5.1. Isolation and Culture of Adult Ventricular Cardiomyocytes. Introduction

Transcription

1 557 Isolation and Culture of Adult Ventricular Cardiomyocytes Klaus-Dieter Schlüter and Hans Michael Piper Introduction The current understanding of the physiological and pathophysiological behaviour of the heart depends on the general features of the interaction of different cell types and the knowledge of the functional behaviour of individual cells. Any analysis of the functional characteristics of ventricular cardiomyocytes with regard to size control, contractility, and biochemical or molecular properties requires the isolation of functionally and morphologically intact cells. This has been a challenge for many years due to the fact that adult ventricular cardiomyocytes are fully differentiated and do not grow by cell division. Techniques for the isolation of cardiomyocytes have been difficult to establish, because the heart muscle cells are firmly connected to each other by intercalated discs and the extracellular matrix network and these connections are difficult to cleave without injuring the cells. The success of any experiments performed on a single cell basis with adult ventricular cardiomyocytes depends on the number of cells isolated from the heart, their purification from other cardiac cell types and the ability to maintain their main characteristics for a reasonable length of time. Normally, cells are cultured for days or weeks to perform experiments requiring long-term exposure of the cells. Although a certain set of experiments can be performed with small cell numbers, i.e. microscopic observations like the analysis of cell contraction or experiments linked to electrophysiological questions, one remaining question will always be whether the cells under investigation represent the normal behaviour of a typical cell. Thus, even in these cases it should always be an aim to isolate large cell numbers and use randomly selected cells. In general, the challenge of isolating adult ventricular cardiomyocytes can be subdivided in two problems; how can we isolate great numbers of calcium-tolerant rod-shaped physiologically intact cells and how can we keep these cells in culture allowing them to maintain their specific characters? The following chapter will give examples as to how these problems can be solved and thus make isolated ventricular cardiomyocytes a suitable model for the study of basic cardiac function.

2 5 558 Cell Culture Techniques Description of Methods and Practical Approach Isolation of Cells from Hearts The rat heart is still the most commonly used model for isolation of ventricular cardiomyocytes for two reasons. First, the size of the heart fits easily into commercially available perfusion systems, it is more or less easy to handle, the animal itself is not expensive and the isolation normally gives reasonable cell numbers that can be used within the next day. Second, the rat is the most commonly used animal model in cardiac biology. Therefore, any data acquired at the single cell level can be compared to a broad field of data available in the literature. Finally, myocytes from in vivo variations, i.e. spontaneously hypertensive rats, genetically modified rats, or rats receiving surgery before use, can also be investigated. In general, ventricular cardiomyocytes can also be isolated from the hearts of other species. However, the protocol has to be modified slightly for the mouse as explained later. For the reasons explained above, we will first describe the isolation of ventricular cardiomyocytes from rat heart. In-Vitro Techniques General Features of Isolation The isolation of ventricular cardiomyocytes from adult rat hearts is performed in several steps. In general, the heart is quickly removed from the animal, connective tissue trimmed away and the heart connected to a Langendorff perfusion system and perfused retrogradely with a buffer to remove any blood. The whole heart is then treated with collagenase for a certain length of time in the absence of exogenous calcium. This step allows firstly the disruption of connections of individual cells with the extracellular network and secondly the dissociation of Ca 2+ -dependent desmosome structures. Thereafter, the heart is removed from the perfusion system and the ventricle cut from the rest of the heart and minced into small pieces. These are further treated with collagenase, and the isolated cells separated from the larger tissue pieces by filtration through a nylon mesh (mesh size 200 µm). The isolated cells can then be re-exposed to physiological calcium concentrations by increasing the calcium concentrations stepwise and separated from non-cardiomyocytes by centrifugation. Figure 1 summarizes these steps. The final cell pellet can be re-suspended in cell culture medium (see below) and cultured. The following description is suitable for male normotensive rats ( g). Preparation of the Heart To remove the heart, animals must be anaesthetized. There are no specific methods known to influence the final outcome of the preparation. Thus, we do not recommend a specific method for anaesthetizing the animals. The thorax should be opened, flooded with ice-cold physiological NaCl (0.9% wt/vol) solution, and the heart removed from the thorax, beginning to cut from the back of the heart. The heart should be removed with parts of the lung and transferred into a Petri dish filled with ice-cold physiological NaCl solution. The adhering lung tissue can now be removed and the

3 Isolation and Culture of Adult Ventricular Cardiomyocytes 559 Figure 1 Schematic drawing of the isolation procedure described in the text for isolation of adult ventricular cardiomyocytes from rats. The heart is adjusted in the Langendorff perfusion system and perfused for approximately 30 min with collagenase. Then the ventricle is separated from the atrium and minced. A second collagenase treatment is performed before filtration. Finally, cardiomyocytes are separated by centrifugation from nonmyocytes and the cell pellet is re-exposed to physiological calcium concentrations aorta cut after the first aortic arch. The heart can then be connected by the aorta to a Langendorff perfusion system. It is important to perform these steps quickly. In order to avoid blood thrombosis during the time the heart is not perfused, the animals can receive heparin with the anaesthetics. People without experience of Langendorff preparations should practise these techniques before attempting the next steps. Perfusion of the Heart The perfusion should be started first (1 drop per second) and then the aorta should be connected to the system. With the heart immersed in saline, open the aortic lumen with two pairs of forceps, lift the heart to the cannula, slip the aorta over the cannula, and fix the aorta with crocodile clamps. Do not insert the cannula too deeply into the heart to ensure that perfusion via the coronary vessels is possible. Once the heart is well perfused, fix the heart to the perfusion system with thread. Continue to perfuse the heart with oxygenated perfusion buffer for 3 5 min to remove blood from the organ. Then add collagenase to the system and continue perfusion for 20 min. When necessary, readjust the perfusion to approximately 10 ml per minute.

4 5 560 Cell Culture Techniques Perfusion buffer (Powell medium): NaCl mmol/l KCl 2.6 mmol/l KH 2 PO mmol/l MgSO mmol/l NaHCO 25.0 mmol/l Glucose 5.0 mmol/l Collagenase buffer Perfusion buffer add Collagenase mg/l All buffers are constantly bubbled with carbogen (95% O 2 /5% CO 2 ). In-Vitro Techniques Collagenase is not a specific term and the amount of collagenase added to the buffer as well as the time to perfuse a heart are not fixed criteria. They depend on the specific quality of the collagenase. It is impossible to give any specific recommendation for collagenase treatment. The best way to start is to test several batches from different distributors and modify the amount of collagenase and the time suggested for perfusion moderately. Post-Perfusion Digestion The collagenase treatment should be stopped when the hearts are smooth. Please note, that the physical behaviour of the heart itself is more important than the time you have perfused the heart. The latter depends on the activity and quality of the collagenase. Then cut the ventricle from the atrium and aorta and transfer the ventricles to a separate glass dish. Cut the heart carefully using a scalpel or, as recommended, a tissue chopper that can be used at a width of 0.7 mm to produce a cell suspension. Put this suspension into a Teflon-coated glass container and add more collagenase buffer as used before, plus 400 mg bovine serum albumin per 30 ml. Bubble with carbogen for the next 10 min. You can improve the success of the post-perfusion digestion by pipetting the solution moderately several times. Separation of Myocytes from non-myocytes After 10 min take the cell suspension and filter the material through a nylon mesh (mesh size 200 µm). The cell suspension is then centrifuged for 3 min at 25 g. The cell pellet contains the myocytes, while the supernatant contains small non-myocytes. This fraction can either be used to isolate endothelial cells or fibroblasts or can be discarded. The myocyte-containing pellet is resuspended in Powell medium (see above) with added 0.2% (vol/vol) CaCl 2 (stock solution 100 mmol/l, final concentration 200 µmol/l)) and centrifuged again as before. The pellet is then resuspended in Powell medium containing 0.4% (vol/vol) CaCl 2 (stock solution 100 mmol/l, final concentration 400 µmol/l) and centrifuged again. The pellet is resuspended in Powell medium containing 1% (vol/vol) CaCl 2 (stock solution 100 mmol/l, final concentration 1 mmol/l) and layered over the following medium:

5 Isolation and Culture of Adult Ventricular Cardiomyocytes 561 Figure 2 Plating of cardiomyocytes: The cell pellet isolated from the heart is loaded on to culture dishes pre-coated with 4% (vol/vol) foetal calf serum. After 2 h for cell attachment, the cells are washed to remove most of the round and nonviable cells BSA gradient Powell Medium see above add bovine serum albumin: 4 g/100 ml CaCl 2 (100 mmol/l): 1 ml/100 ml This gradient is centrifuged again at 15 g for 1 min. The pellet contains rod-shaped and calcium-tolerant cardiomyocytes. These can be plated on culture dishes precoated with 4% foetal calf serum (see Cell Attachment). Figure 2 summarizes these final steps. Success of Isolation Two criteria allow one to quantify the success of the isolation procedure, firstly, the total number of cardiomyocytes isolated and secondly the ratio between rod-shaped calcium-tolerant and round non-tolerant myocytes. Both criteria can only give a gross view of the quality of the cell preparation. The number of initially isolated calciumtolerant cells might decrease during the subsequent recovery period of approximately 2 4 h. On the other hand, rounded cells will not attach to the culture dishes and thus the ratio between calcium-tolerant cells and rounded cells can be improved by washing the culture dishes after 2 4 h. In general the number of cells should be of the order of cells per heart and the ratio of rod-shaped to round cells should be approximately 70:30. We recommend suspending the final cell pellet from one ventricle in 25 ml medium and plating the cells at 1 ml per 3 cm dish (normally called six-well dishes ). In any case, to perform reproducible experiments it is more important to work with a constant quality of the preparation than with the highest quality. In other words, a constant preparation of approximately 10 6 cells with a ratio of 50:50 may be sufficient for most experiments.

6 5 562 Cell Culture Techniques Isolation of Ventricular Cardiomyocytes from Mice In-Vitro Techniques The frequent use of transgenic mice has led to an increased number of experiments using ventricular cardiomyocytes isolated from mice. In general the protocol described above for isolation of ventricular cardiomyocytes from rats can be used with slight modifications. Firstly, the perfusion system and volumes of buffers have to be reduced because of the smaller size of the heart. Second, reconstitution of a physio-logical calcium concentration should be performed more carefully. We suggest a four-step procedure that increases the calcium concentration to 125 µmol/l, 250 µmol/l, 500 µmol/l and 1 mmol/l. The proportion of cardiomyocytes yielded by this procedure is comparable to that from rat hearts. However, due to the smaller size of the hearts, one heart will yield only 10% of the total number of cells normally got from rat hearts. This is a great limitation for biochemical experiments requiring large amounts of cells. The third and last point which differs from the procedure for rat hearts is the more difficult cultivation process. No modifications must be taken into account in regard to the medium. However, cells from mouse hearts do not attach on cell culture dishes precoated by FCS. Therefore, it is essential to use laminin precoating as described below for rat cells. Cultivation of the Cells As pointed out in the introduction, the second challenge in working with isolated ventricular cardiomyocytes is to cultivate the cells. In principle, two different forms of cultures should be kept in mind. First, the use of freshly isolated, attached, rod-shaped cardiomyocytes and second, the use of long-term cultures of cardiomyocytes undergoing a certain amount of differentiation during the cultivation process including morphological differentiation. Nevertheless, the common challenge in both cases is to find a way to get the cells attached to the culture dish. Cell Attachment There are two different types of attachment substrates found suitable. The first protocol requires pre-treatment of the culture dishes for 8 20 h prior to cell plating with cell culture medium supplemented with 4% foetal calf serum (FCS). This procedure has two main advantages over other methods: First, cell attachment via FCS pre-treatment allows the cells to contact via variable components of the serum, resulting in preferentially attachment of rod-shaped calcium-tolerant cells. Therefore, simply washing the culture dishes within the next few hours is sufficient to increase the ratio of rod-shaped to rounded cells. Second, the method is cheap and can thus be widely used even with large amounts of cultures. Culture dishes not specified for primary cells are strongly recommended. As in cell culture procedures, use of FCS depends on batch variations. Therefore, as said before for collagenase batches, several batches of FCS should be screened for suitability prior to the start of experiments. The second method generally used to at-

7 Isolation and Culture of Adult Ventricular Cardiomyocytes 563 tach cardiomyocytes is to pre-treat cell dishes with laminin (1 g/ml) prior to cell plating. Compared to FCS this method cannot be used to increase the ratio of rod-shaped to round cells and is relatively expensive. However, it allows pre-treatment of dishes shortly before cell plating (10 30 min) and is suitable in cases in which FCS pre-treatment does not work properly, i.e. in case of ventricular cardiomyocytes isolated from mice. Cell Culture Medium Ventricular cardiomyocytes represent a finally differentiated cell type that requires complex media. The most commonly used medium is medium 199. However, even this quite complex medium should be modified by addition of creatine (5 mmol/l), carnitine (2 mmol/l) and taurine (5 mmol/l). Also, the recovery of ventricular cardiomyocytes from preparation stress can be further improved by addition of 4% (vol/vol) FCS. As the cells are often isolated under non-sterile conditions, penicillin (100 IU/ml) and streptomycin (100 µl/ml) should be added as antibiotics. Cell Cultures To maintain ventricular cardiomyocytes for a certain period of time in the rodshaped manner, the above medium should be used without addition of FCS. This allows the cells to survive without major morphological changes for approximately 36 h. During this time the amount of protein synthesis is more or less unchanged as well as the total amount of protein per cell (Pinson et al. 1993). Also, the ability of the cell to contract if it is exposed to electric stimulation is quite stable. However, cells cannot be used as confluent cultures under these conditions, because rod-shaped cells cannot attach to the culture dish without leaving spaces in between. Therefore, cell connections between individual cells, which are normal in the native tissue, are rarely present in these cultures. To maintain ventricular cardiomyocytes for a longer period of time in culture, i.e. one or two weeks, the medium must be supplemented with 20% (vol/vol) FCS. The cells then initially undergo a period of atrophy, round up and change their morphology completely (Piper et al. 1988). After six days they reach a stable situation in which the cell spreads around the centre. These cells have obviously lost some of their in vivo characteristics, i.e. rod-shaped morphology. However, energy metabolism and coupling of specific receptors, i.e. that of α-adrenoceptors to the regulation of protein synthesis is still intact (Schlüter et al. 1998). The cells start to build up new cell contacts, as present in native tissue (Schwartz et al. 1985). Also one has to keep in mind that some of the alterations found under these conditions, mimic the situation found during the development of heart failure, i.e. the cells secrete growth factors like TGFβ and such factors can be activated by proteases in the FCS (Taimor et al. 1999). Thus, although some of the changes seen in these spread cells limit their use in cardiovascular experimental biology, this system represents a suitable model for many questions.

8 5 564 Cell Culture Techniques In-Vitro Techniques Examples The success of any work with isolated cardiomyocytes depends on the reproducibility of cell isolation, because the cells do not divide. The morphological and biochemical characteristics of cultured adult ventricular cardiomyocytes from rat have been described previously in great detail (Piper et al. 1982, 1988, 1990). The following examples indicate the yield of cells and the quality of cell preparations based on the ratio of rod-shaped to rounded cells found with different preparations. In addition, approximately 5% of the isolated cells undergo apoptotic cell death (Taimor et al. 1999). As indicated, the number of cells and ratio of rod-shaped cells is quite stable over the next 24 h (Fig. 3). As shown elsewhere, cells maintain their protein content and rate of protein synthesis during this time period (Schlüter and Piper 1992). The preparation is therefore quite stable and suitable for biochemical or physiological analysis. However, the energy metabolism and rate of protein synthesis are the basal values for non-contracting myocytes without the energy demand of a working heart. These non-loaded and non-contracting myocytes can be used to investigate the regulation of protein synthesis by neurohumoral factors independently from mechanical influences. The example shown in Fig. 4 indicates stimulation of protein synthesis by Figure 3 Change in number of cells (right) and percent of rod-shaped cells (left) during 24 h cultivation of cardiomyocytes Figure 4 Influence of phenylephrine (an α-adrenoceptor agonist) on protein synthesis and protein content of cardiomyocytes cultured for 24 h in the absence (basal value) and presence of the agonist (10 µmol/l). *p <0.05 vs. untreated cultures

9 Isolation and Culture of Adult Ventricular Cardiomyocytes 565 Figure 5 Effect of different times of reconstitution of mechanical activity (0.5 Hz). Cells were paced for 0.5, 1, 2, or 4 h starting 4 h after isolation. The rate of protein synthesis was determined by incorporation of 14 C-phenylalanine into cell protein during the next 24 h Figure 6 Cell contraction of cardiomyocytes paced at 0.5 Hz 4 h after the isolation (day 0) and 24 h later (1 day). Cell contraction is expressed as cell shortening relative to the diastolic cell length Figure 7 Influence of phenylephrine (PE), an α-adrenoceptor agonist and hypertrophic stimulus, on the relaxation velocity of isolated cardiomyocytes. Cells were cultured for 24 h in the presence of the agonist (10 µmol/l) and paced at 0.5 Hz thereafter. * p< 0.05 vs. control cultures, which were not treated with phenylephrine α-adrenoceptor stimulation. As indicated, protein synthesis, determined by incorporation of 14 C-phenylalanine, increased by 34%, and in parallel total protein content increased by 24%. The basal value of protein synthesis can be increased by mechanical loading of the myocytes by reconstitution of mechanical activity. As shown on Fig. 5, a brief period of electrical pacing is sufficient to increase the amount of protein synthesis. In a similar way, external loading of the cells increases basal rates of protein synthesis, allowing analysis of the response of the individual cells to passive load. To perform such experiments, cells can be attached to flexible silicone materials by precoating with poly-l-lysine. Loading of the cells must be increased stepwise to allow the cells to adapt to the external load. The contractile activity of isolated cardiomyocytes can be analyzed by cell edge detection techniques, allowing the determination of the mechanical properties. As shown in Fig. 6, cardiomyocytes paced at between 0.5Hz and 2.0 Hz contract regularly under basal conditions when stimulated four hours after preparation. After 24 h, cell contraction is still inducible, however, the basal values are slightly reduced. The isolated cells are able to react to hypertrophic stimuli as they do in the intact heart, i.e. induction of hypertrophy by α-adrenoceptor

10 5 566 Cell Culture Techniques stimulation causes a significant increase in relaxation velocity, caused by an up-regulation of SERCA2A (Fig. 7). These examples clearly indicate the usefulness of the isolated cell system in analyzing the biology of isolated cardiomyocytes. In-Vitro Techniques Troubleshooting During the isolation and cultivation of ventricular cardiomyocytes from adult animals problems may occur at various steps. At the end of the isolation procedure, the yield of cells may be quite large but nearly all of the cells are hypercontracted and round. The problem can arise at different steps. First, the heart might not be smooth at the end of the collagenase treatment period. Mostly the reason is that the blood was not fully removed from the heart initially and coagulated in some of the vessels. Thus, the heart has not been perfused adequately. You can resolve this problem either by reducing the time between the isolation of the heart from the animal and the connection to the perfusion system or alternatively by injecting heparin into the circulation of the animal shortly before starting the preparation to avoid coagulation. The second problem can be that even though the heart has been smoothly, cells are round. In this case, view the morphology of the cells after each step of the calcium gradient. If the cells are initially rod-shaped and round up during the increasing of the calcium gradient, then they are obviously not calcium tolerant. Reduce the time of collagenase treatment or re-establish a physiological calcium concentration more carefully, i.e. as suggested for mouse hearts (see above). If the cells are already round at the beginning of the calcium reconstitution, than repeat with new buffers and new collagenase batches. We found, that the system is very sensitive to the quality of water. Use the highest quality of water purification to prepare your buffers. The next problem may occur after the initial isolation. One problem that often occurs is that the cells looked quite nice initially, but hypercontract during the next few hours. This is often the case, if the cells tend to beat spontaneously. Again, the cells are not perfectly calcium tolerant. Reduce the time of collagenase treatment. Even if you lose a small amount of rod-shaped cells by reducing the time of collagenase treatment, the resulting cells are much more calcium tolerant and survive much better. One of the most common problems is that people focus too much on the initial morphology of the cells. It is more important to have a look at the cells after 4 h, when replacing the medium and removing the round and non-attached cells, than to examine the initial morphology. Another problem that often occurs is that the cells do not attach to the culture dishes. In general, cells should attach to culture dishes pre-treated with 4% (vol/vol) FCS. If not, test different batches of FCS. If the problem persists, then reduce the time of collagenase treatment. Never use culture dishes specially prepared for primary cultures. Falcon dishes (3001 or 3004) were found to be quite useful. If necessary, replace FCS by laminin. When you culture cells in the presence of FCS for a couple of days, they may be overgrown by non-myocytes. This can be avoided by the addition of cytosine-β-darabinofuranoside (Ara-C, 10 µmol/l) for the first 5 days.

11 Isolation and Culture of Adult Ventricular Cardiomyocytes 567 Finally, it should be noted that even in laboratories with long-standing experience of the isolation of cardiomyocytes, the quality of cell preparations declines from time to time. It is important to establish a routine procedure and to repeat experiments with several cell preparations. Some general guidelines may help to shorten the search for the cause of failure. Avoid the use of detergents in cleaning the glassware used for cell isolation. A record should be kept of the use of all materials involved in cell preparations, particularly of all chemicals. If some doubt about the purity of chemicals arises, all should be exchanged for unopened batches. A possible cause of failure is the quality of the water used to make up the solutions. In some places distillation or ion exchange systems are fed with water from a common primary ion-exchange purification system that releases volatile organic impurities. It is also important to keep the perfusion system clean. In general, the system should be flushed with water after the preparation to avoid proteins from the perfusate drying on the glass. Even if the perfusion system does not need to be sterile, it is advisable to flush the glassware after use with 70% ethanol for 30 min and subsequently dry it with a stream of clean gas (e.g. filtered compressed air). References Pinson A, Schlüter K-D, Zhou XJ, Schwartz P, Kessler-Icekson G, Piper HM (1993) Alpha- and beta-adrenergic stimulation of protein synthesis in cultured adult ventricular cardiomyocytes. J Mol Cell Cardiol 25: Piper HM, Jacobsen SL, Schwartz P (1988) Determinants of cardiomyocyte development in long-term primary culture. J Mol Cell Cardiol 20: Piper HM, Probst I, Schwartz P, Hütter JF, Spieckermann PG (1982) Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol 14: Piper HM, Spahr R, Schweickhardt C, Hunnemann D (1988) Importance of endogenous substrates for cultured adult cardiac myocytes. Biochem Biophys Acta 883: Piper HM, Volz A, Schwartz P (1990) Adult ventricular rat heart muscle cells. In: Piper HM (ed) Cell culture techniques in heart and vessel research. Springer Verlag, Berlin, p Schlüter K-D, Goldberg Y, Taimor G, Schäfer M, Piper HM (1998) Role of phosphatidyl 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes. Cardiovasc Res 40: Schlüter K-D, Piper HM (1992) Trophic effects of catecholamines and parathyroid hormone on adult ventricular cardiomyocytes. Am J Physiol Heart Circ Physiol 263:H1739 H1746 Schwartz P, Piper HM, Spahr R, Hütter JF, Spiekermann PG(1985) Development of new intracellular contacts between adult cardiac myocytes in culture. Basic Res Cardiol 80 [Suppl. 1]:75 78 Taimor G, Lorenz H, Hofstaetter B, Schlüter K-D, Piper HM (1999) Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat: Cardiovasc Res 41: Taimor G, Schlüter K-D, Frischkopf K, Flesch M, Rosenkranz S, Piper HM (1999) Autocrine regulation of TGFβ expression in adult cardiomyocytes. J Mol Cell Cardiol 31: