Calcium flux measurements during hypoxia in cultured heart cells

j Mol Cell Cardiol 19, 271 279 (1987)

Calcium Flux M e a s u r e m e n t s During Hypoxia in C u l t u r e d H e a r t Cells Joseph G. Murphy, Thomas W. Smith and James D. Marsh* Cardiovascular Division, Department of Medicine, Brigham and Woman's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA (Received9 October1986, acceptedin revisedform 16 December 1986) J. G. MURPHY,T. W. SMITHANDJ.D. MARSH. Calcium Flux Measurements During Hypoxia in Cultured Heart Cells. Journal of Molecular and Cellular Cardiology (1987) 19, 271-279. We tested the hypothesis that cultured chick embryo ventricular cells grown in monolayer could be used to study calcium fluxes across the myocardial sarcolemmal membrane during simulated ischemia. A specially adapted anaerobic chamber allowed the exposure of heart cells to profound hypoxia (Po 2 < 1.5 Torr) for prolonged periods of time. Ca 2+ flux studies were conducted in this chamber following hypoxia and substrate deprivation to simulate important components of myocardial ischemia. To prove we were measuring cellular rather than interstitial cation contents we conducted 51Cr-EDTA interstitial space marker washout studies and showed that our procedures were sufficient to remove at least 99.7% of the extracellular fluid. The addition of lanthanum (1 raM) to the wash solution reduced nonspecific 45Ca 2+ binding to the polystyrene culture plates to <0.03% of applied counts, but did not alter 45Ca2+ uptake under normoxic conditions. Following 2 h of hypoxia and substrate deprivation the Ca 2+ content of the rapidly exchangeable Ca 2+ pool of cultured monocytes increased 282% compared to control values during normoxia. We conclude that cultured chick embryo ventricular cells grown in monolayer are suitable for investigation of cation fluxes during simulated ischemia. Under the conditions studied, lanthanum displaceable Ca 2+ did not make a major contribution to Ca 2+ influx. The system permitted clear resolution of alterations in Ca 2 + flux kinetics under conditions of profound hypoxia. KEY WORDS: Hypoxia ; Calcium ; Cell culture ; Heart cells ; Ion flux.

Introduction The regulation of mono- and divalent cation fluxes across the myocardial sarcolemmal membrane during ischemia is of theoretical and practical importance [I, 2, 4, 23]. Transmembrane fluxes of Ca 2+, Na +, and K + are of pivotal importance in determining the survival of cells after ischemic injury in a variety of tissues including heart and brain [19]. To test the hypothesis that cultured chick embryo ventricular cells grown in monolayers are a suitable model for the detailed investigation of ion fluxes across the sarcolemmal membrane during simulated ischemia, we developed a new experimental system. In conjunction with modifications of our previously described methods of ion flux measurement in cultured myocardial cells [2] this allowed us to assess C a 2+ fluxes under conditions of profound

1.5 Torr]. The experimental system devised permitted exposure of the cells to a controlled hypoxic environment in a highly reproducible fashion. Using severe hypoxia and substrate deprivation we simulated important components of ischemia and were able to clearly resolve alterations in studied calcium flux kinetics under these conditions. h y p o x i a [-Po 2 <

Methods Tissue culture Monolayer cultures of spontaneously contracting chick embryo ventricular cells were prepared as previously described [2, 3]. Briefly, hearts of lO-day-old chick embryo were sterilely removed and the atria and great vessels removed. The ventricle was cut into

* To whom all correspondence should be sent at the above address. 0022-2828/87/030271

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0.5 m m fragments and placed in Ca 2+- and MgZ+-free Hanks' solution (Gibco Laboratories, Grand Island, NY, USA). The ventricular fragments were transferred to 0.025% trypsin (Grand Island Biological Company, Grand Island, New York) in Ca 2+- and Mg2+-free Hanks' solution and dissociated at 37~ for five cycles of seven minutes each. The supernatant suspensions from cycles 2 to 5 containing dissociated cells were placed in 20 ml of cold trypsin inhibitor medium containing 50% heat inactivated horse serum and 50% C a / + - and Mg2+-free Hanks' solution. The suspension was centrifuged at 150 x g for 10 rains, the supernatant phase discarded and the cells suspended in culture medium consisting of 6% heatinactivated foetal calf serum (FCS), 40% M199 (Grand Island Biological Company) and 0.1% penicillin-streptomycin antibiotic solution and 54% low potassium salt solution. The final concentrations (m~) in the culture medium were Na z+, 144; K +, 4.0; Ca i§ 0.97; Mg a+, 0.8; C1, 131; and HCO~-, 18. The suspension of cells were diluted to 50 000 cells/ml. Cells prepared for Ca 2 + flux experiments were placed in polystyrene tissue culture dishes. Cultures were incubated in a humidified 5% carbon dioxide-95% air atmosphere at 37~ Confluent monolayers in which at least 70% of the cells contracted spontaneously developed by day 3 in culture. All studies were done on day 3-4 in culture. The presence of beating was used as an indicator of cell viability and non-beating cultures were discarded. All ion flux experiments were conducted in HEPES (4 mM) buffered physiological salt solution containing the same ion concentrations as culture medium, except where ion concentrations were adjusted according to the individual experimental protocol. Low oxygen chamber

Initial studies were undertaken using thickwalled Lucite chambers and nitrogen gas washout to exclude oxygen. This technique allowed the achievement of Poz values in the range of 10 to 15 Torr. Lower values could not be achieved due to the contamination of commercial nitrogen with small quantities (0.1 to 0.2%) of oxygen and by oxygen

leakage through tubing and seals. This method was cumbersome, and importantly, did not allow ion flux measurement during the actual hypoxic insult, but rather only during reoxggenation because it was not possible to manipulate the cells within the closed box. The cooling effects of the expanding gas also made precise temperature control difficult. Therefore, a commercially available anaerobic chamber (Forma Scientific, Model 1025, Marietta, OH, USA), originally designed for use in microbiology research for growing fastidious anaerobic organisms, was modified to permit the conduct of cellular physiologic experiments. This chamber was constructed of stainless steel with a transparent Lexan hard front panel into which twin glove ports were installed. A forced draft incubator was built into the cabinet. This provided a stable temperature (-t-0.2) so that investigations would be conducted at physiologic temperature (37~ and also under conditions simulating controlled hypothermia. The work chamber was connected to the exterior via a double-doored interchange chamber. This interchange number was connected to a vacuum p u m p and a 100% N z gas source (99.7% pure; Yankee Oxygen). Air was pumped from the interchange chamber and replaced with pure N2, the cycle being repeated at least 3 times. This procedure reduced the oxygen content of the interchange to less than 0.5% and allowed transfer of material between work chamber interior and exterior without significant contamination of the low oxygen atmosphere. Using only a pure nitrogen flush technique. Oxygen concentrations of between 1% to 2% [7 to 14 Torr] were consistently maintained in the work chamber. T o reduce further the oxygen content of the chamber a gas mixture of t0% hydrogen/90% nitrogen was used. In the presence of a palladium catalyst oxygen scrubber, the mixture reacts non-explosively with oxygen to form water. The water is subsequently absorbed by a dessicant wafer. This method allowed the attainment of oxygen concentrations in the range of 10 to 100 parts million as confirmed by mass spectroscopic analysis. The hydrogen/nitrogen gas mixture was connected to the chamber via a flash back preventer and copper tubing to minimize fire risk. The hydrogen/nitrogen low oxygen

Ion Flux Measurement During Hypoxia

a t m o s p h e r e of the work chamber is nonexplosive so that electrical heating elements and stirrers can be used safely in this environment. A heating plate combined with a precision temperature controller was used to maintain all solutions at physiological temperature within the chamber. The work chamber was also modified to contain connectors to the exterior via two valve controlled gas ports, which facilitated online oxygen concentration monitoring and provided a vacuum line to the chamber interior for aspiration of media from culture plates.

Oxygen concentration measurement Online oxygen concentration analysis was performed using an S-3A low oxygen analyzer (Applied Electrochemistry) connected to the chamber interior via a short length of heavywalled Tygon tubing. There was no significant leakage of oxygen through the Tygon tubing, as confirmed by analysis of gas passed through the tubing from a pure nitrogen source. The use of the hydrogen gas mixture precludes oxygen analysis with this high temperature analyzer as hydrogen and oxygen would combust, giving a spuriously low oxygen content at any actual oxygen level. Therefore, methylene blue [3,7-bis[dimethyl amino]phenothiazin-5-ium chloride] was utilized as a chemical oxygen indicator. It changes from colourless (99.9% reduced) to its blue colour at a redox potential of - 2 3 0 mV, which corresponds to a Po2 < 1.5 T o r r [21], according to the following reaction: methylene blue (reduced) + glucuronic acid [colourless] methylene blue (oxidized) + glucose [blue] The indicator solution contained Tris [tris{Hydroxymethyl}-aminomethane] buffer 20%, glucose 1.33% and methylene blue 0.006% [9]. Glucose reduces methylene blue to the colourless form in an alkaline medium at 37~ which remains stable in this reduced form at Po 2 < 1.5 Torr. Methylene blue is considered to be colourless when the oxidized form is less than one part per thousand of the total present t-9].

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The work chamber atmosphere was continuously bubbled through a methylene blue solution to provide continuous surveillance for any significant oxygen leak into the chamber. Mass spectrometric analysis of chamber gas was used to validate these oxygen monitoring procedures and to quantitate the exact oxygen content.

Calcium ionflux measurement The technique previously reported from this laboratory [2] for measuring ion fluxes in cultured heart cell monolayers utilize cells grown on glass coverslips. This technique was not suitable for use within the low oxygen chamber. A new technique suitable for use within the chamber was developed utilizing general principles of ion flux estimation previously validated in this laboratory for the cultured chick heart cell model [2, 4, 7]. Monolayers were grown in six-welled polystyrene culture plates (Costar) for 3 to 4 days under conditions as described above. Prior to experimentation culture medium was aspirated from culture plates. Cells were preincubated in HEPES buffer [ p H = 7.4] at 37~ for l0 min, then subjected to the experimental intervention. At the indicated time buffer was aspirated and replaced by buffer containing 4SCa2+ 5(~Qi/ml). 45Ca2+ uptake was terminated (after 5 min to label the rapidly exchangeable pool or after 2 h to label the slowly exchangeable pool, vide infra) by removing the #SCaZ+ medium and washing the wells five times for 15 s each with HEPES buffer containing 1 mM La 3 + at 4~ Aspiration of medium and washing of monolayers was carried out simultaneously on all wells using specially constructed manifolds to avoid disturbing the monolayers during exchange of media. Following washing, the culture plates were transferred out of the low oxygen chamber. The cells were dissolved in the culture plates with 1% sodium tetradecyl sulfate and 10 mM sodium borate for 2 h. Aliquots were taken for scintillation spectrometry. Protein content was assayed by the method of Lowry [18], using crystalline bovine serum albumin as standard. To normalize for variation in cell density in each well, for 45Ca 2+ uptake experiments the monolayers were grown in L-[4,5-aH{N}] -

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leucine [0.1 Ci/ml] for 24 h before each experiment. 3H counts i n c o r p o r a t e d into protein p e r m i t t e d cell density correction, since the relationship between radioactive counts a n d protein concentration allowed accurate estimation of protein concentration in each well.

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T h e effect of L a a+ on residual 4SCa2+ was analyzed by p a i r e d t-tests. 5 1 C r - E D T A interstitial space d a t a a n d 45Ca2+ u p t a k e d a t a were a n a l y z e d by analysis of variance.

Results 4 s Ca 2 + f l u x measurements

F o r m e a s u r e m e n t of 45Ca2+ c o n t e n t of cells under control and e x p e r i m e n t a l conditions to be valid, actual cellular 45Ca 2+ content must be measured with m i n i m a l c o n t r i b u t i o n from extracellular 45Ca2+ b o u n d to the polystyrene substrate or localized to the interstitial space. L a 3+ (1 mu) in H E P E S buffer was used to displace 45Ca2+ from substrate a n d interstitial space c o m p a r t m e n t s at the conclusion of the *SCa2+ uptake. W a s h solution [2 ml] was a d d e d simultaneously to all wells in each culture plate for 15 s and then aspirated. This cycle was r e p e a t e d five times to give a total wash time of 75 s. Figure 1 shows the effect of L a a + c o n t a i n i n g wash solution on 45Ca2+ b i n d i n g to polystyrene wells. This shows that L a 3+ reduced non-specific 45Ca2+ b i n d i n g to < 0 . 0 3 % of total a p p l i e d counts. La 3 + also completely stopped 45Ca2 + uptake by cultured heart cells within 5 s of application [2]. T o d e t e r m i n e the degree to which ions in the interstitial space are exchanged d u r i n g the washing p r o c e d u r e that concludes the period of "5Ca2+ u p t a k e by the monolayers, the interstitial space of the monolayers was labelled by i n c u b a t i n g the monolayers for 30 min in buffer containing 2 # C i / m l 51CrE D T A , which has been shown to be a satisfactory m a r k e r for the interstitial space in c a r d i a c p r e p a r a t i o n s [20]. W a s h i n g of monolayers using the manifold r a p i d l y a n d reproducible decreased the residual a m o u n t of interstitial space m a r k e r (Fig. 2). Well to well

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FIGURE I. Effect of 1 inM lanthanum wash solution on *SCa2+ binding to polystyrene culture wells; (a) Polystyrene cultures wells containing no cells. Two millilitres of*SCa2+ buffer were added; and wells were incubated for 5 mins at 37~ The wells were washed with HEPES buffer without lanthanum for 75 s and then aspirated dry (see Methods). SDS/Na-borate was added; wells were incubated for 2 h at 22~ and an aliquot taken for scintillation counting. Ordinate indicates '*SCa2 + remaining in the well after the wash procedure; (b) Same as (a), except wells contained cells; (c) Same as (a), except wash contained La a+ ; (d) Same as (a), except wells contained cells and wash solution contained La 3+. Lanthanum wash solution significantly [P < 0.01] reduced nouspecific 4SCa2+ binding to polystyrene wells both in the presence and absence of cells. (Error bar = 1SD). v a r i a t i o n was < 10% by 75 s and less than 0.3% of the interstitial m a r k e r r e m a i n e d at 75 s [99.9% confidence limit]. T a k e n together with the evidence for essentially complete washout of 4~Ca z+ from the substrate, this observation supports the view that u n d e r the conditions used, the 4SCaZ+ content that is measured actually reflects cellular Ca z + with little ( < 1%) c o n t r i b u t i o n from other compartments. Previous work from this l a b o r a t o r y h a s d e m o n s t r a t e d that for cultured chick e m b r y o ventricutar cells grown in m o n o l a y e r on coverslips, 45Ca2 + is taken up in two kinetically distinct phases. A rapidly exchangeable pool of Ca 2+ is labelled by 4SCa z+ with a half-time of 11 s a n d a slowly e x c h a n g e a b l e pool that reached steady state by 120 min [2]. Alterations in the C a 2+ content of the r a p i d l y e x c h a n g e a b l e pool correlate closely with alterations in cellular contractile properties [2]. T o d e t e r m i n e w h e t h e r u n d e r normoxic conditions a n d using these new techniques similar C a / + pools could be identified, the time course of 45Ca2+ u p t a k e in multiwell plates was studied (Figure 3).

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FIGURE 2. Washout of interstitial space of monolayers of cultured myocardial cells. Monolayers of cells were incubated with culture medium containing the interstitial space marker 51Cr-EDTA for 30 mins at 37~ to fully label the interstitial space. All wells are washed simultaneously using a specially designed manifold with La s+ containing HEPES buffer. Following washing, cells in the wells were dissolved in SDS/Na-borate for 2 h and an aliquot taken for scintillation counting. Abscissa indicates washing time in seconds and the ordinates shows StCrEDTA counts remaining in a well. Standard deviation were less than 10% in each case but are not indicated on the semilogarithmic plot. A wash time of 75 s is adequate to remove 99.7% of the interstitial space marker [99% confidence limits]. R a p i d l y a n d slowly e x c h a n g e a b l e pools similar to those p r e v i o u s l y r e p o r t e d [2] w e r e identified for 4SCa2+ e x c h a n g e . T h e r a p i d l y e x c h a n g e a b l e p o o l filled w i t h a h a l f - t i m e o f a p p r o x i m a t e l y 9 s a n d was fully e q u i l i b r a t e d by 5 min, w h i l e the slowly e x c h a n g e a b l e p o o l

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was at e q u i l i b r i u m by 120 m i n . T h e C a 2+ c o n t e n t o f the r a p i d l y (5 rain) e x c h a n g e a b l e pool was 1.7 ___ 0.1 n m o l C a / m g p r o t e i n , w h i l e the C a 2+ c o n t e n t at 120 rain was 2.7 4- 0.2, (n = 12) n m o l C a / m g protein. L a b e l l i n g to 6 h did n o t l e a d to a significant f u r t h e r increase in 45Ca2+ c o n t e n t . A t 6 h 45Ca2+ c o n t e n t was 2.9 + 0.2. To determine whether reproducible meas u r e m e n t o f 4 5 C a 1+ u p t a k e kinetics w i t h i n a p r o f o u n d l y h y p o x i c e n v i r o n m e n t is possible a n d h o w h y p o x i a alters 45Ca2+ flux a n d c o n t e n t , 4SCaZ+ u p t a k e e x p e r i m e n t s w e r e c o n d u c t e d in the a n a e r o b i c c h a m b e r u n d e r c o n d i t i o n s i d e n t i c a l to the c o n t r o l e x p e r i m e n t , e x c e p t t h a t there was no glucose in the m e d i u m a n d the P o 2 c o n t e n t to w h i c h the cells w e r e exposed was < 1.5 T o r r . C u l t u r e m e d i u m was a s p i r a t e d f r o m the m o n o l a y e r c o n t a i n i n g wells a n d r e p l a c e d w i t h glucosefree H E P E S buffer at p H = 7.40. T h e plates w e r e t h e n transferred to the e x c h a n g e c o m p a r t m e n t of the a n a e r o b i c c h a m b e r . T h e cells a n d m e d i a w e r e exposed t o a v a c u u m o f 20 in. H g for 1 rain followed by inflow of a n i t r o g e n a t m o s p h e r e . T h i s cycle w~ts r e p e a t e d three times a n d the c u l t u r e plates t h e n transferred to the w o r k c o m p a r t m e n t . T h i s p r o c e d u r e did n o t d a m a g e the cells as verified by the p r o m p t r e t u r n of cell b e a t i n g if the cells w e r e i m m e d i a t e l y r e t u r n e d to the outside a t m o s p h e r e . F o l l o w i n g i n c u b a t i o n for 120 m i n at 37~ 4SCa2 + u p t a k e studies w e r e c a r r i e d o u t in the a n a e r o b i c c h a m b e r . A f t e r 120 m i n of sub-

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FIGURE 3. Kinetics of45Ca 2+ uptake by cultured heart cells grown in monolayers during normoxia. HEPES buffer (37~ containing #SCa2+ was added to the monolayer. 45Ca2+ uptake was terminated at the time specified by aspiration of media and washing with i mM lanthanum in HEPES buffer at 4~ Abscissa indicates time in seconds and ordinate shows 45Ca2+ uptake in nmol/mg protein, (Error bar = 1SD). n = 6 for each point. This representative experiment was replicated five times.

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F I G U R E 4. Kinetics of 4SCa 2+ uptake by cultured heart cells during hypoxia. For this experiment, monolayers were exposed to a profoundly hypoxic atmosphere (Po z < 1.5 Torr) for 120 min. 4SCa2+ uptake was then measured during continued hypoxia as described in Methods. n = 6 for each point. This representative experiment was replicated five times with similar results.

strate and oxygen deprivation, measurement of45Ca2 + uptake at the indicated time points was started. The Ca 2 + content at 5 min under control conditions was typically 1 . 7 + 0 . 1 nmol/mg (Fig. 3); after 2 h of hypoxia it was 4.8 nmol/mg (Fig. 4). Thus, this degree of hypoxic insult produced a 282% increase in the size of the rapidly exchangeable Ca 2+ pool. Labelling of the slowly exchangeable Ca / + pool did not reach a plateau within 120 min as occurred during normoxia, but continued to increase at a constant rate. At 120 min, C a 2 + content was 11 +_ 4 nmol/mg and by 6 h had reached 42 4- 11 nmol/mg. The slowly exchangeable Ca 2 + pool thus appears to act as a sink for Ca z + under hypoxic conditions. Its nonsteady-state Ca 2+ content after 2 h exposure to 45Ca2+ was greater than that during normoxia.

Discussion A major experimental problem in the study of ion fluxes during ischemia and hypoxia is the maintenance of sufficiently low oxygen atmosphere so that aerobic cellular metabolism is inhibited. Early investigations suggested that the cultured embryonic chick heart cell was relatively insensitive to hypoxia when compared to mature mammalian cells, but this concept is not well supported by available evidence. Khuri et al. [12], using mass spectrometry have shown that the Po z of working non-ischemic canine left ventricular myocardium is of the order of 20 mmHg while that of ischemic myocardium is < 10 mmHg. Studies by Barry et al. [3] demonstrated that a Poz of

12 m m H g is the threshold necessary to inhibit aerobic metabolism and contraction in nondiffusion limited chick heart cells in monolayers, in good agreement with findings in the mammalian myocardium [12]. Indeed, in the presence of glucose, contraction was not completely inhibited with a Po z = 1 Torr and in the absence of glucose, Po 2 < 5 Torr was necessary to abolish contractions [3]. Our method for generating an atmosphere for the study of Ca 1+ flux during hypoxia offers several advantages. First, it permits generation of adequately low oxygen concentration at the cell surface so that the tissue is reliably and reproducibly hypoxic. The degree of hypoxia is sufficient to abolish contraction which is an invariant finding in animal and human studies of profound ischemia [12]. Secondly, because the preparation is a monolayer with minimal diffusion barriers, the hypoxic insult is well-resolved temporally, and is uniform, which can be a problem in intact heart studies [8, 17]. Thirdly, the hypoxic atmosphere can be generated without high gas flow rates which make precise temperature regulation difficult. C a 2 + uptake by ischemic tissue is highly temperature sensitive [6, 11] so close control of this variable is important. Fourthly, oxygen concentration can be monitored on-line. No assumptions about "anoxia" being generated by a nitrogen atmosphere are necessary. Additional major experimental problems in the study of ion flux during ischemia and hypoxia have been problems in control of the interstitial space ion content [20] and difficulty in measurement of ion flux kinetics

Ion Flux Measurement During Hypoxla during actual hypoxia rather than at time of reoxygenation. Our study tested the hypotheses that cultured chick embryo ventricular cells grown in monolayer (with minimal interstitial space) constitute a suitable model for investigation of calcium flux across the sarcolemmal membrane during simulated ischemia, and that Ca 2 + flux measurements can be conducted during profoundly hypoxic conditions. A low oxygen chamber [ P o 2 < 1.5 Torr] was adapted so that it was indeed possible to conduct ion flux kinetic studies under conditions of hypoxia and substrate deprivation without the necessity of reoxygenation. We modified the techniques of ion flux determination previously validated in our laboratory for cultured chick heart cell monolayers [2]. The initial studies demonstrated that 1 mM La 3+, a trivalent cation, added to the wash solution reduced nonspecific binding of 45Ca2+ to the polystyrene culture wells to < 0.03% of applied radioactive C a 2 + counts. This very low 'background' permitted clear resolution of small changes in 45Ca 2+ flux. Furthermore, La 3+ almost completely inhibits calcium uptake by cultured cells within 5 s and causes a 31% decrease in calcium efflux after 3 mins of La z+ exposure [2]. It is electron dense on electron microscopy and has been reported not to cross intact cell membranes in significant quantities under experimental conditions similar to those described [15, 16]. Thus, using La 3 + in the wash solution, Ca z+ flux can be rapidly inhibited at desired time points. Using 51Cr-EDTA as a marker of the interstitial space, we showed that our technique was effective in washing out 99.7% of this interstitial space marker. Taken together, these experiments demonstrate that we were measuring the kinetics of flux of cellular calcium without a substantial contribution from the substrate and interstitial space compartments. Previous work from this laboratory has shown that 45Ca2+ uptake by chick heart monolayers grown on glass coverslips can be resolved into rapidly and slowly exchangeable pools [2]. We repeated these 45Ca2+ uptake studies on monolayers grown in six-welled polystyrene culture plates, both during normoxia and following 120 min of hypoxia and substrate withdrawal. We showed that the kinetics of 45Ca2 + uptake during normoxia in

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polystyrene culture plates did not differ from those previously reported for cells grown on glass coverslips. For ceils grown on glass coverslips, the rapidly exchangeable C a 2+ pool content was 1.6 + 0.1 nmol Ca/mg protein [2]. For those experiments, Ca 2 + was washed from the interstitial space by immersing the coverslips in HEPES buffered balanced salt solution that did not contain lanthanum. For cells grown in multiwell plates, and for which 1 mM La 3 + was included in the wash medium, the current study shows the Ca 2+ content was 1.7 _ 0.1 nmol Ca/mg protein at 5 rain, in good agreement with the value previously observed for cells grown on glass coverslips. This observation tends to argue against the earlier proposal by Langer and coworkers [13-16] that an important major locus of the rapidly exchangeable Ca 2 + pool is the externally bound C a 2+. Ca 2 + in this pool is bound to the glycocalyx and to acidic phospholipids in the sarcolemma. It has been shown to be displaceable by lanthanum [14]. Thus, when lanthanum is in the wash solution, the rapidly exchangeable Ca 2 + pool size should be substantially reduced if the Ca 2+ is indeed externally bound. Our data support the hypothesis that externally bound C a 2+ (or at least La a+ displaceable C a 2+) does not contribute significantly to the rapidly exchangeable Ca 2+ pool for cultured chick embryo ventricular cells. Primary cultures of heart cells offer several advantages over intact heart muscle preparations for studying myocardial cell physiology and pathophysiology [17]. The cultured heart cell model has minimal diffusion limitations and allows a standard reproducible hypoxic insult to be delivered homogeneously to all cells. All cells are perifused with the same solution and thus changes in blood flow or plasma composition, as may occur with other models, are obviated. The cultured cells are devoid of neural elements and are thus not subject to variation in autonomic control or local neurotransmitter activity. Myocardial ischemia in other models is less uniform and islands of severely ischemic cells may lie adjacent to cells with relatively normal metabolism [8, 10, 11]. Whole heart, isolated septum and papillary muscle preparations are diffusion limited models and considerable heterogeneity ofischemic insult at the cellular level

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has been described [8, 17]. Cells at the periphery of the ischemic zone m a y have relatively n o r m a l m e t a b o l i s m while those at the center m a y be irreversibly d a m a g e d . A n i m p o r t a n t d e t e r m i n a n t of cellular injury pertinent to clinical cardiology is the time of transition from reversible to irreversible cellular d a m a g e . T h e study of this transition time is simplified by using e x p e r i m e n t a l p r e p a r a t i o n in which all cells have sustained a similar degree of insult, and in which it is possible to uniformly a n d a b r u p t l y make m a j o r step changes in oxygen concentration. T h e cultured chick e m b r y o ventricular cell preparation also has limitations: it has an avian rather t h a n m a m m a l i a n system a n d it is i m m a t u r e . T h e t-tubule system, which m a y p l a y an i m p o r t a n t role in regulation of calcium flux, is less developed than in m a t u r e hearts. T h e d e v e l o p m e n t of m o n o l a y e r cultures from a d u l t m a m m a l i a n h e a r t is being p u r s u e d in several laboratories and m a y obviate some of these limitations. S t u d y of the complex elements of m y o c a r d ial ischemia m a y be facilitated by examination of its i n d i v i d u a l components, which include hypoxia, substrate w i t h d r a w a l , acidosis, h y p e r k a l e m i a and adenosine excess. T h e current study demonstrates that the a p p r o a c h of e x a m i n i n g alterations in C a / + homeostasis in a non-diffusion limited system where the elements of ischemia, h y p o x i a a n d substrate w i t h d r a w a l , can be carefully controlled, is feasible. U n d e r the conditions studied, L a 3 + dis-

placeable extracellular Ca 2 + did not contribute significantly to the content of the r a p i d l y exchangeable Ca 2 + pool, either u n d e r normoxic o f h y p o x i c conditions. T w o hours of h y p o x i a p r o d u c e d a 2.8-fold increase in the r a p i d l y e x c h a n g e a b l e Ca 2 + pool content a n d also altered the kinetics of Ca 2 + entry into the slowly e x c h a n g e a b l e pool. I t should be possible to study N a + a n d K + homeostasis in h y p o x i a using a similar a p p r o a c h . This e x p e r i m e n t a l a p p r o a c h m a y be b r o a d l y a p p l i c a b l e to the study of ischemia in cells from variety of tissues a n d m a y offer new insights into mechanisms of ischemic cellular injury and methods of a m e l i o r a t i n g the injury.

Acknowledgements This work was s u p p o r t e d in p a r t by N I H grants KO8 HL0691, HL35681 and HL26215. D r M a r s h is recipient of a Clinical Investigator A w a r d from the N I H . D r M u r p h y is s u p p o r t e d in p a r t by the Ainsw o r t h Scholarship in M e d i c i n e from U n i versity College Cork, I r e l a n d a n d is the recipient of a Postdoctoral Fellowship A w a r d from the A m e r i c a n H e a r t Association, Massachusetts Affiliate. W e a p p r e c i a t e the assistance of Ms Susan M c H a l e in p r e p a r a t i o n of this manuscript.

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