chick car- diomyocytes were mixed with collagen solution and allowed ..... night incubation at 4#{176}C,samples were rapidly filtered over glassfiber filters .... course of [3HJ thymidine incorporation and RNA yield of CMPMs. .... a mechanical.
Three-dimensional cardiomyocytes muscle model
reconstitution in a collagen system
of embryonic matrix: a new heart
ThOMAS ESCHENHAGEN,’ CHRISTINE FINK, UTE REMMERS, HASSO SCHOLZ, JENS WATFCHOW, JOACHIM WElL, WOLFRAM ZIMMERMANN, HANS H. DOHMEN,* HANSJORG SCHAFER,* NANETFE BISHOPRIC,f TETSURO WAKATSUKI,t AND ELLIOT L ELSONt
Abteilung Allgemeine Pharmakologie and *Institut f#{252}r Pathologie, Universit#{227}ts-Krankenhaus Eppendorf, Universit#{227}t Hamburg, Germany; tSRI International, Menlo Park, California 94025-3434, USA; and Department of Biophysics and Molecular Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA
A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of ct-actin and cc-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilainents, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive “staircase”), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant 3-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOl 0.1) to about 50% (MO! 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgemc animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.-Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Well, J., Zimmermann, W., Dohmen, H. H., Schafer, H., Bishopric, N., Wakatsuki, T., Elson, E. L. Three-dimensional reconsti-
ABSTRACT
0892-6638/97/0011
-0683/$01 .50 © FASEB
tution of embryonic matrix: a new heart 11, 683-694 (1997)
cardiomyocytes in a collagen muscle model system. FASEBJ.
Key Words: heart . cell culture noviru.s transgenic model
reconstituted
tissue
ade-
CULTURES OF EMBRYONIC OR NEONATAL cardiac myocytes have been used in cardiovascular research for about 40 years (1) and have become a standard model system to investigate ion channel function, regulation of cardiac gene expression, and molecular mechanisms of cardiac hypertrophy. Widespread use of primary cardiomyocyte cultures is due both to the lack of stable cardiomyocyte cell lines and to the fact that they exhibit stable spontaneous beating for several days and allow pharmacological and genetic manipulations without interference from compensatory mechanisms that operate in animal experiments. Nevertheless, monolayer cultures in general have important methodological and conceptual limitations. For example, skin fibroblasts markedly proliferate and synthesize collagen (up to 15% of total protein) in monolayer cultures but exhibit little proliferative and synthetic activity in normal skin or in a 3-dimensional collagen matrix in vitro (2). Similarly, cardiomyocytes change their pattern of gene expression toward an undifferentiated phenotype in monolayer culture (3-6). Most important, cultured embryonic or neonatal cardiomyocytes do not allow measurement of their major function in the heart, namely, loaded isometric contraction. Now that an increasing number of gene products has been identified whose functional role in a physPRIMARY
Correspondence: Abteilung Allgemeine Pharmakologie, Universit#{227}ts-Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
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iological context is largely unknown, and because molecular techniques allow overexpression or knockout of single genes or gene products, it is important to have methods for assaying the results of these genetic modifications. This is especially true for attempts to define the role of molecular alterations in pathophysiological states such as heart failure that represent subtle quantitative changes of constitutive cardiac genes (e.g., changes in -adrenoceptors, adrenergic receptor kinase, Ci proteins, SR calcium ATPase, phospholamban by ±30-50%; 7). Tissue models for studying functional consequences of genetic manipulations should therefore fulfill the following criteria: stable, reproducible, and simple measurement of the essential heart functions, efficient gene transfer, and a relatively natural and controlled physiological environment. The aim of the present study was to develop and characterize a cell culture system that meets at least some of these criteria. -
MATERIALS Isolation
AND
METHODS
of cardiomyocytes
Ventricles from 9-li day incubated chicken embryos were minced to i mm pieces in Dulbecco’s minimal essential medium (DMEM;2 Gibco-BRL, Eggenstein, Germany), washed once with 0.25% trypsin/0.1% EDTA (Boehringer Mannheim, Germany) in phosphate-buffered saline (PBS), pH 7.45, and then digested in fresh trypsin/EDTA for 15 mm at 37#{176}C. The supernatant was discarded and the pellet was subjected to digestion with 0.1% collagenase (144 U/mg, Bibby Dunn, Asbach, Germany) in PBS, pH 7.45, for 30 mm at 37#{176}C (8). This supernatant was discarded and the pellet digested further with several cycles of collagenase for 1020 mm each until the pellet was completely digested. DNase I (40 p.1, 1 mg/mI in PBS; Sigma, St. Louis, Mo.) was added between cycles depending on the presence of viscous DNA. The isolated cells were kept in petri dishes (Falcon Series 3000) in DMEM supplemented with i5% heat-inactivated fetal calf serum (FCS; Gibco BRL) in the CO2 incubator. After completion of the digestion, cells were incubated for another 30-60 mm in the CO2 incubator (preplating). The cell suspension was centrifuged at 250 rpm (i2Xg). The pellet was resuspended in 10 ml culture medium (DMEM, iO% inactivated horse serum, 2% chicken embryo extract (Gibco BRL), 2 mmol/l glutamine, 100 p.g/ml streptomycm, and 100 U/mI penicillin G (Gibco BRL), recentrifuged at 250 rpm, and finally resuspended in culture medium at 2-3 X 10 cells per ml. Casting
cardiomyocyte-populated
collagen
gels
The principal technique is shown in Fig. la (9). Strips of Velcro were glued with silicone rubber (No. 734, Dow Corn-
2Abbreviations:
essential
me-
dium; PBS, phosphate-buffered saline; MOl, multiplicity fection; CMPM, cardiac myocyte-populated matrices; cacodylate buffer; RT, room temperature; Ln,ax, maximal length; bpm, beats/nsin.
of inCB,
684
ing, Wiesbaden, ERG) to glass tubes (13 mm length, 3 mm outer, 2 mm inner diameter). Pairs of Velcro-coated tubes, kept at a fixed distance by a stainless steel wire spacer, were placed in rectangular wells (l5X17X4 mm) cut intoalayer of silicone rubber in a 100 mm polymethylpentene petri dish (Nalgene, Nalge Co., Rochester, N.Y.). This assembly was autoclaved before use. For each gel, 1 ml of an ice-cold collagen/cell mixture was poured into each well between the Velcro-coated glass tubes. This mixture had the same composition as the culture medium and contained in addition 1 mg neutralized collagen I from rat tail (Upstate Biotechnology Inc.), 1 X 106 cardiomyocytes, the acetic acid in the collagen solution, and the NaOH used to neutralize it. The mixture was allowed to gel at 37#{176}C for 60 mm before culture medium was added to the dish. Medium changes were performed after overnight incubation and then every other day.
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Dulbecco’s
minimal
force
b Force transducer
-
-
-
-
-
S.
Cell matrix Pacing electrode
S. Fixed pole
Figure
1. Illustration of the experimental arrangement. a) Photograph of a culture dish with six cardiomyocyte-populated collagen gels (day 5). The bottom of the dish contains a layer of silicone rubber with six rectangular wells that have been used to cast the CMPMs. The CMPMs show the typical concave shape of the free edges. b) Schematic representation of the force measurement technique. After 10 days in culture, one end of a CMPM was attached to a lower fixed pole, which also serves as an electrode; the other, via a noncompliant filament, was attached to an isometric force transducer. The transducer measurements were recorded on a thermal array recorder. Preload of the CMPMs was adjusted by raising the force transducer. CMPMs were paced with a Grass stimulator. The CMPM isimmersed in continuously gassed (95% 02/5% C02) Tyrode’s solution in a thermostatted organ bath.
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Isometric
force
measurements
After 6-11 days in culture, the gels were removed from the culture dish, the spacers were withdrawn, and one of the glass tubes was mounted on a fixed electrode (Fig. 1 b); the other tube was connected by an inelasticsilkstring to an isometric force transducer (Scientific Instruments, Heidelberg, Germany) attached to a Wekagraph thermal array recorder (FOhr Instruments, Heidelberg, Germany). The preparation was adjusted to its original (spacer) length before it was immersed in a conventional organ bath filled with modified Tyrode’s solution (NaCI 119.8, KCI 5.4, CaCI2 1.8, MgCI2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, glucose 5.0 mmol/l) maintained at 35#{176}C and continuously gassed with 95% 02 + 5% CO2, as described for papillary muscle previously (10). After a 30-60 mm equilibration period without pacing, force and frequency reached a stable value that we define as baseline force/frequency. Gels were then electrically stimulated with rectangular pulses (10 ms, 20-40 V) at a standard frequency of 1.5 Hz. Preload was stepwise adjusted to L, the length at which the preparation developed maximal force. After equilibration, the pacing frequency was varied from 0.8 to 2.5 Hz for 3 mm each and then returned to 1.5 Hz, which was held constant throughout the experiment. Inotropic interventions were performed as indicated in the Results section. The following stock solutions were used: 2.25 mol/l CaCl2, 10 mmol/l isoprenaline in 4 mmol/l HC1, 1 mmol/l propranolol, 30 mmol/l forskolin (Calbiochem) in 100% ethanol (giving a final maximal concentration in the bath of 0.01%), 10 mmol/l carbachol inTyrode’s solution, 2 mmol/l cytochalasin D in dimethyl sulfoxide (DMSO, final concentration in bath 0.1%). Stock solutions were prepared freshly (carbachol, ouabain, norepinephrine, propranolol) and stored for up to 2 wk at 4#{176}C (isoprenaline, forskolin) or at -20#{176}Cfor up to 6 months (cytochalasin D). The KC1-rich Tyrode’s solution was prepared by replacing 119.8 mmol/I NaC1 with 125.2 mmol/l KCI. All gels were exposed to a concentration-response curve (CRC) for calcium (1.8-12.6 mmol/l) and to one or two additional inotropic stimuli. The length of contraction experiments was 3 to 5 h. In that time basal force of contraction decreased by less than 30%. Histology,
immunohistochemistry,
and
electron
microscopy
Gels were either removed directly from the culture dish or carefully removed from the holding electrode after contraction experiments. For light microscopy, specimens were fixed overnight at 4#{176}C in 4% formaldehyde in PBS, pH 7.4, with the metal wire spacer attached. After dehydration in graded concentrations of ethanol and paraffin infiltration according to standard procedures, the specimens were cut from the Velcrocoated glass tubes, cut in half, and embedded in paraffin blocks in either of the three orientations (perpendicular and parallel to the glass tubes and flat). Four-micrometer sections were cut and stained with hematoxylmn-eosin or trichrome stain according to Masson and Goldner (11). For immunohistochemistry, 4 p.m sections were mounted on Superfrost Plus slides (Menzel-Gl#{227}ser, Hamburg, Germany) and dried overnight at 55#{176}C. After deparaffinization and rehydration through xylene and graded concentrations of ethanol, antigen retrieval was performed by boiling the slides in DAKO ChemMate buffer for antigen retrieval (DAKO A/S, Glostrup, Denmark) in a domestic pressure cooker for 4 mm at 120#{176}C. Endogenous peroxidase activitywas blocked by incubation in 3% hydrogen peroxide for 5 mm at room temperature (RT). Nonspecific binding was masked by incubation in normal goat serum (DAKO) diluted 1:5 in TBS, pH 7.6, for 20 mm at RT. Primary antibody incubation was done in a moist chamber for 1 h at 37#{176}C. The following antibodies were used: anti-muscle-actin (monoclonal, mouse, clone HHF 35, Enzo
CARDIAC
MYOCYTE-POPULATED
COLLAGEN
MATRIX
Diagnostics, Farmingdale, N.Y., purchased in ready-to-use dilution), anti-sarcomeric-tropomyosin (monoclonal, mouse, clone CH1, SIGMA, dilution 1:50), and anti-c-sarcomeric-actin (monoclonal, mouse, clone 5C5, SIGMA; in TBS, pH 7.6, with 3% BSA). The detection system consisted of the DAKO Duett streptavidin/horseradishperoxidase kit used according to the manufacturers instructions. Secondary biotmnylated goat-anti-mouse/rabbit antibody (1:100) was incubated for 30 mm at RT, the streptavidin-biotin complex for 30 mm. The sections were developed in a solution of SIGMA FAST diaminobenzidine and urea-H202 tablets in water for 5 mm at RT. Sections were counterstained with hematoxylin, dehydrated, and mounted in EUKITT. Negative and positive controls were obtained by omission of primary antibody, sections from chicken bowel, and whole heart. For electron microscopy, cardiac myocyte-populated matrices (CMPMs), with the spacer attached, were fixed overnight in a 2.5% solution of glutaraldehyde in 0.1 mol/l sodium cacodylate buffer, pH 7.3 (CB), rinsed in GB containing 7.5% sucrose, postfixed for 2 h in 1% Os04 in GB, rinsed with GB with sucrose, dehydrated in graded series of ethanol, and incubated for 1 h each in propylene oxyde and 1:1 mixture of propylene oxyde/epon. After infiltration and embedding in epon 812, ultrathin sections were obtained in an Ultracut S (Reichert, Austria), double-stained by uranyl acetate and lead citrate according to standard procedures, and visualized in a Philips EM 400 electron microscope.
[3H]Thymidine
incorporation,
cell
count,
and
RNA
preparation One microliter of [3H]thymidine (NEN DuPont, Bad Homburg, Germany; 20 Gi/mmol, 1 p.Ci/pi) was added to the GMPMs in 1 ml culture medium in the absence or presence of 5-bromo-2’-deoxyuridine (BrdU; 0.1 mM) at cultivation days 0, 1, 2, 3, 4, 5, and 6. After 6 h of incubation at 37#{176}C, CMPMs were washed 3 X 10 mm with 2 ml PBS, dissected from the Velcro, and dissolved in 1 ml 0.1% acetic acid. After overnight incubation at 4#{176}C, samples were rapidly filtered over glassfiber filters (GE 50, Schleicher & Schuell, Keene, N.H.). Filters were washed twice with 6% trichloric acid and once with 95% ethanol. Radioactivity was measured after overnight incubation in scintillation liquid. [3H]thymidine incorporation in the presence of BrdU amounted to less than 5% of that in the absence of BrdU and was defined as nonspecific binding. The number of intact nuclei was determined in individual samples by adding 0.001% Hoechst 33258 (Sigma) to the 0.1% acetic acid-solubilized CMPM and counting the number of intact nuclei under fluorescent light. Total RNA yield was determined in parallel samples by RNA extraction according to Chomczynski and Sacchi (12). RNA was quantified photometrically and checked by gel electrophoresis. Adenovirus-mediated
gene
transfer
A -galactosidase-encoding recombinant replication-deficient adenovirus type 5 (Ad5LacZ) was a kind gift from Dr. Harold Prentice, Glasgow, U.K. The virus was propagated in human embryonic kidney 293 cells (HEK 293), and the medium of infected cultures was used for infection of CMPMs. The virus titerwas determined by plaque assays on HEK 293 cellsand, to biologically compare different preparations of viruses, by infection of cardiac myocytes in monolayer cultures. Normal titers were 1-5 X iO pfu/ml. To define the optimal transduction efficiency, CMPMs were infected on day 4, 6, 8, 10, or 12 with 1, 10, 100, and 1000 p.1 virus-containing medium for 18 h at 37#{176}C and then incubated for an additional 48-72 h with complete medium. Control gels were subjected to the
685
Figure 2. Light microscopy of GMPMs (days 9-10). a) Paraffin section of a CMPM parallel to the plane of the tissue, showing the characteristic concentration of longitudinally oriented cells at one of the two free edges of the gel. Note the continuous cell layer at the surface and several cell layers that form cell-cell contacts (Masson-Goldner, ref ii). b) Same as panel a at higher magnification. Brown immunohistochemical staining of muscle actin (c, d), a-sarcomeric actin (e), and sarcomeric tropomyosin in paraffin sections similar to those in panels a and b counterstain with hematoxylmn. More than 80% of cells stain positive with each antibody. The photograph in panel d shows a prominent concentration of highly cross-striated cells at a free edge of the CMPM that is often found close to the Velcro-coated glass tubes. Magnifications: 128x (a), 256x (b c, e), 470x (d), 354X (f).
(/)
same conditions without the virus. Gels were thoroughly rinsed with PBS pH 7.4 (2X20 mm; essential to get rid of galactosidase in the virus-containing medium), fixed for 30 mm in PBS-buffered 2% formaldehyde, 0.2% glutaraldehyde at RT, washed 2 X 5 mm with PBS, and then incubated for 118 h in 2 mmol/l MgCl2, 20 mmol/l KFe(CN)6 II, 20 mmol/ I KFe(CN)6 III, 0.02% Ipegal (Sigma), 0.01% deoxycholate (Sigma), and 1 mg/ml X-gal at 37#{176}C. The reaction was terminated by washing with PBS and overnight incubation in 10% formaldehyde at 4#{176}C. Four-micrometer sections were cut from paraffin-embedded CMPMs in three different orienta-
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tions as described and stained with hematoxylin-eosmn, nuclear fast red, or according to Masson-Goldner (11). In parallel, galactosidase activity was determined by a colorimetric assay. In short, CMPMs were rinsed 3 X 10 mm with PBS, dissected from the Velcro, mechanically homogenized in 1 ml PBS at 4#{176}C, and subjected to one freeze/thaw cycle. Fifty microliters were incubated in 250 p.160 mmol/l sodium phosphate buffer, pH 7.5, 1 mmol/l MgCl2, 45 mmol/l mercaptoethanol, and 0.8 mg/ml 2-nitrophenyl-fl-D-galactopyranosid (ONPG; Boehringer Mannheim) for 30 mm at 37#{176}C. The reaction was stopped by addition of 350 p.1 1 mol/l Na2CO2. OD at 420 nm
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sion and reshaping of the gel from an original size of (thickness X width X length) 5 X 13 X 15 mm (1 ml drop) to biconcaval CMPMs, with an average size of 0.18 ± 0.06 mm (n=25) X 6-10 X 15 mm. Thus, the initial drop formed a stable tissue spanning the gap between the two glass rods (see Fig. 1 a, Fig. 11 a). As seen in fibroblast-populated collagen lattices, the free edges are concave, perhaps due to the balance of forces in this region (9). Spontaneous contractions of single cells were seen after 1-2 days, coordinated contractions of the entire CMPM after 3-5 days. CMPMs remained stable for at least 2 wk. After 6-8 days they did not change their gross macroscopic or microscopic appearance, nor did they stop beating. Light microscopy revealed a reproducable pattern of cell growth (Fig. 2). Sections parallel to the plane
a
incorporation
3H-thymidine
8 a a a
6
1 x 4.
E 0 C)
2
0
6
9
5
0123456
Figure 3.
Electron microscopy of a CMPM (day 10). a) Typical myocardial cell presenting cross-striation with Z-clisks (Z), transversal and longital tubules, and mitochondria (M); 16,500x. b) Adjacent myocardial cells presenting intercellular contact organelles. Note desmosomes (arrow) and tight junctions (arrowhead) adjacent to the peripheral part of a T tubule (T); 30,100x. c) Formation of a typical intercalated disk (arrow); 23,000x.
I
II
b
RNA yield per CMPM 15
*
*
10 was referred (Boehringer
to a standard curve of purified Mannheim, 300 U/mg).
*
*
3-galactosidase
5
Statistics All values presented are arithmetic means ± SEM. Student’s I test for paired observations was used in all cases to compare force of contraction, resting tension, or beating frequency before and after other interventions. Student’s I test for unpaired observations was used for all other experiments. A P value of less than 0.05 was considered significant.
0
0
1
2 Time
*
3
3 4
3 5
3 6
(days)
p‘ 0
C
a) a) I.IL
Ctr *p
