Cell cultures of adult cardiomyocytes as models of the myocardium

J MoI Cell Cardiol 18, 661-678 (1986)

REVIEW ARTICLE Cell Cultures of Adult Cardiomyocytes of the Myocardium

as Models

(Received 26July 1985, accepted in revisedform 24 January 1986) Review Outline (table of contents)

(]) Introduction A. Problems posed by intact cardiac tissue.

B. In Vitro preparations. 1. Tissue cultures of immature myocardium. 2. Cell cultures of immature myocardium. 3. Newly isolated adult cardiomyocytes. 4. Cultured adult cardiomyocytes. a. Redifferentiated model. b. Rapid attachment model.

(I1) Methods for culturing adult cardiomyocytes A. Cell isolation. 1. The role of Ca z+ 2. Enzymes and mechanical treatments. B. Criteria ofcell condition. 1. Dye exclusion. 2. Morphological criteria. 3. Biochemical criteria. C. Culture substrata. 1. Role of the substratum. 2. Specific properties of substrata. D. Culture media. 1. Defined culture media. 2. Supplements to defined media. E. Control of non-myocytes, chemical and mechanical methods.

(II1) Properties of cultured cardiomyocytes A. Morphology. 1. Rapid attachment model. 2. Redifferentiated model. B. Thymidine uptake and mitosis. 0022-2828/86/070661 + 17 $03.00/0

C. Metabolism. 1. Rapid attachment model. 2. Redifferentiated model. D. Electrophysiology. 1. Rapid attachment model. 2. Redifferentiated model.

(IV) Prospects andproblems A. The state of the art. B. Conclusions.

I. Introduction The complex organization and interactivity of cells in cardiac tissue have limited the extent to which individual myocardial ceils could be characterized. Progress in cellular cardiology requires experimental preparations that circumvent problems such as :

(a) Cellular heterogeneity Myocytes comprise 80% of the heart mass, but they represent only about 20% of cardiac cells [6, 33, 82]. Fibroblasts, neurons, endothelial cells and cells of the epicardial and endocardial layer comprise the balance. The variety of cells makes it difficult to attribute properties of tissue to any one cell type and various types interact in a way that cannot be analyzed in situ.

(b) Intercellular coupling Electrical and mechanical intercellular coupling can make it impossible to distinguish effects due to intrinsic cellular properties from those due to cell interactions [63, 101]. 9 1986AcademicPress Inc. (London) Limited

>.. co

o

e~

~2

9 I '>"0

o4

~

~5

=-

=

~

o~

aU

a~

aU

o~

+~.

o .~ Xg

+~

X-~ ~

g

~-~

~oo

~0

~0

a~

a.g

a

dg

:~&

ci ~

~=.= ><

"12

.>'a=

~-~.~ ~ >~

~.=

~

~ - ~ 9, - ~ . ~. ~ -~=-~ ~

"~

~

.~. "

0o

~z

<'~z

o4

~ o = op.

~oo

~

o

--,:.

g-~ ~ . ~ z

~o

~ ~.~

L~..,4

oi

do'

"0

~

em

dg

"12

=,-~ o

t:m

.

ca

I

[

I

kO

+-t-~

+ I X~

~Lf

0

I

I

(

o'r~ tmo 9

9 o

r~ 0em

o

0 em O0

0 em

oo

o

0 r ~0

r, 0 em ~0

o

E

~

o ~

F.-~

0

.~ ~ r ~

~

~ Oe~ emO

0 0"~

o

v

ttb

o,i

A

8

4.a

~ u

A

0

em o,i

V I

0,1

V+

VV

++

I

I

0 0

Z

0~ e~

664

S . L . J a c o b s o n and H. M. Piper

Notes (a) Numbers in ( ) are literature citations, see bibliography section. (b) Redif. = Redifferentiated model; Rap. Att. = Rapid attachment model. (c) H = human; R = rat, G.P.= Guinea Pig; F = female; M = male; S-D = Sprague-Dawley. (d) MEM(+) = Minimal E s s e n t i a l Medium (supplemented) ; M199 = Medium 199. (e) FBS=Fetal bovine serum; HS=horse serum; CS = Calf serum; P = Penicillin; S = Streptomycin. (f) 4% FBS 1st 3 h of culture, then 0% FBS. (g) M = from minced tissue; L = by Langendorff perfusion. (h) T = trypsin; C = collagenase; H = hyaluronidase; E = elastase.

(c) Multicompartmentation M a n y experiments require controlled rapid exchange between interstitium a n d b a t h i n g media. I n the perfused heart, the capillary wall provides a barrier a n d arterio-venous gradients, which m a y vary within the heart, must be considered. Even in small tissue specimens, diffusion pathways are not negligible. T h u s the m y o c a r d i u m is a complicated multic o m p a r t m e n t system [63] a n d precise control of the interstitial milieu is not possible. A variety of experimental preparations are used by cellular cardiologists. Tissue cultures from i m m a t u r e hearts, d a t i n g back 75 years [14, 46, 126], are a m o n g the earliest in vitro preparations. T h e use of enzymes, by Dulbecco [35] a n d Moscona [83] to isolate single cells for cultures are more recent notable events. T h e i r method applied to chick embryo heart, in 1955, by C a v a n a u g h [18] a n d to n e o n a t a l rat heart by H a r a r y and Farley [53], in 1957, produced the first cultures from isolated cardiac cells. Subsequently m a n y other workers used cultured chick embryo a n d n e o n a t a l rat cells in culture leg. 31, 66, 75, 115, 123], as well as cells from embryonic a n d neonatal, mouse [9, 45] hamster [7] a n d h u m a n embryo [51]. Cultures of cardiomyocytes of i m m a t u r e animals gained wide acceptance as models for studies of cellular cardiology. I m m a t u r e cells, however, differ from adult myocardial cells leg. see 23], and a more accurate model of adult m y o c a r d i u m was sought, at first, in isolated cells from adult animals. T h e isolation of adult cardiomyocytes that remained vital in media of physiological Ca z +

(i) Sedim= density sedimentation; ARA-C = cytosine1-/~-D-arabinofuranoside; Glut = glutamine, S.P. = selectiveplating. (j) TR. GL. = treated glass; PL=plastic; Colag= collagen; Gelat = gelatin. (k) THY < 4%24h = max. % cells which incorporate thymidine in 24 h period. (1) Oct = octanoate, glu = glucose,lact = lactate. (m) value calculated from author's published data. (u) 1BMX = 1-isobutyl-3-methylxanthine; Epi = epinephrine; DCA = dichloroacetate. (o) Spont. =contracts spontaneously; Elec. Stim. = responds to electric stimulation. (p) TTX-tetrodotoxin; D600= methoxyverapamil; Epi = epinephrine; + = sensitive. (q) Trans = transient; dely'd = delayed; out. = outward.

c o n c e n t r a t i o n a n d temperature i.e., Ca 2+ tolerant cells, proved to be a problem. T h e early work of K o n o [71], Berry, et al. [6] a n d V a h o u n y , et al. [128] led to that of Powell a n d Twist a n d others [33, 34, 37, 105, 107, 108], a n d to preparations of Ca2+-tolerant isolated adult m a m m a l i a n cells. Newly isolated cells are increasingly used for short-term experiments, b u t there is evidence that they can have a b n o r m a l metabolic properties for several hours after isolation [27]. This, a n d the inevitably declining condition of isolated cells, argues for the use of cultured preparations whose cells, after a period of time, recover a relatively n o r m a l stable state. Cultures offer additional advantages of economy of time a n d sparing of experimental animals. A n a n i m a l n o r m a l l y used for one experiment c a n provide cell cultures for several days' experiments. T h e earliest report, k n o w n to us, of in vitro growth of cardiac tissue from an adult a n i m a l appeared in 1974 [47]. Explanted rabbit tissue was used. No m e n t i o n is made of contractility of outgrowing cells, b u t observation of striated cells is reported. I n several attempts, isolated cardiomyocytes from a d u l t animals either failed to establish in culture [50, 51, 54, 67] or produced cells lacking convincing myotypic properties [90]. A culture of isolated CaZ+-tolerant a d u l t cardiomyocytes was described by J a c o b s o n in 1977 [59]. W h e n established in culture, the cells contracted spontaneously for up to 60 d. C l a y c o m b a n d Palazzo in 1980 [29] a n d Nag a n d C h e n g in 1981 [88] also described cultures of cardiomyocytes from adult rat ventri-

Cell Cultures of Adult Cardiomyocytes cle, and Cantin, et al. in 1981 [16] reported culturing atrial cells from adult rat. In the aforementioned systems of adult cell culture, cardiomyocytes undergo, stereotypically, a gradual morphologic transition from the elongate in vivo cell shape to a spheroidal shape. Many cells in these cultures contract spontaneously during the transition, which takes several hours to days, and is distinct from hypercontracture, the fatal rounding of damaged isolated cardiomyocytes that occurs in seconds. During the gradual transition, cells lose much of their myotypic structure, but myotypy is regained during a subsequent flattening and spreading phase of cell development that follows attachment of cells to the culture substratum. Myotypic order returns in a way that resembles its original development during cellular differentiation in vivo. Therefore we will refer to this type of culture as the redifferentiated model. A problem with this model concerns its accuracy as a model of terminally differentiated cells in vivo. A second culture system of adult cardiomyocytes introduced by Piper, et al. in 1982 [98] contrasts sharply with the redifferentiated model. In this system, isolated Ca 2 +-tolerant cardiomyocytes attach rapidly to serum pretreated culture dishes. Attached cells do not contract spontaneously and they maintain the characteristic morphologic features and energetic stability of intact cardiomyocytes for periods of about 1 week. We will refer to this type of culture as the rapid attachment model. A problem with this model is that with time in culture the number of adherent cells decreases, limiting, for many purposes, the useful life of this system. The two models provide complementary systems with redifferentiated cells taking 10-14 d to reach a stage with stable myotypic features and rapid attachment cells being useful primarily for earlier, shorter-term experiments. With so few years of experience, rigorous assessment of the full potential of these adult cell culture preparations is premature. Data are incomplete but those from similar preparations studied in different laboratories can be lumped to gain a broader picture. The use of different techniques and conditions for cell isolation and culture, however, requires that conclusions based on

665

lumped data be considered as tentative, pending full data on individual preparations. Before considering properties of cells in the two culture models, we will discuss cell isolation and other methodology for culturing adult cardiomyocytes.

II. M e t h o d s f o r c u l t u r i n g a d u l t cardiomyocytes Progress in adult cardiomyocyte cultures required suitable methods to prepare Ca 2+tolerant cardiomyocytes. This is reviewed elsewhere [33, 34, 37], and we consider only special aspects of the subject here. The role of Ca 2+ in cell isolation is of central importance and .has been extensively investigated [cf 33]. Cell cohesion depends upon the prfsence of Ca 1+. Therefore it is necessary to expose tissue to unphysiologically low Ca 2+ concentrations, on the order of 10 -5 to 10 -4 M, to separate cells [87]. With adult myocardial cells, this necessity poses a problem. After isolation, when they are again exposed to media containing physiologic levels of Ca 2+, 10% to 20% of cells, in even the best preparations, are Ca / + intolerant; ie. they undergo irreversible hypercontraction within a few seconds to a few minutes. It is suggested [21, 37] that the failure of early attempts to isolate Ca2+-tolerant cardiomyocytes might be due to a too assiduous effort to lower Ca/+ concentrations during the cell separation process. This suggestion arises from the idea that Ca 2+ withdrawal might elicit the 'Ca 2+ paradox' [131]. Thus, Powell hypothesizes [106] that his success in isolating Ca 2+-tOlerant cells is due to avoiding too low Ca 2+ concentrations [30]. However, isolated myocardial cells are not subject to the Ca 2+ paradox. In contrast to perfused myocardium, isolated cells can be incubated 30 rain at 37~ in the presence of 1 mM EGTA without undergoing irreversible hypercontracture on re-exposure to normal Ca 2+ levels [103]. Instead of the Ca 2+ pardox, the necessity for avoiding too low a Ca 2 + concentration may arise from a requirement for a minimal Ca 2+ concentration to heal small sarcolemmal disruptions inflicted on intercalated discs during cell separation [6, 32]. In any case, Ca 2 + concentrations not

666

S.L. Jacobson and H. M. Piper

lower than 2 to 5 x 10 -SM appear to be necessary for obtaining Ca2+-tolerant adult cardiomyocytes. In cases where some Ca z + is not added to isolation media, the tissue probably supplies the critical Ca 2 § [120]. All methods for isolating Ca2+-mlerar~t adult cardiomyocytes, in a quantity sufficient for cultures, employ enzyme treatment of tissue with concomitant or subsequent mechanical treatment such as trituration by repeated pipetting, shaking, stirring or abrading. Exposure of tissue to enzymes is accomplished in one of two ways. (a) Retrograde perfusion of the coronary circulation of an intact heart, via the aorta, with a saline enzyme solution (Langendorff method); (b) Immersion of small pieces of cardiac tissue in a saline enzyme solution. In the former case, tissue is subsequently treated mechanically to disperse cells. In the latter case, enzymic and mechanical treatment are concomitant. Both methods give adult cardiomyocytes suitable for preparation of cultures. The Langendorff method usually gives a somewhat greater cell yield while the immersion method facilitates maintenance of sterility and enables isolation of cells from small pieces of tissue, as with tissue from human heart surgery [60]. Various enzymes including trypsin, hyaluronidase, elastase, crude collagenase and non-specific proteases have been used to isolate adult cardiomyocytes leg. 4, 15, 16, 29, 88, 107]. Only some of these have been used for adult myocytes established in culture. With Langendorff methods, collagenase is always used, either alone [29, 98] or with other enzymes [4, 88, 127]. Atrial myocytes have been isolated by immersion with trypsin [16] or with trypsin plus collagenase [60], and ventricular cells have been isolated with trypsin plus collagenase [59, 60]. Isolation of ventricular cells by immersion requires exposure to both trypsin and collagenase [60, 128], otherwise very few cells are obtained. In some cases a slimy material containing DNA [2, 125] is produced, entrapping isolated cells and reducing cell yield. Addition of DNAase to the cell isolation media alleviates the problem

[79]. Two methods for isolation of Ca 2 +-tOlerant adult cardiomyocytes do not fit the usual pattern. One [5] lowers Ca 2 + to 250/~M and uses pronase, a crude protease mixture. The

second [8] uses Ca z +-free medium containing Ba 2 + or Sr 2 + and no enzyme. Unfortunately, cell yields are not reported for these methods and little is said about cell integrity, leaving doubt whether cell quantity and quality are adequate for. cel.l~cuItures.. The large variety of currently used methods suggests that success in cell isolation is achieved mainly by trial and error. Regardless of the specific method used, when setting up to isolate and culture adult cardiomyocytes, the efficacy of every batch of chemicals and supplies must be tested.

B. Criteria of cell condition There is no technique of choice for evaluating the condition or predicting survival of adult cardiomyocytes in culture. Cells' ability to exclude a dye such as trypan blue [95] is regarded as the simplest viability test. But this is probably unreliable for adult cardiomyocytes. In two redifferentiated preparations, 80% to 90% of newly isolated cells [89] or 90% of cells after 24 h [29] took up trypan blue, yet in each case more than 50% survived in culture. Furthermore, Cheung et al. [20], examining several means of assessing condition of adult cardiomyocytes, conclude that trypan blue exclusion is unreliable. A T P content, ability to contract in response to electrical stimulation, and maintenance of elongated, striated morphology are judged to be the most useful measurements. Consistent with that, Piper, et al. [98] note a high correlation of content of high energy phosphate with electrical excitability and maintenance of elongated, striated morphology. All things considered, the percentage of cells which maintain in vivo-like morphology and electrical excitability seems the most dependable and easily measured criteria of cell condition.

C. Culture substrata Understanding the interaction of cardiomyocytes with the culture substratum may lead to superior culture models. Attachment to a substratum influences cell morphology and function. For example, in redifferentiated model cultures, some cells attach to the substratum before rounding up. These develop directly in a spread elongate form, maintaining a high degree of myotypy for periods of several weeks [Jacobson, unpublished, 89].

Cell Cultures o f Adult C a r d l o m y o c y t e s

Maintenance of myotypic order in these early adhering redifferentiated cells and in rapid attachment cells is evidence that substratum contact stabilizes internal structure, and the mechanism seems to be more than simple mechanical support. During the elongate-toround transition, myotypic structure of redifferentiated model cells degenerates. But this is reversed [48, 84, 89] once the cells attach to the substratum. Attachment appears to trigger a shift from breakdown to synthesis of cell structural elements. Various substrata can be used to grow redifferentiated cells ( s e e Table). These include cell-culture plastic [4, 29, 127], gelatin [16], rat tail collagen [27, 88], hepatocyte monolayers [121] and glass treated [59] to change surface charge [111]. Cells on all of these surfaces follow the pattern of morphological change characteristic of redifferentiated cells, although rat tail collagen and hepatocytes tend to promote spreading of cells directly, without rounding up. Studies of attachment of adult cardiomyocytes to surfaces are beginning to appear. One study compares interaction of adult and neonatal rat cardiomyocytes with substrata coated by extracellular matrix molecules [10]. Neonatal cells attach to fibronectin, laminin, collagen types I, II, III, IV and V, denatured collagen, and reconstituted collagen fibers. Fibronectin, laminin and collagen IV are their preferred surfaces. Adult cardiomyocytes attached efficiently to laminin and collagen IV, and weakly to fibronectin. Attachment of adult cells occurs first at their ends, in the area of the intercalated disk. In further studies [79, 80], adult cardiomyocytes are shown to adhere to surfaces treated with collagen IV, foetal bovine serum, laminin, and fibronectin, in order of decreasing preference, with very little difference between serum and laminin. However, after 2 weeks in culture substrata coated with laminin or collagen IV retained nearly 10-fold more cells than one treated with serum. Another study [104] presents data on adhesion of rapidly attached adult cardiomyocytes to a variety of surfaces including those treated with ten different types of serum, ten extracellular matrix components and several materials known to promote cell attachment. Only materials which selectively bind intact

667

cells were judged satisfactory. Several practical conclusions can be drawn from this study: (a) There are large variations in efficiency of cell attachment to commercial culture plastics. (b) Foetal bovine serum and laminin are the most efficacious surface treatments. (c) Pretreatment with foetal bovine serum counteracts variability in culture plastics and must be used at concentrations of 2% to 10% for optimal results. (d) A modified plastic which covalently binds serum proteins gives good attachment in the presence of 4% foetal bovine serum without serum pretreatment. Extension of this study to follow-up cell development in culture would be useful. In a recent study, a reconstituted basement membrane gel is described [69] which might be a promising substratum for adult cardiomyocytes. This material is a blend of extracellular matrix components including laminin, collagen IV, heparin sulfate proteoglycan, nidogen and enactin. Sertoli testicular cells, grown on this substratum, exhibit morphological and functional features remarkably like those of Sertoli cells in vivo

[49]. D. Culture media Redifferentiated cells can be cultured in various defined media, eg, Minimum Essential Media [16, 29, 88], F12K [127], Medium 199 [59, 98, 121] and Weymouth's or William's media [98]. Each defined medium is supplemented by 4% to 20% serum of one or more type i.e., foetal bovine, calf, or horse serum. In one case, NuSerum, a commercial serum substitute containing 25% bovine serum, was used, in rat cell cultures, to replace serum ; but neither homologous nor analogous adult rat serum was suitable [60]. In addition to serum, Claycomb and Lanson [28] use a conditioned medium from corneal epithelial cells as a medium supplement. With redifferentiated model cells, defined culture media must be supplemented with serum. If it is omitted, myocytes do not attach to the substratum, and they degenerate after a few days. In contrast, rapid attachment cells can be cultured for several days in Medium 199 without serum after an initial 4 h attachment period with 4% foetal bovine serum in the medium. When serum is supplied for a time after attachment, the cells round up by

668

S.L. Jacobson and H. M. Piper

the first two days in culture. This observation was reproduced with 25 different batches of foetal bovine serum. Furthermore, neither heat inactivation nor addition of fatty acids, phospholipids or cholesterol changed the result [96]. In another study [78] where rapid attachment cells are kept in medium with serum, the cells begin to spread out earlier than would be expected if serum were absent. It appears that sera contain factor(s) which promote morphological instability, as well as factors which help establish cells in culture. This suggests that some undesired properties of cultured cells may arise from the influence of serum factors. It is known for example, that serum influences contractile activity of redifferentiated cells [61], and serum lipoproreins are known to affect action potentials of cultured immature cardiomyocytes [112]. The use of defined factors to replace serum in adult cultures would represent a significant improvement. Though not yet achieved for adult cardiomyocytes, there are defined media for long-term cultures of immature cardiomyocytes [24, 52, 68, 73].

new culture vessel [16, 29, 88, 127] leaving the attached non-myocytes behind. A third method for control is the addition of cytosine-l-fl-D-arabinofuranoside (ARA-C) to culture medium for the first 5-7 d [60, 86]. Afterwards, medium without ARA-C is used. Over 99% of non-myocytes are eliminated after 3 weeks in culture (Jacobson, unpublished). A fourth method, omission of glutamine from growth medium, has been used in cultures of cells from immature animals [22]. In adult cell cultures, it has been used only in conjunction with ARA-C or with a selective plating technique [29]. Therefore its effectiveness, per se, is unknown.

HI. Properties o f cultured cardiomyocytes

A. Morphology

Rapid attachment model cells adhere to serum pretreated dishes [98] within 3 h of plating. Attachment is mediated by sarcoplasmic projections that contact the subE, Controlof non-myocytes stratum at intervals equal to sarcomere Techniques for isolation of adult cardio- length. By 19 h foldings of the intercalated myocytes also yield non-myocytes. Mitosis in disk are reduced and remnants of adherent or cultured ventricular myocytes is slow or internalized gap junctions have disappeared absent, but non-myocytes have a high rate of [118]. By 2 d, the sarcolemma at the ends of mitosis. Unless proliferation of non-myocytes cells is smooth and has a continuous glycois controlled, they become the dominant cell calyx [98]. By this time the ends of many cells population in culture. enlarge and extend pseudopodia. However, a Four methods are used for control of non- general spreading and flattening does not myocytes in adult cardiomyocyte cultures. occur as is the case for redifferentiated cells. The first is density sedimentation of the newly Occasionally a clump of isolated cells are isolated cells before plating [60, 98, 121]. The plated and after 4 h they interact as evisedimentation medium of choice is 3% to 6% denced by formation of close membrane conbovine serum albumin in a saline medium tacts with the appearance of gap junctions [108]. In cultures of rapidly attached cells, [121]. These junctions form at end to side connon-myocytes are removed by sedimentation tacts between cells as well as with the usual prior to plating, while periodic flushing of cul- end to end contacts seen in vivo. A similar phetures with fresh medium gives a virtually nomenon has also been observed in a suspenhomogeneous myocyte population since sion of isolated cells [129]. damaged myocytes do not remain attached to The ultrastructure of cells of the rapid the substratum and are washed away [98]. attachment model, for the first 7 d in culture, A second method, selective plating, can be characterized as normal. Myofibrillar, depends upon the fact that non-myocytes sarcomeric, mitochondrial, sarcoplasmic retiattach to a substratum sooner than culum and T-tubule structure is maintained. redifferentiating myocytes. With this method, Abnormalities during the first days, in rapid at a predetermined time non-adherent cells, attachment cultures, are internalized gap predominantly myocytes, are decanted into a junctions and development of pseudopodia.

Cell Cultures of Adult Cardiomyocytes

By 6 d there are signs of beginning rarefaction of myofibrillar structure resembling atrophia in denervated skeletal muscle [,118]. After 7 d cells round up in increasing numbers and are lost when medium is changed. Cells which remain, spread out during the second week of culture (Piper, unpublished). From this it might be suggested that rapid attachment cells undergo, in essence, the same morphological development as redifferentiated cells but with the morphological transition from the in vivo form occuring at a later time. Cells of the redifferentiated model undergo profound morphological changes which have been described [-84, 86, 89, 120]. They eventually develop myotypic properties, but the timecourse of development varies from cell to cell [-89, 120] and the extent to which cells become developmentally homogeneous after some time in culture is unclear. Newly isolated myocytes with the elongated morphology of cells in vivo gradually take on a spheroidal shape. Rounding starts at one or both ends and moves centrally with the cell forming a spheroid. Fifty percent of cells undergo the elongate-to-round transition in 5 d, with the range of times 1 to 9 d [,120]. In rounding, the intercalated disk region is the site of earliest changes [-84]. Myofibrils at that time lose their parallel, closely packed organization, and disruption of mitochondria, sarcoplasmic reticulum, and T-tubules is apparent. In one preparation, observed in phase contrast microscopy [,120], cells in transition were characteristically free of blebs and other damage. However, in an electron microscopic study [84] of a different preparation, membranes became blebbed and the cells were permeable to trypan blue. These differences may only reflect the higher resolution of the second study, but they may also indicate the extent of damage sustained by the cells during isolation. Rounding of redifferentiated model cells generally precedes attachment, but some bypass the rounding and attach when still elongated. At first attachment, contacts with the substratum are focal [89]. Later, contact becomes more uniform as cells grow polymorphically, extending processes and flattening. Fifty percent of cells complete this second transition in 12 d with a range of 8-19 d

[,1203.

669

By 28 d in culture, reconstruction ofmyotypic features is essentially complete [86]. Cells from 28 to 63 d in culture are filled with organized, in-register sarcomeres. In some cells, polymorphic growth produces bundles of myofibrils running at angles. Sarcoplasmic reticulum and T-tubules are well developed. Where cells grow into apposition, intercalated disks with intermediate, desmosomal, and gap junctions are formed. The observation that apposed redifferentiated cells contract synchronously is evidence that the gap junctions are functional [31, 59, 88, 127]. Adult atrial cells can also be cultured. Regardless of whether [4] or not [,16, 60, 85] they are treated to promote rapid attachment, they still undergo the elongate-round-spread transitions characteristic of ventricular redifferentiated cells. However for atrial cells the transition is faster than for ventricular cells. This transition and the failure of rapid attachment to stabilize in vivo structure show that adult atrial cells are morphologically less stable, possibly due to their less abundant myofibrils.

B. Thymidine uptake and mitosis Rat ventricular cardiomyocytes, in vivo, are amitotic after 17 to 21 d postpartum [93, 113]. Rapid attachment model cells are amitotic [98] and there are no data for thymidine uptake by them. In redifferentiated cells data on mitotic activity and thymidine uptake in different preparations are not consistent. In one preparation with mitotic activity [26], up to 25% of myocytes incorporate thymidine. In this preparation, newly isolated cells lack DNA polymerase ~, as would be expected for rat cardiomyocytes in vivo [25]. After 12 d in culture, however, these cells have 33% of their D N A polymerase in the form. Furthermore, myocytes with up to 10 nuclei are reported [-26], but another study [-86] of this same preparation, reports cells to be binucleate. T h a t these redifferentiated cells are binucleate agrees with observations on similar preparations [59, 88]. In a second preparation [59], cells do not undergo mitosis if isolated from rats older than 22 d [120] and they take up thymidine at a low rate. The number of cells which incorporate thymidine during the first 43 d in culture ranges from 0% to 4 % (Jacobson,

670

S.L. Jacobson aad H. M. Piper

unpublished). Differences in thymidine incor- stimulated heart muscle [11]. Cells of rapid attachment cultures are poration and mitotic frequency for different preparations may be related to the degree of mechanically at rest and have minimal damage done during the isolation of cells. It is oxygen demand. Therefore one would expect known that adult atrial myocytes in vivo them to behave metabolically like hyporespond to damage with thymidine incorpo- dynamic or arrested, aerobically perfused myocardium. In the hypodynamic myocarration [92, 1141. Consistent with their behaviour in vivo dium, higher ratios of N A D H / N A D and of [114], redifferentiating atrial adult myocytes acetyl-CoA/CoA SH favour the inactive state in culture show intense thymidine incorpo- of pyruvate dehydrogenase [38, 58, 110]. In ration and a low level of mitotic activity [16]. these cultures, with 5 mR glucose as the sole Maximum thymidine uptake is 63% of cells at exogenous substrate, 10% of the enzyme is in 5 d in culture. In spite of the high percentage the active form [109]. Therefore, one would of atrial cells which incorporate thymidine, expect fatty acid to be preferred over glucose only 0.5% are mitotic. Once these cells and lactate. Indeed, in rapid attachment culdevelop myofilaments and mature Z bands, tures, cells oxidize exogenous palmitate to CO2 at a rate 3-fold greater than exogenous they stop incorporating thymidine. glucose [122]. This agrees with the finding C. Metabolism that with glucose as sole substrate in the The metabolic state of ventricular cells in medium, metabolism of endogenous lipid rapid attachment cultures is characterized for accounts for about 80% of energy demand. aerobic and anoxic conditions [98-102, 109, When oxygen demand is calculated from con119, 122, 124]. These cells contract only if sumption rates of exogenous and endogenous stimulated electrically and they have a meta- substrates under these conditions, again a bolic state comparable to cells in diastolic value of 20 #1/min/gww is obtained [96, 109]. arrest. Oxidation of glucose and lactate is simultaNewly isolated myocytes, in a thin neously impaired by palmitate and, as in the unstirred layer of medium, consume oxygen at beating organ, lactate is preferred to glucose. a rate of 20 #1 per min per gram wet weight The inability, in various studies [19, 27, 39, 77, (#l/min/gww). The Qa0 is 1.8 [98], a value in 81], to demonstrate a preference of the cardiogood agreement with the O~0 of 1.4 in the myocyte for fatty acids as oxidative substrates arrested heart [1] and of 1.6 in slices of myo- probably is due to carnitine depletion of cardium [40]. Oxygen consumption increases freshly isolated cells. Evidence is that carni5-fold if measured in a stirred cuvette [98]. tine supplementation substantially increases Mechanical stimulation of oxygen consump- fatty acid oxidation in newly isolated cells tion may underly the very low tolerance of [44, 77], but not in cultured cells [109]. In medium equilibrated with air or 100% isolated adult cardiomyocytes to mechanical oxygen, when 2 to 10 mM glucose is the only treatment [60]. Oxygen consumption rates have only been exogenous substrate, rapid attachment cells reported for mechanically agitated newly iso- produce lactate and CO2 linearly for hours at lated Ca 2 +-tolerant cells [12, 13, 21, 39, 56, 81, a constant molar ratio of 2.5 [122]. Insulin, at 130]. These cells are usually described as a saturating concentration of 10 -7 M, leads to quiescent. However reported values of oxygen a 4.5-fold stimulation of lactate production consumption, 50 to 100 #l/min/gww , are dis- and stimulates CO 2 formation by 60%. A half tinctly higher than for quiescent cells, if maximal effective dose of insulin is 10-9M adjusted for the usual 60% to 70% of cells [122], which agrees with results on the effect with rod shape [124]. On the other hand, of insulin on glucose uptake of newly isolated there is agreement on the maximal oxygen myocytes [36, 55, 76]. The high rate of lactate consumption rates obtained by respiratory production apparently is caused by the inacuncoupling. Corrected for the percentage of tivity of pyruvate dehydrogenase and prerod-shape cells, they range from 400 to 600 dominantly reduced mitochondrial redox #l/min/gww [124]. These values are consistent systems, as is typical for inactive muscle. Since with the oxygen consumption of maximally- glycolytic flux is only loosely coupled to mito-

Cell Cultures o f Adult C a r d i o m y o c y t e s

chondrial oxidation [58, 70], cytosolic N A D H is preferentially oxidized by formation of lactate. Higher rates of lactate production with reduced mechanical activity have also been observed for the aerobic, glucosepcrfused heart [58]. With deprivation of oxygen and substrate, metabolic changes in rapid attachment cells resemble those of the anoxic perfused heart in the absence of exogenous substrates [57, 72]. However, the course of changes is comparatively long, probably due to the fact that these cells, like cells in arrested hearts, are at diastolic rest. After 60 min of anoxia, A T P falls from 6 to 2 #mol/gww and glycolysis almost stops, although only half of total glycogen reserve is consumed [97, 100]. The gradual decrease of metabolic activity during the first hour of anoxia is accompanied by a gradual release of cytosolic enzymes related to reversible injury [100, 102, 119]. After more than 60 min of anoxia, there is an increase of hypercontracted round cells. These exhibit morphological features of fatal damage but ruthenium red does not penetrate the cell surface [101, 119]. Upon reoxygenation, the cells that retained a polygonal shape recover, but round cells do not. Reoxygenation [101] does not lead to an abrupt enzyme release nor to ultrastructural signs of the 'oxygen paradox' [41]. Damage associated with the Ca 2 + paradox and with the oxygen paradox in heart tissue cannot be evoked in rapid attachment cultures [103]. T h a t this cell behaviour in tissue differs from behaviour in the isolated state is in accord with the hypothesis that cell interactions are critical to generation of these paradoxa [41, 42]. Another factor, supposed to be involved in the mechanism of the oxygen paradox is apparently absent in rapid attachment cultures [116]. In this system the cascade of adenine nucleotide degradation stops at the level of inosine [43], probably because only endothelial cells contain xanthin oxidase [62]. Therefore in a pure myocyte preparation one mechanism required for peroxidative reactions upon reoxygenation [64] is missing. These results demonstrate how a cell culture model can help distinguish between effects related to intrinsic properties of cells and those arising out of cell interactions.

671

Only one study of metabolic properties of redifferentiated model cells is published [27]. When newly isolated, these cells prefer glucose to octanoate as an exogenous substrate. After 12 h in culture, this order is reversed and a preference for octanoate remains for the subsequent 1 4 d period. Since oxidation of octanoate does not depend on the carnitine Acyl-CoA transferase reactions, impairment of newly isolated cells in oxidizing octanoate indicates an even more substantial damage [44] than just carnitine depletion (see above). From data of Figure 1 in [27], and assuming protein to be 16% of wet weight, we calculate ratios of oxygen consumption equivalents, in 14 d cultures at 37~ for 0.1 mM octanoate, 5.5 mM glucose and 1.0 mM lactate to be, respectively, about 50, 30 and 15 pl/min/gww. These values do not take into consideration a possible contribution from endogenous substrates (see above). These results indeed demonstrate a preference for fatty acid over non-lipid substrates at 14 d. Compared to resting newly isolated or to cultured rapid attachment cells, these oxygen demands are elevated. Perhaps this is because these redifferentiated myocytes were likely to be contracting spontaneously and that measurements were made on agitated monolayer cultures [98]. A T P content of these cells [27], assuming protein to be 16% of wet weight, can be calculated as 4pmol/gww of tissue. Unfortunately, only a single value is given and time in culture is not specified. Other A T P measurements have not been reported for redifferentiated model cultures. However, 4/~mols of ATP/gww is lower than in rapid attachment cultures [98] and in intact myocardium [110]. In conclusion, rapid attachment cells provide a good model of in vivo cardiomyocytes in a basal metabolic state. Because rapid attachment cultures are comprised of essentially 100% intact, metabolically stable cardiomyocytes, they provide a superior system for metabolic studies. Defects of newly isolated preparations of cardiomyocytes (e.g., with respect to fatty acid metabolism) suggest that several existing studies on them may be invalid. Results from any study of metabolism of cells in well-defined states of contractile activity, spontaneous or electrically driven

672

S.L. Jacobson and H. M. Piper

[56] are of great interest. However mechanical agitation should be avoided because it may exert a poorly defined and difficult-toreproduce influence on metabolism.

D. Electrophysiology Newly isolated and cultured cardiomyocytes are attractive for electrophysiological studies. They circumvent the problems of cellular heterogeneity, interactivity, and compartmentalization of intact tissue and afford visual control and accessibility for intracellular, patch clamp and microinjection studies. Newly isolated cells have already been used in many electrophysioLogical studies [cf, 8, 33, 34, 115]. However, their metabolic state is usually poorly defined, and membrane polarization, action potentials, ionic transport, and excitation-contraction coupling are all sensitive to cellular metabolic state. Therefore, cultures of adult cardiomyocytes with a normalized, well defined metabolic state might provide a better preparation than newly isolated cells. There are m a n y examples of the use of cultured cardiomyocytes from immature animals for electrophysiological and pharmacological studies. (For reviews, see 115, 123). However, there are few reports of measurements on adult cardiomyocytes in culture [4, 61, 91]. Rapid attachment cultures, at present, probably provide the best available substitute for newly isolated adult cells in electrophysiological studies. Specific electrophysiological data on them are not yet available, but they have a normal resting potential when newly isolated [94, 117], and do not contract spontaneously after the first few hours in culture, suggesting that they maintain a normal polarization. Spontaneous contractility of ventricular myocytes can be taken as a sign of membrane depolarization [17]. Mammalian and avian myocytes of the sinoatrial node and of the Purkinje system are expected to show spontaneous activity, but myocytes from other areas of the heart develop spontaneous activity only when depolarized. Cultured adult atrial and ventricular cardiomyocytes in redifferentiated cultures and adult atrial myocytes in rapid attachment culture are spontaneously contractile after several days [4, 16, 29, 59, 88, 91,

121, 127]. Data

on spontaneous

contractility

and

membrane polarization are available for one preparation of redifferentiated model ventricular cells [60, 61, 120]. Fifty percent are contractile when plated a few hours after isolation [120]. Newly isolated cells have a membrane polarization of - 8 0 mV, but many depolarize within a few hours and by 9 d their mean polarization is - 2 5 mV. From 9-18 d, as cells flatten and spread, 75% are contractile. The mean polarization of flattened (12 to 28 d) cells is - 53 inV. With increased time in culture, fewer cells contract (Jacobson, unpublished), and membrane polarization rises, reaching --70 mV by 60 d [60]. Contractile cells can be stopped by a hyperpolarizing current and can then be paced by depolarizing pulses. Myocytes of this same preparation [61] with a membrane potential more positive than - 6 0 mV have action potentials with a m a x i m u m rate of depolarization (Vm,x) of less than 15 V/s. Methoxyverapaxnil (D600), a blocker of inward Ca z+ current, abolishes these action potentials, providing evidence that Ca 2 + carries charge inward during the depolarization phase of the action potential. These low Vmax action potentials are insensitive to Tetrodotoxin (TTX), a blocker o f N a + channels. Apparently, this is because Na § channels are inactivated at membrane potentials in the --60 to - 5 5 mV range. Hyperpolarization apparently reactivates the Na § channels because Vm,x increases (80 V/s at --80 mV) and T T X then reduces Vmax to less than 15 V/s. Furthermore, D600 shortens these TTX-sensitive action potentials with little effect on their amplitude. This suggests than an inward, D600-sensitive Ca 2 + current prolongs the action potential. Electrophysiological data are available for two other preparations of cultured cardiomyocytes from rabbit sinoatrial node [91] and Guinea Pig atrium [3]. Sinoatrial cells are spheroidal and contract spontaneously after 1 to 2 d. These cultures have two.types of cells. One has action potentials with V,~,• 3 to 8 V/s and has a TTX-insensitive inward current that activates at a membrane potential of --30 mV. The other type has action potentials with l~m~X of 50 V/s and has an inward current that is blocked by T T X and activates at a membrane potential of - 4 1 mV. Guinea pig atrial cells even though adher-

Cell Cultures of Adult Cardiomyocytes ent 8 to 24 h after plating, do not show the morphologic stability of rapidly attached ventricular cells. They round up in 2 to 3 days and 50% contract spontaneously. After 6 to 10 d in culture, these cells begin to flatten. They continue to contract spontaneously for up to 60 days and, while they are in the spherical state, are claimed to be excellent for patch clamp studies [3]. IV. P r o s p e c t s a n d p r o b l e m s A. The state of the art The table summarizes much of the available data for cultured adult cardiomyocytes. No single preparation has properties fully defined at the present time. In the case of rapid attachment cultures, available ultrastructural and metabolic data for the first 4 to 7 d in culture show that cells retain m a n y of their in vivo properties, but are in a resting state. Electrophysiological and pharmacological data, fundamental in evaluating these cells as a model of myocardium, are not available at present. It is clear that this culture system, with its maintenance of in vivo properties, is useful primarily for the first several days after plating. Present evidence (Piper, unpublished) suggests that with extended time in culture, rapid attachment cells undergo morphological changes reminescent of those of redifferentiated model cells. Both culture systems may be fundamentally the same with conditions in rapid attachment cultures simply slowing the course of in vitro development. The table illustrates, for redifferentiated cultures, that few metabolic and electrophysiological data are available for any single preparation and, taken together, the data for all preparations present an incomplete and sometimes contradictory picture. Data on ultrastructural changes associated with cell rounding and redifferentiation are consistent. But there are few data describing the distribution of developmental states among cells in a single culture. Metabolic data are available for only one preparation. They are marginal with respect to the early period in culture, and are insufficient to evaluate the consequences of long-term culturing. The apparently different behaviour with respect to thymidine incorporation, mitotic frequency and nuclear

673

number of different redifferentiated preparations is puzzling. Resolution of the puzzle may give information useful in improving this preparation. The depolarized electrophysiological state of some redifferentiated cells requires further examination. Changes of membrane ionic permeability or intracellular ionic activity are the likely cause, but important questions are whether it can be avoided and whether it is a consequence of cell-isolation trauma or arises from changes triggered by removal from in vivo conditions. B. Conclusions Experience with rapid attachment cells and with the effect of specific culture conditions on in vitro properties of other cells [e.g., 49] suggests that an appropriate combination of physical and biochemical factors will produce a more in vivo-like, longer-term culture system for adult cardiomyocytes. The great potential benefit, for fundamental and applied cardiac research, of a better and better-defined long-term culture model of adult myocardium, demands continued efforts for improvement. Areas whose exploration is especially timely include effects of culture substrata and of serum factors on behaviour of cultured cardiomyocytes. The effect of cell interactions in adult cardiomyocyte cultures is a virtually unexplored area of both fundamental and practical importance. Reduction of serum used [65] and introduction of multicellular reaggregates [74, 123] in cultures of myocytes from immature hearts, are paths to the preservation of more highly differentiated properties in those cultures. Experience with cultured adult cardiomyocytes is in a nascent state. It is no surprise, therefore, that there are many questions and, as yet, few answers. Application of present techniques for simultaneous dynamic measurements of force, electrical activity, sarcoplasmic Ca / +, and histochemical responses of cultured cells can dramatically advance cardiac cellular research. The future is promising; the rapid attachment model already provides an excellent system for short-term experiments not possible with intact tissue nor with suspensions of isolated cells; and the redifferentiated model already provides a useful system for fundamental studies ofdevel-

674

S . L . J a c o b s o n and H. M. Piper

o p m e n t , cell i n t e r a c t i v i t y , e l e c t r o p h y s i o l o g y and pharmacology. When the p r o b a b l e results o f e v o l u t i o n o f c u l t u r e m o d e l s is cont e m p l a t e d , the prospects are truly exciting.

F a c u l t y o f G r a d u a t e Studies a n d R e s e a r c h ; a n d to H . M . P. f r o m the D e u t s c h e F o r schungsgemeinschaft, SFB 89-Kardiologie Giittingen and SFB 242-Koronare Herzkrankheit.

Acknowledgements W e are g r a t e f u l to D o r o t h y L. J a c o b s o n for i n v a l u a b l e e x p e r t assistance w i t h p r e p a r a t i o n of the m a n u s c r i p t . T h i s w o r k was assisted by g r a n t s to S. L . J . from The Heart and Stroke Foundation of O n t a r i o a n d f r o m the C a r l e t o n U n i v e r s i t y

S. L. J a c o b s o n I a n d H. M . P i p e r z

x Department of Biology, Carleton University, Ottawa, Ontario, Canada, K1S 5B6, and 2Physiologisches Institut I, Universitfit Dfisseldorf, Moorenstrasse 5, D-4000 Dfisseldorf 1, FRG

KEY WORDS: Culture technique cardiomyocyte; Heart; Cardiac myocyte; Cell culture; Myocardium.

References 1 ARNOLD,G., LOCHNER,W. Die Temperaturabhangigkeit des Sauerstoffverbrauchs stillgestellter kunstlich perfundierter Warmbluterherzen zwischen 34~ und 4~ Pfliigers Arch 284, 169-175 (1967). 2 BASHOR,M. M., Dispersion and disruption of tissue. In Methodsin Enzymology,vol. 58, W. B. Jakoby, and I. H. Pastan, (Eds), pp. 119 131. New York: Academic Press (1979). 3 BECHEM,M., GLITSCH, H. G., POTT, L. Properties of an inward rectifying K channel in the membrane of guinea-pig atrial cardioballs. Pfliigers Arch 399, 186-193 (1983). 4 BECHEM,M. and POTT, L. Atrial muscle cells from hearts of adult guinea-pigs in culture: a new preparation for cardiac cellular electrophysiology. EurJ Cell Bio131,366-369 (1983). 5 BENDUKIDZE,Z., ISENBERO,G., KLOCKNER,U. Ca 2+ tolerant guinea pig ventricular myocytes as isolated by pronase in the presence of 250 micromolar free calcium. BasicRes Cardio180[Suppl 1] 13 17 (1985). 6 BERRY,M. N., FRIEND,D. S., SCHEUER,J. Morphology and metabolism of intact muscle cells isolated from adult rat heart. Circ Res 26, 679 687 (1970). 7 BESTER,A.J., GEVERS,W. The synthesis of myofibrillar and soluble proteins in cell-free systems and in intact cultured muscle cells from newborn polymyopatbic hamsters.J Mol Cell Cardiol 7, 325-344 (1975). 8 BKAILY,G., SPERELAKIS,N., DOANE,J. A new method for preparation of isolated single adult myocytes. Am J Physio1247, H1018-H1026 (1984). 9 BODER,G. B., ELLIS,L. F. Uhrastructure of mouse heart cell culture. Proc Indiana Acad Sci 81,103--104 (1972). 10 BORG,T. K., RUBIN,K., LUNDOREN,E., BORG,K., OBRINK, B. Recognition ofextracellular matrix components by neonatal and adult cardiac myocytes. Dev Bio1104, 86-96 (1984). 11 BRETSCHNEIDER,H.J., HELLIOE,G. Pathophysiologie der Ventrikelkontraktion-Kontraktilitat, Inotropie, Suffizienzgrad und Arbeits~Jkonomie des Herzens. Verh Dtsch Ges Kreisl-Forsch 49, 14-30 (1976). 12 BURNS,A. H., MONTINI,J. Myocardium in hypertrophy: oxygen consumption by isolated cardiac myocytes and working hearts from spontaneously hypertensive rats. Life Sci 30, 23-37 (1982). 13 BURNS,A. H., REDDY,W.J. Amino acid stimulation of oxygen and substrate utilization by cardiac myocytes. Am j Physio1235, E461 E466 (1978). 14 BURROWS,M. T. The cultivation of tissues of the chick embryo outside the body. J Am Med Assoc 55, 2057-2058 (1910). 15 BUSTAMANTE, J. O., WANTANABE,T., MCDONALD,T. Non-specific proteases: a new approach to the isolation of adult cardiomyocytes. CanJ Pbysiol Pbarmaco160, 997-1002 (1982). 16 CANTIN,M., BALLAK,M. BEUZERON-MANOINA,J., ANA~D-SRIVASTAVA,M. B., DNA synthesis in cultured adult cardiocytes. Science, 214, 569-570 ( 1981 ). 17 CARStELIET,E., VEREECKE,J. Electrogenesis of the action potential and automaticity. In Handbookof Physiology, Berne, R. M., Sperelakis, N. and Geiger, S. R., (Eds), Section 2, vol. I, pp 269-334, Bethesda, Md. American Physiological Society, (1979). 18 CAVANAUOI-I,M. W. Pulsation, migration and division in dissociated chick embryo heart cells in vitro.J Exp Zool 128, 573 589 (1955). 19 CHEN, V., BAOB7, G. J., SPITZER,J. J. Exogenous substrate utilization by isolated myocytes from chronically diabetic rats. Am J Physio1245, C46-C51 (1983). 20 CHEUNO,J. Y., LEAF, A., BONVENTRE,J. V. Determination of isolated myocyte viability: staining methods and functional criteria., Basic Res Cardio180 [Suppl 1], 23-30 (1985). 21 CLARK, M. G., GANNON,B. J., BODKIN, N. PATTEN, G. S., BERRY, M. N. An improved procedure for the high-yield preparation of intact beating heart cells from the adult rat. Biochemical and morphologie study. J Mol Cell Cardiol 10, 1101-1121 (1978). 22 CLARK,W. A., JR. Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro. Dev Bio152, 263-282 (1976).

C e l l Cultures of Adult Cardiomyocytes 23

675

CLAVCOMB,W. C. Cardiac muscle cell proliferation and cell differentiation in vivo and in vitro. In Myocardial Injury, Spitzer, J.J., (Ed.), pp. 249 265. New York: Plenum Press (1983). 24 CLAYCOMB,W. C. Culture of cardiac muscle cells in serum-free media. Exp Cell Res 131, 231 236 (1980). 25 CLAVCOMB,W. C. DNA synthesis and DNA enzymes in terminally differentiating cardiac muscle cells. Exp Cell Res 118, 111-114 (1979). 26 CLAYCOMB,W. C., BRADSrIAW, H. D. JR. Acquisition of multiple nuclei and the activity of DNA polymerase cr and reinitiation of DNA replication in terminally differentiated adult cardiac muscle cells in culture. Dev Bio199, 331 337 (1083). 27 CLAVCOMB,W. C., BURNS,A. H., SIaEPnERD, R. E. Culture of terminally differentiated ventricular cardiac muscle cells. Characterization of exogenous substrate oxidation and the adenylate cyclase system. FEBS Lett 169, 261 266 (1984). 28 CLAYCOMB,W. C., LANSON, N., JR. Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell. In Vitro 20, 647 651 (1984). 29 CLAYCOMB,W. C., PALAZZO,M. C. Culture of the terminally differentiated adult cardiac muscle cell. A light and scanning electron microscope study. Dev Bio180, 466-482 (1980). 30 CREVEY, B. J., LANGER, G. A., FRANK,J. S. Role of Ca 2+ in maintenance of rabbit myocardial cell membrane structure and functional integrity.J Mol Cell Cardiol 10, 1081 1100 (1978). 31 DEHAAN,R. L., HmAKOW, R. Synchronization of pulsation rates in isolated cardiac myocytes. Exp Cell Res 70, 214 220 (1972). 32 DEMELLO, W. C. Membrane sealing in frog skeletal muscle fibers. Proc Nat Acad Sci USA 70, 982-984 (1973). 33 Dow, J. W., HARO1NG, N. G. L., POWELL, T. Isolated cardiac myocytes. I. Preparation of adult myocytes and their homology with the intact tissue. Cardiovasc Res 15, 483-514 (1981 ). 34 Dow, J. w . , HARmNG, N. G. L., POWELL, T. Isolated cardiac myocytes. II. Functional aspects of mature cells. Cardiovasc Res 15, 549 579 (1981). 35 DULBECCO,R. Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proc Natl Acad Sci USA 38, 747 752 (1952). 36 ECKEL,J., PAr~DALXS,G., REXNAUER,H. Insulin action on the glucose transport system in isolated cardiocytes from adult rat. BiochemJ 212, 385 392 (1983). 37 FARMER,B. B., MAUCIMA,M., WILLIAMS,E. S., WATANABE,A. Isolation of calcium tolerant myocytes from adult rat hearts: review of the literature and description of a method. Life Sci 33, 1-18 (1985). 38 FATH,P. A., KAKO, K.J. Glycolytic and tricarboxylic acid cycle intermediates during cardiac arrest and recovery in eu-, hyper- and hypothyroid rats.J Mol Cell Cardiol 5, 359 373 (1973). 39 FRAN~AKIS,C. J., BArIL, J. J., MCDANIEL, H., BRESSLER, R. Tolerance to physiological calcium by isolated myocytes from the adult rat heart; an improved cellular preparation. Life Sci 27, 815-825 (1980). 40 FUHRMAN,G. J., FUHRMAN, F. A., FIELD, J. Metabolism of rat heart slices with special reference to effects of temperature and anoxia. A m J Physiol 163, 642 647 (t950). 41 GANOTE, C. D. Contraction band necrosis and irreversible myocardial injury. J Mol Cell Cardiol 15, 67 73 (1983). 42 GANOTE,C. D., SIMS, M. A., VAN hER HEIDE, R. S. Mechanisms of enzyme release in the calcium paradox. Eur Heart J 4 [Suppl HI 63-71 (1983). 43 GEISBUHLER,T., ALTSCHULD, R. A., TREWYN, R. W., ANSEL, A. Z., LAraKA, K., BmERLEV, G. P. Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells. Circ Res 54, 536-546 (1984). 44 GLATZ,J. F. C., JACOBS, A. E. M., VEERKAMP,J. H. Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems. Biochem Biophys Acta 734, 454-465 (1984). 45 GOSmMA,K., TONOMURA,Y. Synchronized beating of embryonic mouse myocardial ceils mediated by FL cells in monolayer culture. Exp Cell Res 56, 387 392 (1969). 46 Goss, C. M. 'Slow-motion' cinematographs of the contraction of single cardiac muscle cells. Proc Soc Exp Biol Med 29, 292-293 (1931). 47 GRIGOR'EV, L. M., SHELKUNOV,A. N. Growth and secondary differentiation of explanted heart muscle of adult animals. (Transl. from Russian) Dokl Biol Sci 214, 12-14 (1974). 48 Guo, J-X.,JAcoBsoN, S. L., BROWN, D. L. Rearrangement oftubulin, actin and myosin in cultured ventricular cardio myocytes of the adult rat. Cell Motility Cytoskel (in press). 49 HADLEY,M. A., MYERS, S. W., SUAREZ-QUIAN,C. A., KLEINMAN,H. K., DYM, M. Extracellular matrix regulates sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J Cell Biol 101, 1511 1522 (1985). 50 HALBERT, S. P., BRUDERER, R. Studies on the in vitro growth of dissociated beating rat and human heart cells. Circulation 44 [Suppl II], 174 (1971). 51 HALBERT, S. P., BRUDERER,R., THOMPSON,A. Growth of dissociated beating human heart cells in tissue culture. Life Sci 13, 969-975 (1973). 52 HALLE,W., WOLLENBERGEI~,A. Differentiation and behavior of isolated embryonic and neonatal heart cells in a chemically defined medium. A m J Cardio125, 92 299 (1970). 53 HARARY, I., FARLEY, B. In vitro studies of single isolated beating heart cells. Science 131, 1674-1675 (1960). 54 HARARY, I., FARLEY, B. In vitro studies on single beating rat heart cells. I. Growth and organization. Exp Cell Res 29, 451-465 (1963). 55 HAWORTH, R. A., HUNTER, D. R., BERKOFF, H. A. Insulin stimulates deoxyglucose transport in adult rat heart cells in the absence of Ca z+. FEBS Lett 141, 37M-0 (1982).

676 56 57 58 59 60 61 62 63 t54 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

S.L. Jacobson and H. M. Piper HAWORTH,R. A., HUNTER, D. R., BERKOrr, H. A., MOSES, R. L. Metabolic cost of the stimulated beating of isolated rat heart cells in suspension. Circ Res 52, 342 351 (1983). HEARSE,D.J., CHAIN, E. B. Effect of glucose on enzyme release from, and recovery of, the anoxic myocardium. In Myocardial Metabolism: Recent Advances in Studies of Cardiac Structure and Metabolism, Dhalla, N. S., (Ed.) vol. 3, pp. 763-772, Baltimore, Md. : University Park Press (1973). HtLTUNEN,J. K., HASSINEN,1. E. Energy linked regulation of glucose and pyruvate oxidation in isolated peffused rat heart. Role ofpyruvate dehydrogenase. Biochim Biophys Acta 440, 377 390 (1976). JACOBSON, S. L. Culture of spontaneously contracting myocardial cells from adult rats. Cell Structure and Function 2, 1-9 (1977). JACOBSON,S. L., BANFALVI, M., SCHWARZFELD,T. A. Long term primary cultures of adult human and rat cardiomyocytes. Basic Res Cardio180 [Suppl 1], 79-82 (1985). JACOBSON,S. L., KENNEDY,C. B., MEALINO,G. A. R. Evidence for functional sodium and calcium ion channels in the membrane of cultured cardiomyocytes of the adult rat. Can J Physiol Pharmaco161, 1312-1316 ( 1983). `JARASC/-I,E. D., GRUND, C. BRUDER,G., HELD, H. W., KENNAN, T. W., FRANKE, W. W. Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium. Cell 25, 67 8 2 (1981 ). JOHNSON,E. A., LIEBERraAN,M. Heart: excitation and contraction. Am Rev Physio133, 479 532 (1971). .JOLLY,S. R., KANE, W.J., BAILIE,M. B., ABRAMS,G. D., LUCCttESl,B. R. Canine myocardial reperfusion injury. Its reduction by the combined administration ofsuperoxide dismutase and catalase. Circ Res 54, 277--285 (1984). ,JONES,J. L., LEI'ESCHKIN, E.,.JONES, R.. E., RUSH, S. Response of cultured myocardial cells to countershock-type electric field stimulation. A m J Physiol 235, H214-H222 (1978). KASTEN,F. H. Rat myocardial cells in vitro: mitosis and differentiated properties. In Vitro, 8, 128 150 (1972). KASTEN,F. H. Mammalian myocardial cells, pp. 73 81. In Tissue Culture: Methods and Applications. Kruse, P. F. and Patterson, M. K.`jr, (Eds) New York: Academic Press (1973). KESSLER-ICEKSON,G., SeERLtNO, O., ROTEM, C. WASSERMAN,L. Cardiomyocytes cultured in serum-free medium: growth and creatine kinase activity. Exp Cell Res 155, 113-120 (1984). KLEINraAN,H. K., MCGARVEY, M. L. HASSELL,J. R., STAR, V. L., CANNON,F. B., LAURIE, G. W., MARTIN, G. R. Basement membrane complexes with biological activity. Biochemistry, 25, 312-318 (1986). KOBAYASHt,K., NEELY,J. R. Control of maximum rate ofglycolysis in rat cardiac muscle. Circ Res 44, 166-175 (1979). KoNo, T. Roles of collagenases and other proteolytic enzymes in the dispersal of animal tissues. Biochim Biophys Acta 178, 397-400 (1963). KUBLER,W., SPmCKERraANN, P. G. Regulation of glycolysis in the ischemic and the anoxic myocardium. J Mol Cell Cardiol 1,351-377 (1970). LmBY, P. Long-term culture of contractile mammalian heart cells in a defined serum-free medium that limits non-muscle cell proliferation.J Mol Cell Cardio116, 803-811 (1984). LIEBERMAN,M., HORRES, C. R., SHmETO, N., EmttARA, L., ALTON,J. F., JOrlNSON, E. A. Cardiac muscle with controlled geometry. In : Excitable Cells in Tissue Culture. Nelson, P. G. and Lieberman, M. (Eds) pp 379 408. New York: Plenum Press (1981). LIEBERMAN,M., SANO,T., (Eds) Developmental and Physiological Correlates of Cardiac Muscle. New York: Raven Press (1976). LINDC~REN,C. A., PAULSON,D.J., SHANAHAN,M. F. Isolated cardiac myocytes. A new cellular model for studying insulin modulation of monosaccharide transport. Biochim Biophys Acta 721,385-393 (1982). Ltu, M. S., SPITZER,J. J. Oxidation of palmitate and lactate by beating myocytes from adult dog heart. J Mol Cell Cardiol 10, 415-426 (1978). LUNI)OREJ~,E., BORG, T., MARDH, S. Isolation, characterization and adhesion of calcium-tolerant myocytes from the adult rat heart.J Mol Cell Cardio116, 355-362 (1984). LUNDGREN,E., TERRACtO, L., BORn, T. K. Adhesion of cardiac myocytes to extraceilular matrix components. Basic Res Cardiot BO [Suppl 1], 69-74 (1985). LUNDGREN,E., TERRACIO, L., MARDH, S., BORG,T. K. Extracellular matrix components influence the survival of adult cardiac myocytes in vitro. Exp Cell Res 156, 371-381 (1985). MONTiNI,,J., BAGBY,G.J., SPITZER,J..J. Importance of exogenous substrates for the energy production of adult rat heart myocytes..J Mol Cell Cardio113, 903-911 (1981). MORKIN,E., ASHFORD,T. P. Myocardial DNA synthesis in experimental cardiac hypertrophy. Am J Physio1215, 1409-1413 (1968). MOSCONA,A. Cell suspensions from organ rudiments of the early chick embryo. Exp Cell Res 3, 535-539 (1952). MosEs, R. L., CLAYCOMB,W. C. Disorganization and reestablishment of cardiac muscle cell ultrastructure in cultured adult rat ventricular muscle cells.J Ultrastruct Res 81,358-374 (1982). MOSES,R. L., CLAYCOMB,W. C. Ultrastructure of cultured atrial cardiac muscle cells fi'om adult rats. A m J Anat 171,191-206 (1984). MOSES, R. L., CLAYCOMB,W. C. Ultrastructure of terminally differentiated adult rat cardiac muscle cells in culture. A m J Anat 164, 113-131 (1982). Mum, A. R. Further observations of the cellular structure of cardiac muscle. J Anat 99, 27-46 (1965). NAO, A. C., CnENG, M. Adult mammalian cardiac muscle cells in culture. Tissue and Cell 13, 515-523 ( 1981). NAG,A. C., CHENG, M., FtSCHMAN,D. A., ZA~, R. Longterm cell culture ofadult mammalian cardiac myocytes.

Cell Cultures of Adult C a r d i o m y o c y t e s

677

Electron microscopic and immunofluorescent analysis of myofibrillar structure. J Mol Cell Cardiol 15, 301-317

(1983). 90 NAG,A. C., FlSGHMAN,D. A., RABINOWITZ,M., ZAK,R. Cell culture of adult rat cardiac muscle. J Cell Biol 63, 91

92 93 94 95 96 97 98 99 00 Oi O2 ~03 04 05

238a (1974). NATHAN,R. D. Two electrophysiologically distinct types of cultured pacemaker cells from rabbit sinoatrial node. Am J Physio1250, H325-H329 (1986). OBERPRILLER,J. O., FERRANS,V. J., CARROLL,R.J. DNA synthesis in rat atrial myocytes as a response to left ventricular infarction. An autoradiographic study of enzymatically dissociated myocytes. J Mol Cell Cardiol 16, 1119-1126 (1984). OVERV,H. R., PRIEST, R. E. Mitotic cell division in postnatal cardiac growth. Lab Invest 15, 1100 1103 (1966). PELZER,D., TRUBE, G., PIPER, H. M. Low resting potentials in single isolated heart cells due to membrane damage by the recording microelectrode. Pflligers Arch 4100, 197-199 (1984). PmLLXPS,H. J. Dye exclusion tests for cell viability In Tissue Culture: Methods and Applications. Kruse, P. F. and Patterson, M. K.Jr, (Eds), pp. 406-408. New York: Academic Press (1973). PAPER,H. M. Isolierte aduhe Herzmuskel als Myokardmodell--Eigenschaften und Anwendungen. Stuttgart: Thieme Verlag (1985). PIPER,H. M., HUTTER,J. F., SPIEeKERMANN,P. G. Relation between enzyme release and metabolic changes in irreversible anoxic injury of myocardial cells. Life Sci 35, 127-134 (1984). PIPER, H. M., PROBST, I., SCHWARTZ,P., HOTTER,J. F., SPIECKERMANN,P. G. Culturing of calcium stable adult cardiac myocytes.J Mol Cell Cardiol 14, 397 412 (1982). PIPER, H. M., SCHWARTZ,P., HUTTER,J. F., PROBST, I., SPIECK~RMANN,P. G. Anoxia ofcuhured heart cells as a model for myocardial ischemia. In Protection of Tissues Against Hypoxia, Wauquier, A., Borgers, M. and Amery, W~ R., (Eds) pp 437-442, Amsterdam:Elsevier (1982). PIPER, H. M., SCHWARTZ, P., HUTTER,J. F., SPIECKERMANN, P. G. Energy metabolism and enzyme release of cultured adult rat muscle cells during anoxia.J Mol Cell Cardiol 16, 995-1007 (1984). PIPER, H. M., SCHWARTZ,P., SPAHR, R., HUTTER,J. F., SPIECKERMANN,P. G. Absence ofreoxygenation damage in isolated heart cells after anoxic injury. Pflfigers Arch 401, 71 76 (1984). PIPER, H. M., SCHWARTZ, P., SPAHR, R., HUTTER, J. F., SPIECKERMArCN, P. G. Early enzyme release from myocardial cells is not due to irreversible cell damage. J Mol Cell Cardio116, 385-388 (1984). PIPER, H. M., SPAHR, R., HUTTER, J. F., SPtECKERMANN, P. G. The calcium and the oxygen paradox: nonexistent on the cellular level. Basic Res Cardio180 [Suppl 2], 150-163 (1985). PIPER, H. M., SPAnR, R., PROBST, I., SPIECKERMANN, P. G. Substrates for the attachment of adult cardiac myocytes in culture. Basic Res Cardio180 [Suppl 2], 175-180 (1985). POWELL, T. Isolated heart cells. The development of a new experimental model. Basic Res Cardio180 [Suppl 1],

8-12 (1985). O6 POWELL, T. Isolation of cells from aduh mammalian myocardium. J Moi Cell Cardiol I1, 511 513 (1979). O7 POWELL, T. Methods for the preparation and characterization of cardiac myncytes. In : Methods in Studying Cardiac Membranes, Dhalla, N. S. (Ed.) pp. 41-62, vol. I, Boca Raton, Fla: CRC Press (1984). 08 POWELL, Y., TWIST, g. W. A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium. Biochem Biophys Res Commun 72, 327-333 (1976). 09 PROBST,I., SPAHR,R., SCHWEICKARDT,C., HUNNEMAN,D. PIPER, H. M. Carbohydrate and fatty acid metabolism of adult cardiac myocytes maintained in short term culture. A m J Physio1250, H853 H860 (1986). 10 RANDLE,P.J., TUBBS,P. K. Carbohydrate and fatty acid metabolism. In: Handbook of Physiology, Section 2, Vol. I, Berne, R. M., Sperelakis, N. and Geiger, S. R., (Eds) pp 805-844, Bethesda, MD.: American Physiological Society (1979). 11 RAPPAPORT,C., BISHOP, C. B. Improved method for treating glass to produce surfaces suitable for the growth of certain mammalian cells in synthetic medium. Exp Cell Res 20, 580-584 (1960). I2 RENAUD,J. F., SCANU, A. M., KAZAZOOLOU,T., LOMBET, G., ROMEV, G., LAZDUNSKX,M. Normal serum and [ipoprotein-deficient serum gives different expressions of excitability, corresponding to different stages of differentiation, in chicken cardiac cells in culture. Proc Nat Acad Sci USA 79, 7768 7772 (1982). 13 RU~VANTSEV,P. P. A morphological and autoradiographical study of the peculiarities of differentiation rate, DNA synthesis and nuclear division in the embryonic and postnatal histogenesis of cardiac muscle of white rats. Folia Histochem Cytochem (Krakow) t, 463-471 (1963). 14 RUUVANTSEV,P. P. Interrelations of the proliferation differentiation process during cardiac myogenesis and regeneration. Int Rev Cytol 51,187 273 (1977). 15 SCHAN~E,O. F., BKAILY,G. Explanted cardiac cells: a model to study drug actions ? C a n J Physiol Pharmaco159, 443-467 (1981). 16 SCHRABER,J. Mechanisms ofischemic injury in the heart. Basic Res Cardio180 [Suppl 2], 135-139 (1985). 17 SCHREIBMAYER;W., TRITTHART, H. A., ZERNIG, G., PIPER, H. M. Single voltage-dependent and outward rectifying K + channels in isolated rat heart cells. Eur BiophysJ I1,259-263 (1985). 18 SCHWARTZ,P., PIPER, H. M., SPAHR, R., SPIECKERMANN, P. G. Adaptation phenomena of the adult cardiac myocytes in culture. Basic Res Cardio180 [Suppl 2], 181-185 (1985). 19 SCHWARTZ,P., PIPER, H. M., SPAHR, R., SPIECKERMANN,P. G. Uhrastructure of adult myocardial cells during anoxia and reoxygenation. A m J Patho1115, 349-361 (1984).

678

S . L . J a c o b s o n and H. M. Piper

120 SCHWARZFELD,T. A., JACOBSON,S. L., Isolation and development in cell culture of myocardial cells of the adult 121 122 123 124 125 126 127

128 129 130 131

rat.J Mol Cell Cardiol 13, 563 575 (1981). SPAHR, R., PIPER, H. M., SCHWARTZ, P., PROBST, I., SPIECKERMANN,P. G. Morphological dedifferentiation of adult cardiac myocytes in coculture with hepatocytes. Basic Res Cardio180 [Suppl 1], 83 86 (1985). SPAHR,R., PROBST, 1., PIPER, H. M. Substrate utilization of adult cardiac myocytes. Basic Res Cardiol 80 [Suppl 1],53 56 (1985). SPERELAKIS,N. Cultured heart cell reaggregate model for studying cardiac toxicology. Environ Health Perspect 26, 243 267 (1978). SPIECKERMANN,P. G., PIPER, H. M. Oxygen demand of calcium tolerant adult cardiac myocytes. Basic Res CardiolS0 [Suppl 2], 71 74 (1985). STEINBERG,M. S. 'ECM': its nature, origin and function in cell aggregation. Exp Cell Res 30, 257 279 (1963). SZEPSENWOL,J. A comparison of growth, differentiation, activity and action currents of heart and skeletal muscle in tissue culture. Anat Rec95, 125 146 (1946). TERRACIO,L., INGEBRETSEN, C. C., INGEBRETSEN,W. R. Isolation and culture of adult rat heart myocytes. Anat Rec 204, 403 --404 (1982). VAHOUNY,G. R., WEI, R., STARKWEATHER,R., DAVIS, C. Preparation of beating heart cells from adult rats. Science 167, 1616 1618 (1970). WHITE, R. L., MAZET, F., SPRAY, D. C., CARVALHO, A. C., WITTENRERC, B. A., BENNETT, M. V. L. Gap junctions between isolated adult rat myocytes: Electrophysiological properties and morphological changes. Circulation [Suppl I I I], I I 1-24 [Abstract 95] (1983). WITTENBERG,B. A., ROBINSON,T. F. Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats. Cell Tiss Res 216, 231 251 (1981). ZXMMERMAN,A. N. E., HULSMANN,W. C. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature 211,646 647 (1966).