In addition, chronic culture is facilitated by the ability of oxygen and metabolic substrates to diffuse to all of the cells within the preparation, which is not possible or is very difficult to achieve with other cardiac multicellular preparations2.

In summary, myocardial slices provide substantial advantages over other commonly used cardiac preparations and can be produced from a large variety of species. The protocols used to date vary greatly, and there are a number of critical steps that must be addressed in order to produce myocardial slices with high viability and preserve structure and function. This protocol describes a robust and reproducible technique for producing myocardial slices and aims to standardize the methodology by which they are produced, reducing the variability in the quality of myocardial slices produced in different laboratories.

Experimental design

In addition to the PROCEDURE below, Supplementary Videos 1, 2, 3, 4, 5, 6, 7 illustrate various parts of the procedure.

When designing experiments using myocardial slices, the number of slices that can be produced must be considered. Smaller mammals, including mice and rats, can yield 3–6 slices per heart, whereas large mammals, including canine or human biopsies, can yield 20–30 slices per tissue block, and several tissue blocks can be prepared from the left ventricle. As multiple slices can be produced from each animal, many experiments can be conducted, ultimately reducing the number of animals used in studies. The age and phenotype of the animal can also contribute to the thickness of the ventricular wall and these should be considered. Another important consideration is the region of the ventricular wall from which the slice has been derived. The endocardium, midmyocardium and epicardium have differing electrophysiological and contractile properties39, and also display varied responses to pharmacological stimuli40. This can be overcome by using slices from specific regions or by randomizing slices and increasing the number of repeats. The timings of the experiments should also be considered carefully. Slices should be used as swiftly as possible, but they can be kept in ice-cold, oxygenated Tyrode's solution for up to 4 h before being used. Exceeding this time period may result in degradation of the myocardial slices.

Limitations and expertise required

Although myocardial slices have a number of benefits over other cardiac preparations, they also have their limitations. The biggest determinant of myocardial slice quality is the method by which the cardiac tissue block is dissected and prepared. Owing to the manual nature of this process, there is a relatively steep learning curve, and a number of weeks and animals (10–15 hearts) should be dedicated to practicing before high-quality tissue blocks can be consistently prepared. The quality of the tissue block preparation directly correlates with the quality of data that can be collected (see 'Anticipated Results—Viability'). Access to fresh cardiac tissue/animal facilities, and the expense of equipment (see 'Equipment') may also be prohibitive for some laboratories.

Materials

REAGENTS

  • Fresh mammalian cardiac tissue. Rodent, human heart failure, porcine and canine cardiac tissues have been successfully used to produce myocardial slices

    Caution

    All animal experiments must comply with institutional and national regulations. Our use of living cardiac tissue was approved by Imperial College London. The procedures that we describe here were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). Human samples were provided by the NIHR Cardiovascular Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The inferior thirds of explanted hearts were obtained from end-stage heart failure patients undergoing cardiac transplantation. The results shown here were obtained from samples obtained while conforming to the principles outlined in the Declaration of Helsinki, and the investigation was approved by a UK institutional ethics committee (ref: 09/H0504/104+5 CBRU Biobank)

    Caution

    For the results shown here, informed consent was obtained from each patient involved in this study. Porcine hearts were obtained from the Translational Biomedical Research Centre, University of Bristol. The procedures were approved by the University of Bristol. 18-month-old healthy beagle dog hearts were donated by GlaxoSmithKline, after the animals were necessarily sacrificed at the end of pharmacology/toxicology studies. Only control animals were used.

  • 2,3-Butanedione monoxime (BDM; Acros Organics, cat. no. AC150375000)

  • Agarose (Sigma-Aldrich, cat. no. A9539)

  • BSA (Sigma-Aldrich, cat. no. A7030)

  • Calcium chloride (1 M solution; VWR, cat. no. E506-500ML)

  • D-Glucose, anhydrous (Fisher Chemical, cat. no. D16-1)

  • Sodium pentobarbital, 200 mg/ml solution (Sigma-Aldrich, cat. no. P3761)

  • Ethanol (VWR, cat. no. 64-17-5)

  • FBS (Gibco, cat. no. 10270)

  • Formaldehyde solution (Sigma-Aldrich, cat. no. F8775)

  • Heparin sodium, 1,000 IU/ml (Fannin, cat. no. PL 20417/0109)

  • HEPES (Sigma-Aldrich, cat. no. H3375)

  • Horse serum (heat inactivated; Gibco, cat. no. 26050088)

  • ITS liquid media supplement (100×; Sigma-Aldrich, cat. no. I3146)

  • Magnesium chloride hexahydrate (VWR, cat. no. 97061-356)

  • Magnesium sulfate (Sigma-Aldrich, cat. no. M2643)

  • Medium-199 (Sigma-Aldrich, cat. no. M5430)

  • Penicillin–streptomycin (Sigma-Aldrich, cat. no. P4333)

  • Potassium chloride (Sigma-Aldrich, cat. no. P9541)

  • Phosphate-buffered saline (Oxoid, cat. no. BR0014G)

  • Sodium chloride (VWR, cat. no. 470302-522)

  • Sodium bicarbonate (Sigma-Aldrich, cat. no. S5761)

  • Sodium hydroxide (Fisher Chemical, cat. no. S/4880/60)

  • Sodium phosphate monobasic monohydrate (Sigma-Aldrich, cat. no. S3522)

  • Triton X-100 (Sigma-Aldrich, cat. no. X100)

  • 100% Medical-grade oxygen gas (BOC, cat. no. 101)

  • Fluo-4 AM (Thermo Fisher Scientific, cat. no. F14201)

  • Pluronic F-127, 10% solution in water (Thermo Fisher Scientific, cat. no. P6866)

EQUIPMENT

  • 150 × 15-mm Petri dish (Falcon, cat. no. 351058)

  • 28-mm Syringe filter (0.2 micron; SFCA+PF membrane; Corning, cat. no. 431219); other 0.2-micron filters are appropriate

  • 40-μm Cell strainer (Corning, cat. no. 352340)

  • 6-Well plate (customized with holes in the base of each well—see Fig. 2; Corning, cat. no. CLS3506)

    Figure 2: Apparatus required for the production of myocardial slices.
    figure2

    (a) Customized 6-well dish with holes drilled into the base of each well. (b) Specimen holder covered with 4% agarose. Both the base and the back of the holder should be completely covered. (c) Myocardial slice holding bath (without lid). It is filled with cold slicing solution and bubbled with filtered 100% O2. The bath is surrounded with ice to maintain a temperature of 4 °C. (d) Suggested dissection area setup. (e, right) Vibratome with a vibratome bath in place. The outer section of the bath is filled with ice and the inner section is filled with cold slicing solution, bubbled with filtered 100% O2. (Left) Myocardial slice holding bath covered with lid. The holding bath is placed on ice.

  • Ceramic vibratome blades (Campden Instruments, cat. no. 7550-1-C); stainless-steel blades can be used for only a single use, as they wear out quickly

  • Disposable sterile scalpel–surgical steel blades (Swann-Morton, cat. no. 05XX)

  • Electronic balance

  • Flat spatula

  • Gauze

  • Glass beaker (1 liter/0.25 liter; Duran, cat. no. Z231894/Z231843); other glass beakers are appropriate

  • Glass Petri dish, 100 × 20-mm, soda-lime (VWR, cat. no. CLS70165101); other glass Petri dishes are appropriate

  • Graefe forceps (0.8 × 0.7 mm; Fine Science Tools, cat. no. 11050-10); other small forceps are appropriate

  • Histoacryl surgical glue (Braun, cat. no. 1050052)

  • Incubator; 37 °C and 5% CO2 required

  • Laboratory bottles (1 liter/0.5 liter)—high-density polyethylene, wide mouth (Nalgene, cat. no. 10547341/10633141); other laboratory bottles are appropriate

  • Laminar flow cabinet with UV light

  • Large glass dish ± lid (Fig. 3c)

    Figure 3: Preparation of a small mammalian (rat) left ventricular tissue block.
    figure3

    These images show dissection with a dry heart to aid visualization of the method. However, always dissect tissue in a Petri dish with the tissue submerged in cold slicing solution. For further details, please see Step 2A. (a) Using a surgical scalpel/razor blade, dissect off the lungs and other tissues. (b) Visualize the atria and make an incision slightly inferiorly, through the base of the ventricle. (c) Visualize the right ventricle, which has a thinner wall and is more cresentic in shape. Hold the free wall of the right ventricle with tweezers. (d) Using microscissors, cut along the right ventricular–septal junction on one side. Cut toward the apex. (e) Lift the cut border of the right ventricle away from the heart. (f) Cut along the other ventricular–septal junction to remove the right ventricle. (g) Place one edge of the microscissors inside the left ventricle, and cut along the septum toward the apex. (h) The ventricle should now open. (i) Make small incisions along the borders of the tissue block to allow the ventricle to fully flatten. Fibrotic tissue and large papillary muscles will also need to be cut. (j) Turn the tissue block over so that the epicardium is facing upward. Visualize the edges of the septum at the lateral edges of the tissue block. Remove them with a surgical scalpel/razor blade. (k) Flat left ventricular tissue block. (l) The tissue block is 20 × 12 mm. Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).

  • Magnetic stirring bars

  • MS-H-S circular-top analog hotplate stirrer (Scilogex, cat. no. 811121029999); other hotplate stirrers are appropriate

  • Noyes spring scissors (14-mm blade; Fine Science Tools, cat. no. 15012-12); other small dissecting scissors are appropriate

  • Parafilm 'M' laboratory film (Bemis, cat. no. PM992)

  • Pasteur pipette (sterile; VWR, cat. no. 612-1685)

  • pH meter

  • Polythene bag

  • Rapid-flow sterile-disposable filter unit (PES membrane—0.2 μm; Nalgene, cat. no. 167-0045); other 0.2-μm filters are appropriate

  • Razor blade (Fisher Scientific, cat. no. 11904325)

  • Scissors, Iris straight, 11.5 cm (4.5-inch) (Rocialle, cat. no. RSPU100-093); other surgical scissors are appropriate

  • Self-seal sterilization pouch (Westfield Medical, cat. no. WPSS135255)

  • Stainless-steel plain washer (RS, cat. no. 189-636)

  • Sterilin polystyrene containers (Thermo Scientific, cat. no.11319143)

  • Transwell polyester membrane cell culture inserts (Corning, cat. no. CLS3450)

  • Vibrating microtome, 7000smz-2 (Campden Instruments, cat. no. 7000smz-2); alternative vibratomes can be used

    Caution

    Alternative vibratomes must be able to check and calibrate z-axis error, be capable of cooling the slicing solution to < 8 °C (ideally the solution should be kept at 4 °C) and be capable of advancing at slow speeds (0.03 mm/s).

  • Vibrating microtome bath (Campden Instruments, cat. no. 7000-3-1A)

REAGENT SETUP

Preparation of 1 liter of slicing solution (Tyrode's solution + 30 mM BDM)

  • Cardiac tissue is sliced in Tyrode's solution with the addition of an excitation–contraction uncoupler (BDM). To create this solution, add the following to 1 liter of dH2O while mixing using a magnetic stirrer: 3.00 g of BDM (30 mM), 8.18 g of sodium chloride (140 mM), 0.45 g of potassium chloride (6 mM), 1.86 g of glucose (10 mM), 2.38 g of HEPES (10 mM), 1 ml of 1 M magnesium chloride solution (1 mM) and 1.8 ml of 1 M calcium chloride solution (1.8 mM; see also Table 1 for details of the components required). Wait for the reagents to dissolve and then measure the pH. Add drops of 2 M sodium hydroxide solution, until the solution is at a pH of 7.40. Filter the solution with a 0.2-micron-pore filter, and transfer it to the previously sterilized 1-liter container. Cool the solution to 4 °C

    Table 1 Preparation of 1 liter of slicing solution (Tyrode's solution + 30 mM BDM).

    Caution

    For large mammalian hearts, >1 liter may be required.

    Critical

    The solution must be freshly prepared on the day of the experiment.

    Critical

    Prepare the slicing solution first. This must be cooled to 4 °C before starting the PROCEDURE.

    Critical

    If myocardial slices are to be cultured, all solutions must be filter-sterilized before starting the PROCEDURE.

Preparation of hot and cold heparinized slicing solution

  • Take two 60-ml containers, and fill them with slicing solution. Add 100 IU of heparin sodium (1,000 IU/ml) to each container (100 μl in 50 ml of slicing solution). Cool one container to 4 °C and warm the other to 37 °C.

    Critical

    The solution must be freshly prepared on the day of the experiment.

Preparation of 1 liter of cardioplegia solution

  • This is required only for the cardioplegic arrest of large mammalian hearts in situ, or the transportation of cardiac tissue specimens. Alternatively, premade clinical-grade cardioplegia solution can be purchased and used.

  • To make the solution, add the following to 1 liter of dH2O while mixing using a magnetic stirrer: 0.99 g of glucose (5.50 mM), 0.13 g of magnesium sulfate (0.50 mM), 1.79 g of potassium chloride (24.00 mM), 1.68 g of sodium bicarbonate (20.00 mM), 6.37 g of sodium chloride (109.00 mM), 0.12 g of sodium phosphate monobasic monohydrate (0.90 mM) and 1.8 ml of 1 M calcium chloride solution (1.8 mM; see also Table 2 for details of the components required). Wait for the reagents to dissolve, and then measure the pH. Add drops of 2 M sodium hydroxide solution until the solution is at a pH of 7.40. Filter the solution with a 0.2-micron-pore filter, and transfer it to the previously sterilized 1-liter container. Cool the solution to 4 °C.

    Table 2 Preparation of 1 liter of cardioplegia solution.

    Critical

    The solution must be freshly prepared on the day of the experiment.

Preparation of 4% agarose

  • Dissolve 4% agarose (4 g in 100 ml) in dH2O. Heat the solution and stir it until the agarose has melted (80 °C). Pour the hot agarose solution into large glass Petri dishes to a height of 2–3 mm. Leave the solution to cool on a flat surface. Once the agarose has cooled and solidified, cover it with Parafilm, and keep it at 4 °C for up to 1 month.

    Critical

    It is critical that the agarose be as flat as possible.

EQUIPMENT SETUP

Sterilization of tools and equipment

  • This is required only for chronic studies that require myocardial slice culture.

  • To preserve sterility and prevent infections during culture, it is important to keep the vibratome and tools as clean as possible. Place all the tools, including small tweezers, microdissecting spring scissors, surgical scissors and the vibratome specimen holder in a self-sealing sterilization pouch and autoclave them. Transfer the nonautoclavable pieces of equipment (1-liter container + lid, vibratome bath, glass dish ± lid, and customized 6-well plate) to a sterile laminar flow hood, spray them with 70% ethanol and expose them to UV light for 1 hour.

    Critical

    Sterilization takes 1 h. Sterilize the equipment before setting up other pieces of equipment.

Vibratome calibration

  • Ensure that the vibratome is clean by spraying it with 70% ethanol and wiping it dry. To work in complete sterility, place the vibratome in a sterile laminar flow hood. Mount a new ceramic blade. It is critical to adjust the blade alignment until the z-axis error is <1.0 μm. If it is not possible to achieve a z-axis error <1.0 μm, remove the blade and wipe it clean with ethanol before remounting. If achieving a z-axis error <1.0 μm is still not possible, the blade may be damaged and must be replaced. Once you are satisfied with the blade alignment, mount the blade protector to avoid injuries. See Troubleshooting section.

Mounting of agarose on a specimen holder

  • Remove the 4% agarose from the refrigerator. Cut a 2.5-cm2 piece of agarose. Place a large drop of Histoacryl glue in the center of the base of the specimen holder. Using tweezers, carefully pick up the square of agarose and place it on the base of the specimen holder. Wait 30 s for the agarose to attach. Cut another 2.5-cm2 piece of agarose. Apply Histoacryl glue to the back of the specimen holder and place the second square here. Wait 30 s for it to attach. The agarose-covered specimen holder should be as shown in Figure 2b. Place the specimen holder in a polythene bag and keep it at 4 °C. The specimen holder should be prepared on the day of the experiment.

    Critical

    Make sure that the agarose is completely flat, as the tissue will be mounted on top. Uneven agarose can result in poor alignment of myocardial fibers and subsequent tissue damage.

Preparation of the tissue dissection area

  • The tissue dissection area must be prepared in advance to minimize delays. Choose an area of the bench or a sterile laminar flow hood close to the vibratome. The following will be required for dissection: 15-cm Petri dish, sterile surgical scalpel/razor blade, small tweezers, microscissors, Histoacryl surgical glue, surgical scissors (large mammal only) and a specimen holder with agarose. Organize the dissection area as shown in Figure 2d.

Preparation of vibratome and holding bath

  • Mount the vibratome bath on the vibratome. Connect the waste tubing to a 500-ml container. Fill the outer part of the vibratome bath with ice, or set the bath cooling system to 4 °C. Remove the slicing solution from the refrigerator and pour it into the inner part of the vibratome bath. Bubble the solution with filtered 100% oxygen, making sure that the tip of the tubing has been sterilized. The myocardial slice holding bath, which can hold slices before experiments, should also be prepared. Place the glass dish on ice. The 6-well dish with holes (Fig. 2a) should be placed inside, and a cell strainer should be inserted into each well. Fill the dish with enough cold slicing solution to cover all the wells. Oxygenate the solution, and cover the container with a lid. The setup should be as shown in Figure 2c,e.

Procedure

Excision of mammalian heart

Caution

Experiments involving live animals must conform to relevant institutional and national guidelines.

  1. 1

    Follow option A if using a small mammalian heart (for example, a rodent heart) or option B, C or D if using a large mammalian heart (for example, a human, porcine or canine heart, respectively).

    1. A

      Excision of a small mammalian heart (rodents) • TIMING 5 min

      1. i

        Sedate the rodent with isoflurane (4% isoflurane and 4 liters/min oxygen).

      2. ii

        Sacrifice the rodent following institutional and national guidelines. Our laboratory performs cervical dislocation, and death is confirmed by dissection of the carotid arteries.

      3. iii

        Place the rodent in the supine position.

      4. iv

        Locate the sternum. Hold the overlying skin with tweezers, and make a small incision distally.

      5. v

        Once the sternum is exposed, hold it with tweezers. Make an incision along the entirety of the costal margin using scissors.

      6. vi

        Once the diaphragm has been exposed, make another incision along the costal junction to remove the diaphragm and allow access to the mediastinum.

      7. vii

        Hold the lung parenchyma with your tweezers (pulling the heart and lungs away from the posterior wall of the thorax) and gently move your scissors behind the heart and lungs. Cut through the aorta and venae cavae.

      8. viii

        Remove the heart and lungs from the animal by pulling the lungs. Never directly hold the heart with the tweezers.

        Critical Step

        The excision of a small mammalian heart should take no longer than 30 s after the sacrifice of the animal. The heart should still be vigorously beating after excision.

        Troubleshooting

      9. ix

        Rapidly transfer the heart and lungs to your warm (37 °C) heparinized slicing solution.

      10. x

        With two fingers, gently and repeatedly compress the heart for 5–10 s to eject the remaining blood from the ventricles.

      11. xi

        Transfer the heart and lungs to the cold (4 °C) heparinized solution, and gently compress the heart to wash the remaining blood for 5 s. Keep the cold solution on ice, and quickly transfer it to the dissection area.

      Critical Step

      The following procedure minimizes the pain, suffering and distress of rodents, while allowing the rapid excision of the rodent heart after sacrifice. This is critical to obtain highly viable slices.

    2. B

      Excision of a large mammalian heart (human) • TIMING excision, 5 min; transport to the laboratory, 1–4 h

      1. i

        Perfuse the heart with cardioplegia before explantation.

      2. ii

        After explantation and dissection of the failing heart, place the specimen in a 1-liter container with freshly made, cold (4 °C) cardioplegia solution.

      3. iii

        Place the container on ice, and transfer it to the laboratory.

        Pause point

        The heart remains viable for further studies if it is kept on ice for up to 4 h.

    3. C

      Excision of a large mammalian heart (Porcine) • TIMING 5 min

      1. i

        Carry out the procedure in a controlled manner in a theater, under general anesthesia, with ongoing full monitoring and with antibiotic cover.

      2. ii

        Perform sternotomy, and isolate the aorta and venae cavae.

      3. iii

        Administer heparin (300 IU/Kg), and allow it to circulate for 2 min.

      4. iv

        Clamp the aorta and venae cavae.

      5. v

        Deliver 2 liters of cold cardioplegia into the aortic route at a delivery pressure of 300 mmHg. Open the inferior vena cava at the junction of the right atrium to vent excess cardioplegia from the coronary sinus.

      6. vi

        Cool the ventricular surface with cold saline/ice.

      7. vii

        Carefully remove the heart by transecting the venae cavae and pulmonary veins.

      8. viii

        Place the whole heart or biopsies in sterile containers with cold cardioplegia solution, and put them on ice.

        Pause point

        The heart remains viable for further studies if it is kept on ice for up to 4 h.

    4. D

      Excision of a large mammalian heart (Canine) • TIMING 5 min

      1. i

        Kill a dog with an overdose of sodium pentobarbital (200 mg/ml solution for injection).

      2. ii

        Confirm its death.

      3. iii

        Remove the rib cage to expose the thoracic cavity. Remove the heart immediately, and place it into a 1-liter container with freshly made, cold (4 °C) cardioplegia solution.

      4. iv

        Place the container on ice, and transport it to a laboratory.

        Pause point

        The heart remains viable for further studies, if it is kept on ice, for up to 4 h.

Preparation of the left ventricular tissue block

Timing 5–10 min

  1. 2

    Follow option A and Supplementary Videos 1, 2, 3 if using a small mammalian heart, or option B if using a large mammalian heart.

    1. A

      Preparation of a small mammalian (Rat) left ventricular tissue block

      1. i

        Carefully transfer the heart and lungs to a large Petri dish at the tissue dissection area.

        Critical Step

        See Supplementary Video 1 to visualize Step 2A (i–vii).

      2. ii

        Fill the Petri dish with enough 4 °C slicing solution to cover at least half the heart and keep it cold.

      3. iii

        Using a scalpel/razor blade, dissect off the lungs and other tissues to leave an intact heart (Fig. 3a).

      4. iv

        Visualize the atria, and make an incision slightly inferiorly (through the base of the heart) (Fig. 3b).

      5. v

        From the superior position, visualize the left and right ventricular walls and the septum. Locate the right ventricle, which is cresentic in shape and has a thinner wall. With small tweezers, hold the right ventricle by its free wall (Fig. 3c).

      6. vi

        With microscissors in your other hand, make an incision along the right ventricular–septal junction toward the apex (Fig. 3d).

      7. vii

        Continue to hold the right ventricle by its free wall (Fig. 3e) and cut along the other ventricular–septal junction to remove it (Fig. 3f). Some papillary muscles may also have to be cut to remove the right ventricle.

      8. viii

        You should now have the left ventricle and the septum. Locate the septum, and make an incision down toward the apex using microscissors (Fig. 3g).

        Critical Step

        See Supplementary Video 2 to visualize Step 2A (viii–x).

      9. ix

        Open the left ventricle and allow it to flatten (Fig. 3h).

      10. x

        If further flattening is required, make small incisions along the superior and inferior borders of the tissue block. If necessary, also cut the papillary muscles (Fig. 3i).

      11. xi

        Turn the block over, and use a scalpel/razor blade to remove the remaining septal tissue from the edges of the tissue block (Fig. 3j). You should now have a relatively flat block of left ventricular tissue (Fig. 3k,l).

        Critical Step

        See Supplementary Video 3 to visualize this step.

        Critical Step

        It is critical to make the tissue as flat as possible. Use microscissors to cut large papillary muscles from the endocardial surface, and remove any fibrous tissue or subvalvular tissue from the basal part of the ventricle. It may also be necessary to make very small incisions along the borders of the tissue block to make it truly flat. During tissue handling, it is important to keep the tissue cold to prevent the myocardium from contracting.

        Troubleshooting

    2. B

      Preparation of a large mammalian (human) left ventricular tissue block

      1. i

        Transfer the heart from the container of cold cardioplegia solution to the tissue dissection area.

      2. ii

        Remove the heart from the container, and place it in a large Petri dish.

      3. iii

        Using the scalpel/razor blade, make an incision inferior to the atria and atrioventricular valves to leave the left and right ventricles and the septum.

      4. iv

        Identify the left ventricle, and locate the area of the left ventricular free wall with the least epicardial curvature.

      5. v

        Dissect out a 1.5-cm2 tissue block from this area by making incisions through the full thickness of the ventricular wall with a scalpel/razor blade.

        Critical Step

        Human specimens may contain regions of pathology (e.g., infarct scarring). Unless they are specifically required for experiments, these areas should be avoided to produce highly viable myocardial slices.

        Troubleshooting

        Critical Step

        See Supplementary Video 4 to visualize Steps 3–8.

        Critical Step

        Tissue blocks are attached to the agarose-coated specimen holder with the epicardial surface facing down. The epicardium is substantially flatter than the endocardium, and this helps us to align the myocardial fibers in the same plane within the tissue. This is critical to obtaining a myocardial slice with high viability.

  2. 3

    Lightly blot the epicardial surface of the tissue block with tissue paper to remove excess solution from its surface.

  3. 4

    Apply a drop of Histoacryl glue to the agarose attached to the base of the specimen holder. Spread the glue to ensure a thin and even coating over the entire surface.

  4. 5

    Using small tweezers, hold and pick up the tissue block by one of its lateral edges.

  5. 6

    Move the left ventricle so that it is positioned directly over the specimen holder. Slowly lower the ventricle until the free edge of the ventricle makes contact with the agarose.

    Critical Step

    The tissue will attach to the surface immediately upon contact. It is not possible to remove the tissue once it is stuck.

  6. 7

    Smoothly roll the rest of the ventricle onto the agarose, until the entire epicardial surface is in contact.

  7. 8

    Using a flat spatula, apply gentle pressure to the endocardial surface to ensure that the tissue attaches.

    Critical Step

    The sample should not be placed too close to the back of the specimen holder, as this can cause slices to become trapped during slicing.

    Troubleshooting

  8. 9

    Rapidly transfer the specimen holder to the inner part of the vibratome bath.

Slicing

Timing 0.5–1.5 h

Critical Step

See Supplementary Video 5 to visualize Steps 10–14.

  1. 10

    Once the tissue block is in position, move the blade to the correct starting position. Move the blade forward until it reaches the anterior border of the tissue block. Adjust the height of the blade to the superior border of the tissue block. The following settings have been optimized to maintain tissue viability while slicing. The blade should be set to vibrate at a frequency of 80 Hz and an amplitude of 2.00 mm. The section thickness should be 300 μm (although slices of a thickness of 100–400 μm can be produced). While slicing, advance the blade at 0.03 mm/s. Ensure that the z-axis error is <1.0 μm.

  2. 11

    Start slicing. The blade should advance slowly and make contact with the tissue.

  3. 12

    Once the blade reaches the posterior border of the tissue block, stop the advancing of the blade.

  4. 13

    Return the blade to the starting position. Once the blade has returned to this position, it will automatically lower by 300 μm (or the selected slice thickness).

  5. 14

    Start slicing. Repeat Steps 11–13 until you have sliced through the full thickness of the ventricle.

    Troubleshooting

Handling and storage of myocardial slices

Timing 1 min

Critical Step

See Supplementary Video 6 to visualize Steps 15 and 16.

  1. 15

    Once the blade has completely cut through the tissue block, a slice will detach from the superior surface of the tissue block. It takes 5–10 min to cut a slice (depending on tissue block dimensions) because of the slow speed at which the blade advances. Use a Pasteur pipette to transfer a slice to the holding bath. Move the pipette tip under the slice and gently lift it. The slice should drape over the pipette tip.

    Critical Step

    It is important to avoid sucking the slice into the Pasteur pipette, as this creates tissue damage.

    Troubleshooting

  2. 16

    Submerge the pipette in one of the cell strainers in the holding bath to release the slice. Make sure that the slice is flat on the cell strainer gauze. Place a circular gauze over the top of the slice, and hold it down using a washer.

    Pause point

    Slices can be kept in the holding bath for up to 4 h. Slices must be used for acute experiments or must be put in culture within this time period.

  3. 17

    Repeat slicing to generate more slices.

  4. 18

    Ceramic blades can be used to produce myocardial slices from several animals. To avoid damage to the blade, we do not recommend removing it between experiments. Thoroughly clean the blade with 70% ethanol after each experiment. If multiple tissue blocks are sliced in a single day, clean the blade after each slice, and recheck the z-axis error.

Culture, fixing and staining of myocardial slices

  1. 19

    Follow option A for culture or option B for fixing and staining. If desired, the two options can be carried out consecutively.

    1. A

      Culture—air–liquid interface method • TIMING 10–15 min for 6 slices

      1. i

        Transfer a myocardial slice from the holding bath to a sterile Petri dish using a Pasteur pipette. Gently pipette some cold Tyrode's solution onto the slice surface to keep it cool and prevent it from drying out. Quickly transfer the slice to a flow hood.

        Critical Step

        Handle myocardial slices, as described in 'Handling and storage of myocardial slices' (Steps 15–18).

      2. ii

        Wash the slice with PBS + 3% penicillin–streptomycin at room temperature (23 °C).

      3. iii

        Myocardial slices can be cultured on an air–liquid interface using Transwell membrane cell culture inserts. Use a Pasteur pipette to transfer the slice onto the surface of the Transwell membrane, and pipette some cold Tyrode's solution into the insert.

      4. iv

        Gently remove the solution from the insert using a Pasteur pipette. Make sure that the slice is lying flat on the Transwell membrane.

      5. v

        Remove as much liquid as possible from the edges of the slice using a Pasteur pipette.

      6. vi

        Place 1 ml of medium (Medium-199 + 3% penicillin–streptomycin + 0.001% ITS liquid media supplement) into the well in which the insert is placed.

      7. vii

        Cover the 6-well plate with a lid, and transfer the plate to an incubator. The slices should be incubated in humidified air at 37 °C with 5% CO2.

      8. viii

        Replace the medium every 24 h. Penicillin–streptomycin concentration can be reduced to 1% after 24 h. An additional wash in PBS + 3% penicillin–streptomycin can be carried out after 24 h to prevent infections.

      Caution

      The following steps must be carried out in a sterile manner in a laminar flow hood. Spray all items with 70% ethanol before placing them in a flow hood, and observe standard cell culture practices. See Supplementary Video 7 to visualize Step 19A (i–viii).

    2. B

      Fixing and staining • TIMING fixing, 15 min; staining, 20 h

      1. i

        Fix myocardial slices in 4% formaldehyde solution for 15 min at room temperature.

        Pause point

        Fixed myocardial slices can be kept in PBS at 4 °C for several months.

      2. ii

        If permeabilization is required, permeabilize the fixed slice in 1% Triton X-100 for 1 h at room temperature.

      3. iii

        Block the slice using 10% FBS, 5% BSA and 10% horse serum in PBS solution (for several antibodies, a weaker blocking (1% BSA) is sufficient) for 1 h at room temperature.

      4. iv

        Incubate the slice in a primary antibody in PBS at 4 °C overnight.

      5. v

        Wash the slice in PBS for 30 min. Repeat this step three times.

      6. vi

        Incubate the slice in a secondary antibody in PBS for 3 h at room temperature.

      7. vii

        Wash the slice in PBS for 30 min. Repeat this step three times.

        Pause point

        Stained myocardial slices can be kept at 4 °C covered in aluminum foil for up to one week. Please be aware that secondary antibody fluorescence decreases with time, and imaging samples directly after staining is recommended.

Troubleshooting

Troubleshooting advice can be found in Table 3.

Table 3 Troubleshooting table.

Timing

Total time required: 4 h (excluding Step 19)

Reagent Setup: 1 h

Equipment Setup: 1 h

Step 1, excision of the heart: 5 min (if excision cannot take place in the laboratory, additional tissue transport time of 1–4 h will be needed)

Steps 2–9, preparation of the left ventricular tissue block: 5–10 min

Steps 10–18, slicing and storage: 0.5–1.5 h (dependent on thickness of ventricle)

Step 19, culture: 10–15 min to put 6 slices into culture; fixing and staining: 24 h

Anticipated results

In this section, we present typical data that we collected from healthy rat (small mammal) and failing human hearts (Human HF; large mammal) myocardial slices. When we present N values, this indicates the number of slices/number of hearts (e.g., 10/5 = 10 slices derived from 5 hearts). All data are presented as mean ± standard error.

For structural and functional studies, only aligned areas (myocardial fibers running parallel to each other) should be used. To visualize the aligned areas, place the myocardial slice on a Petri dish and remove excess solution. Visualize the structure of a slice using a macroscope, and determine the aligned area of the slice (Fig. 4). Using a scalpel/razor blade, trim the slice to isolate the aligned area, and use it for structural and functional studies.

Figure 4: Rat myocardial slice on a 1-mm grid visualized using a macroscope.
figure4

The aligned area, in which myocardial fibers are parallel, is highlighted in green. This area is appropriate for structural and functional studies. Nonaligned areas are highlighted in red. Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).

Viability

The viability of the surface of myocardial slices can be assessed using live/dead staining (Thermo Fisher Scientific). Live cells are stained green by calcein-AM, and the nuclei of dead cells are stained red by ethidium homodimer-1. The surface of myocardial slices can be imaged with wide-field microscopy (Fig. 5a) or confocal microscopy (Fig. 5b). The percentage area of living cells on the surface can be quantified using Image J (National Institute of Health, https://imagej.nih.gov/ij/download.html). We found that 59.94 ± 4.35% of cells were alive on the surface of rat myocardial slices (N = 11/6, Fig. 5c) and 47.42 ± 4.59% of cells were alive on the surface of human HF slices (N = 9/4, Fig. 5d).

Figure 5: Myocardial slice viability.
figure5

Viability was assessed using a live/dead staining. Live cells are stained green (calcein-AM), and the nuclei of dead cells are stained red (ethidium homodimer-1). (a) Representative image of live/dead staining of a rat myocardial slice imaged using wide-field microscopy. Scale bar, 1,000 μm. (b) Representative image of live/dead staining of a human HF myocardial slice imaged using confocal microscopy. Scale bar, 300 μm. (c) 59.94±4.35% of the surface of rat myocardial slices was alive (N = 11/6, K–S distance = 0.1677, P > 0.10). (d) 47.42±4.59% of the surface of human HF myocardial slices was alive (N = 9/4, K–S distance 0.1773, P > 0.10). Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). Data are presented as mean ± standard error if normally distributed, or median and 95% confidence intervals if not normally distributed. Mean ± standard error is shown on graphs if data are normally distributed. N = number of slices/number of hearts. Kolmogorov–Smirnov (K–S) test was used to test the normality of data (K–S distance and P-value are reported). Biological replicates are shown as different symbols on graphs. Technical replicates have the same symbol.

Imaging of the layer of cardiomyocytes directly below the slice surface revealed that almost all of the cardiomyocytes were alive (Fig. 6a), indicating that tissue damage was limited to the slice surface. It was not possible to image the full thickness of the myocardial slice using confocal microscopy, as the tissue is too optically dense. Calculations were used to estimate the viability of the myocardial slices. These revealed that the damaged cardiomyocytes on the myocardial slice surface account for 3% of the total cardiomyocyte population within the slice, demonstrating preserved viability (see Box 1 for full calculations).

Figure 6: Understanding the viability of myocardial slices.
figure6

(a) 50–60% of cardiomyocytes on the surface of myocardial slices were alive. Living cardiomyocytes were stained green (calcein-AM), whereas the nuclei of dead cardiomyocytes were stained red (ethidium homodimer-1). Images were acquired using confocal microscopy. Deep imaging of the layers of cardiomyocytes below the surface revealed that almost 100% of cardiomyocytes below the surface were alive. The representative schematic shows live cells in green and dead cells in red. Schematic of the slice surface shows that 50% of the cells were alive. Schematic of the layer of cardiomyocytes just below the slice surface shows that almost all cardiomyocytes are alive. (b) The tissue block should never be mounted endocardium-down. When this is the case, the tissue block has an increased curvature, which results in large portions of the tissue being damaged. Damage usually occurs on the lateral portions of slices. (c,d) When the tissue block is not flat, myocardial fibers can be transected. Slices were stained with α-actinin (see 'Table 4'; further information on fixing and staining of myocardial slices can be found in Step 19B) and imaged using confocal microscopy. The images show areas of undamaged cardiomyocytes alongside areas in which cardiomyocytes have been transected. Cardiomyocytes lying in the same plane as the blade are undamaged, whereas those lying perpendicular to the blade are transected. Scale bars, 100 μm. Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).

There are a number of critical steps that must be followed to produce slices of high viability. The method by which the tissue block is prepared is particularly important, especially for small mammals. The method described in this protocol ensures that the left ventricle of small mammals is completely opened and consequently flattened. This is critical, as the ventricles of small mammals have a pronounced curvature. Flattening of the ventricles ensures that the long axes of the myocardial fibers lie in the same plane as the blade. When this is the case, the layers separate during slicing, resulting in minimal damage to cardiomyocytes within the slice. Owing to the reduced curvature of the large mammalian ventricle, less dissection and flattening is required. However, with both small and large mammals, it is important to mount the tissue epicardium-side down (epicardial surface in contact with agarose). The epicardium is substantially flatter than the endocardium, which is covered with papillary muscles, and this helps to correctly orientate the fibers. When the tissue is mounted endocardium-side down, it will have an increased curvature, and more damage will occur at the edges of slices (Fig. 6b). When the tissue block is not flat, myocardial fibers will be transected, reducing slice viability (Fig. 6c,d). It is not possible to prepare highly viable myocardial slices by simply sticking biopsies onto agarose when the fiber direction is ambiguous. This also applies to embedding biopsies in agarose, which disregards fiber direction.

There are a number of additional factors that must also be considered to preserve viability. An important determinant is the way in which the cardiac tissue is explanted. Small mammalian hearts can be directly removed from the animal after sacrifice, whereas large mammalian hearts require cardioplegic arrest before explantation. The faster the heart is explanted and cooled, the higher the viability of myocardial slices. Therapeutic hypothermia is cardioprotective41,42, reducing the tissue's metabolic demand and the initiating protective signaling pathways43. The time taken to slice is also critical. Oxygen is unable to diffuse through the entirety of the ventricular wall, and the tissue becomes increasingly ischemic with time. This is minimized by tissue cooling, but any delays will have an impact on slice viability. Studies have shown that diffusion distances >200 μm are generally poorly tolerated by tissues33. 300-μm slices have a maximum diffusion distance of 150 μm from either surface, and allow the rapid diffusion of substrates to their innermost cells34. Thinner slices (100–250 μm) can be prepared, but they are more difficult to handle and, because of the lower number of cell layers, the proportion of damaged cardiomyocytes is increased. The use of an excitation–contraction uncoupler (BDM) prevents the cardiac tissue from contracting during slicing, and its omission results in poor slice viability. The vibratome settings are also important and were optimized in our laboratory, as described in Camelliti et al5. Other studies investigating optimal slicing solutions have been carried out, and the solutions described in this protocol yielded the highest viability.

Contractility

We used a force transducer (Harvard Apparatus) to assess the maximum contractility of myocardial slices. Rat and human HF slices were field-stimulated at 1 Hz (Fig. 7a) and 0.5 Hz (Fig. 7c; 10–30V), respectively. The maximum contractility was recorded by stretching the slices in a stepwise manner. Custom PTFE-coated silver rings were attached to opposite ends of myocardial slices orthogonal to the longitudinal fiber direction using Histoacryl surgical glue. The slices were stretched by 5% and were then left to contract for 2 min. This was repeated until isometric contraction was achieved, and maximum contractility was recorded. Rat slices had a maximum contractility of 11.55±1.30 mN/mm2 (N = 10/10, Fig. 7b). Human HF slices had a maximum contractility of 13.78±3.52 mN/mm2 (N = 9/9, Fig. 7d).

Figure 7: Contractility of rat and human HF myocardial slices.
figure7

(a) Representative contractility traces from rat myocardial slice field stimulated at 1 Hz. (b) Maximum contractility of rat myocardial slices was 11.55±1.30 mN/mm2 (N = 10/10, K–S distance 0.1497, P > 0.10). (c) Representative contractility traces from human HF myocardial slices field stimulated at 0.5 Hz. (d) Maximum contractility of human HF myocardial slices was 13.78±3.52 mN/mm2 (N = 9/9, K–S distance 0.1373, P > 0.10). Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). Data are presented as mean ± standard error if they are normally distributed, or median and 95% confidence intervals if they are not normally distributed. Mean ± standard error is shown on graphs if data are normally distributed. N = number of slices/number of hearts. Kolmogorov–Smirnov (K–S) test was used to test the normality of data (K–S distance and P-value are reported). Biological replicates are shown as different symbols on graphs. Technical replicates have the same symbol.

See Supplementary Video 8 to visualize a human heart failure and rat myocardial slices contracting on a force transducer.

Rat slices were paced at 1 Hz, whereas human HF slices were paced at 0.5 Hz. Rat slices are derived from healthy animals and can follow a field stimulus of >5 Hz (resting heart rate of 300 bpm44). Human HF slices can be paced at >0.5 Hz, but at these rates, the tissue cannot fully relax, reflecting the disease state of the tissue (end-stage HF, New York Heart Association (NYHA) class IV). Myocardial slices can also be point-stimulated (1 V; Supplementary Video 7).

Ca2+ handling

To assess Ca2+ handling, myocardial slices were loaded in Medium-199 with Fluo-4 AM (5.56 μg/ml) (Thermo Fisher Scientific) and Pluronic F-127 (0.001%) (Thermo Fisher Scientific). A circular gauze and a washer were placed over the slice (as in Step 17; Supplementary Video 6) to prevent contraction during loading. Rat myocardial slices were loaded for 10 min at 37 °C, and then directly transferred to the optical mapping setup. Human HF slices were loaded for 20 min, and then left to de-esterify for a further 10 min in Tyrode's solution with 30 mM BDM at 37 °C. BDM prevents movement artifact during calcium transient acquisition. Rat and human HF slices were field stimulated at 1 Hz (Fig. 8a) and 0.5 Hz (Fig. 8b) (30 V), respectively. The average time to peak, time to 50% decay and time to 90% decay were analyzed (Fig. 8c). Rat myocardial slices have a time to peak of 30.27±1.27 ms, time to 50% decay of 61.95±2.54 ms and a median time to 90% decay of 139.3 ms; 95% confidence intervals: 135.7–170.9 (all N = 24/6, Fig. 8d). Human HF myocardial slices had a time to peak of 0.134±0.017 s, time to 50% decay of 0.409±0.039 s and time to 90% decay of 0.982±0.098 s (all N = 10/6, Fig. 8e).

Figure 8: Ca2+ handling of rat and human HF myocardial slices was assessed by loading slices with Fluo-4 AM and performing optical mapping.
figure8

(a) Representative Ca2+ transient from a rat myocardial slice field stimulated at 1 Hz. (b) Representative Ca2+ transient from a human HF myocardial slice field stimulated at 0.5 Hz. (c) Visual description of the aspects of Ca2+-handling kinetics assessed. (d) Ca2+-handling kinetics of rat myocardial slices field stimulated at 1 Hz. Rat myocardial slices had time to peak of 30.27±1.27 ms (N = 24/6, K–S distance 0.1582, P > 0.10), time to 50% decay of 61.95±2.54 ms (N = 24/6, K–S distance 0.1693, P = 0.0733) and median time to 90% decay of 139.3 ms; 95% confidence interval (CI) 135.7–170.9 (N = 24/6, K–S distance =0.2293, P = 0.0021). (e) Ca2+-handling kinetics of human HF myocardial slices field stimulated at 0.5 Hz. Human HF myocardial slices had time to peak of 0.134±0.017s (N = 10/6, K–S distance 0.1783, P > 0.10), time to 50% decay of 0.409±0.039s (N = 10/6, K–S distance 0.1574, P > 0.10) and time to 90% decay of 0.982±0.098s (N = 10/6, K–S distance 0.2393, P > 0.10). Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). Data are presented as mean ± standard error if they are normally distributed, or median and 95% confidence intervals if they are not normally distributed. Mean ± standard error is shown on graphs if data are normally distributed. N = number of slices/number of hearts. Kolmogorov–Smirnov (K–S) test was used to test the normality of data (K–S distance and P value are reported). Biological replicates are shown as different symbols on graphs. Technical replicates have the same symbol.

See Supplementary Video 9 to visualize Fluo-4 flashing on the surface of a rat myocardial slice.

Conduction velocity

The conduction velocity of myocardial slices can be assessed using multielectrode arrays (Multichannel Systems), which allow noninvasive, synchronous and multifocal recording of extracellular field potential. Data were acquired with a 60-microelectrode plate with the microelectrodes arranged in an 8 × 8 matrix with a 700-μm interelectrode distance, providing a recording area of 4.9 × 4.9 mm2. Representative conduction maps show the propagation of electrical activity in rat and human HF slices longitudinal (Fig. 9a,d) and transverse (Fig. 9b,e) to myocardial fiber direction. Rat myocardial slices had a median longitudinal conduction velocity of 51.00 cm/s, 95% confidence intervals: 44.26–77.52 and a transverse conduction velocity of 21.88±2.14 cm/s (both N = 14/6, Fig. 9c). Human HF myocardial slices had a longitudinal conduction velocity of 55.53±9.75 cm/s and a median transverse conduction velocity of 14.73 cm/s; 95% confidence intervals: 13.19–22.05 (both N = 11/4, Fig. 9f).

Figure 9: Conduction velocity of rat and human HF myocardial slices.
figure9

(a,b) Representative conduction maps from rat myocardial slices, with electrical propagation longitudinal (a) and transverse (b) to myocardial fiber direction. (c) Rat myocardial slices have a median longitudinal conduction velocity of 51.00 cm/s, 95% CI 44.26–77.52 (N = 14/6, K–S distance 0.2567, and P = 0.0130) and a transverse conduction velocity of 21.88±2.14 cm/s (N = 14/6, K–S distance 0.1888, P > 0.10). (d,e) Representative conduction maps from human HF myocardial slices, with electrical propagation longitudinal (d) and transverse (e) to myocardial fiber orientation. (f) Human HF myocardial slices have a longitudinal conduction velocity of 55.53±9.75cm/s (N = 11/4, K–S distance 0.1965, P > 0.10) and a median transverse conduction velocity of 14.73cm/s; 95% CI 13.19–22.05 (N = 11/4, K–S distance 0.2773, P = 0.0179). Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU). Data are presented as mean ± standard error if they are normally distributed, or median and 95% confidence intervals if they are not normally distributed. Mean ± standard error is shown on graphs if data are normally distributed. N = number of slices/number of hearts. Kolmogorov–Smirnov (K–S) test was used to test the normality of data (K–S distance and P value are reported). Biological replicates are shown as different symbols on graphs. Technical replicates have the same symbol.

Structural studies

The structure of myocardial slices can be investigated using various techniques. We have used immunohistochemistry and confocal microscopy. As myocardial slices can be stained and imaged either fresh or fixed, and do not require sectioning, they are an ideal model for assessing cardiac structure. Sectioning requires tissue freezing, handling and processing, and can result in sample damage. Owing to tissue density and light scattering, imaging is limited to the surface of the preparation (2–3 cell layers). A number of antibodies were used to visualize fundamental cardiac structures (see Table 4 for further details). Caveolin 3 antibody was used to stain cardiomyocyte cell membranes, and intercalated disks were stained with connexin-43 antibody (Fig. 10a). The structure of the sarcomeric apparatus was assessed with α-actinin staining (Fig. 10b). The microvasculature was stained with isolectin, and the cardiac stromal cell population was stained with vimentin (Fig. 10c). More detailed assessment of myocardial slice ultrastructure can be carried out using scanning electron microscopy (Fig. 10d). To image the full thickness of the myocardial slice, optical clearing methods were used, as described by Perbellini et al25. Second harmonic generation imaging was used to visualize collagen distribution within a human HF slice (Fig. 10e). The addition of antibody labeling for large blood vessels (vimentin) and high-definition 3D reconstruction provided a precise map of the macrovascular network (Fig. 10f).

Table 4 Antibodies and staining methods.
Figure 10: Cardiac structure studied using myocardial slices.
figure10

(ac) Immunohistochemical staining and confocal microscopy of rat myocardial slices. (a) The cell membranes of cardiomyocytes have been stained with caveolin 3 antibody, the intercalated disk is stained with connexin-43 antibody and nuclei are labeled with Hoechst 33342. Scale bar, 50 μm. (b) The sarcomeric apparatus is stained with α-actinin antibody, and nuclei are labeled with Hoechst 33342. Scale bar, 50 μm. (c) Microvasculature is stained with isolectin antibody, the cell membranes of cardiomyocytes are stained with caveolin 3 antibody, fibroblasts are stained with vimentin antibody and nuclei are labeled with Hoechst 33342. Scale bar, 50 μm. (d) Scanning electron microscopy of human HF myocardial slice. Transverse section. Gray structures are cardiomyocytes and the smaller, darker substructures are mitochondria. Scale bar, 20 μm. (e,f) Optical clearing was used to image the full thickness of myocardial slices (300 μm)25. Second harmonic generation imaging was used to visualize collagen distribution within a human HF myocardial slice (e); scale bars, 130 μm. The macrovascular network was imaged using this method and the addition of a vimentin antibody (f); scale bars, 100 μm. Further information on the antibodies used can be found in Table 4. The fixing and staining protocol used is described in Step 19B. Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed in accordance with guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).

Cultured myocardial slices

Using the air–liquid interface method, as described in Step 19A, myocardial slices can be kept in culture for up to 1 month2. However, as slices are kept in an unloaded condition and do not beat due to a lack of electrical stimulation or intrinsic pacemaker activity in adult ventricular tissue, they start to dedifferentiate, and a gradual decline in their contractility occurs with time2,3. However, the viability of the slices is preserved for 7 d using this technique3.

Conclusions

Myocardial slices provide a unique platform that facilitates the study of myocardial structure and function at the cellular level in vitro. Myocardial slices have been produced for a number of years, but the protocols used have varied greatly2,5,6,15. This protocol describes a robust and reproducible method for producing slices with high viability, and addresses a number of critical steps that must be followed. The data presented represent a fraction of the studies that can be performed using myocardial slices. Data collected using myocardial slices have the advantage of being derived from native adult myocardium with preserved multicellularity and architecture. These aspects have important roles in myocardial function28,29 and cannot be ignored. Owing to their unique advantages and their ability to be chronically cultured, myocardial slices will be an important platform for future cardiovascular research.

Additional information

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Acknowledgements

We thank the British Heart Foundation for funding our work, particularly the BHF Centre for Regenerative Medicine award at Imperial College London (RM/13/1/30157) and the MBBS PhD studentship to S.A.W. (FS/15/35/31529). We thank The Facility for Imaging by Light Microscopy (FILM) at Imperial College London, in particular S.M. Rothery. Human samples were provided by the NIHR Cardiovascular Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. Canine samples were provided by GlaxoSmithKline. Porcine samples were provided by the Translational Biomedical Research Centre, University of Bristol.

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Contributions

S.A.W. wrote the manuscript, collected data and contributed to the optimization of the protocol. M.S. collected data and contributed to the optimization of the protocol. I.B. contributed to the optimization of the protocol. R.A. provided porcine specimens. C.M.T. contributed to the optimization of the protocol. F.P. collected data and contributed to the optimization of the protocol. All authors proof-read the manuscript.

Corresponding authors

Correspondence to Cesare M Terracciano or Filippo Perbellini.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Original vs. optimized protocol for rat (small mammal) & human HF (large mammal) myocardial slices.

Comparison between original and optimized protocols. Original protocol was based on several methods from previously published manuscripts(2,6,13). A) Rat myocardial slices produced using optimized protocol had a significantly higher maximum contractility compared to those produced with original protocol (0.39±0.05mN/mm2 vs. 11.55±1.30mN/mm2, Original: N=24/24, Optimized: N=10/10, Unpaired t-test). B) Human HF myocardial slices produced using optimized protocol had a significantly higher maximum contractility compared to those produced with original protocol (3.44±0.38mN/mm2 vs. 13.77±3.52mN/mm2, Original: N=20/20, Optimized: N=9/9, Unpaired t-test).Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed following guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).Data is presented as mean ± standard errorN = number of slices/number of hearts

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1. (PDF 183 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 1 (video depicts Step 2A(i–vii)).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24480 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 2 (video depicts Step 2A(viii–x)).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24867 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 3 (video depicts Step 2A(xi).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24584 kb)

Mounting of left ventricular tissue block on agarose-coated specimen holder.

Video depicts how to handle, dry and mount tissue block correctly. (MP4 18702 kb)

Slicing of left ventricular tissue block.

Video depicts bringing blade to 'starting position' and various stages of slicing. (MP4 26143 kb)

Handling and storage of myocardial slices.

Video depicts how to move a myocardial slice from a vibratome bath to a holding bath. Video also shows how myocardial slices should be kept in a holding bath. (MP4 9422 kb)

Culturing myocardial slices using air–liquid interface method.

Video depicts how to culture myocardial slices on an air–liquid interface using Transwell membranes as described by Brandenburger et al. (ref. 2). (MP4 24327 kb)

Myocardial slice contraction.

Video depicts a human HF myocardial slice contracting (field stimulation, 0.5 Hz, 30 V) and a rat myocardial slice contracting (point stimulation, 1 Hz, 10 V). (MP4 5001 kb)

Ca2+ handling of myocardial slices.

Rat myocardial slice was loaded with Fluo-4 AM (as described in 'Anticipated Results—Ca2+ handling'). Myocardial slice was field stimulated at 1 Hz, 10 V. Cardiomyocytes at the slice surface flash with each calcium transient. (MP4 9282 kb)

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Watson, S., Scigliano, M., Bardi, I. et al. Preparation of viable adult ventricular myocardial slices from large and small mammals. Nat Protoc 12, 2623–2639 (2017). https://doi.org/10.1038/nprot.2017.139

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