BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 38, 195-204 (1987)

Effects of Energy Deprivation and Hydrogen Peroxide on Contraction and Myoplasmic Free Calcium Concentrations in

Isolated Myocardial Muscle Cells

MICHAEL SHEPHERD, MARTIN BRUENING, AMANDA M. AULD, AND

GREGORY J. BARRITT

Department of Biochemistry and Chemical Pathology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia

Received October 20, 1986, and in revised form March 6, 1987

One of the major intracellular consequences of ishemia of the heart is the accummulation of Ca*+ by myocardial muscle cells (l-5). Early intracellular events induced by the ischemic state and subsequent reperfusion are a decrease in cellular ATP concentrations (1,6-10) and the generation of free radicals (1 l- 13). It has been proposed that these changes lead to net Ca” inflow across the sarcolemma and cell damage which arises directly or indirectly from high con- centrations of intracellular Ca2+ (1,5,10,13-18). The mechanisms by which a decreased intracellular ATP concentration or the generation of free radicals lead to increased intracellular Ca2’ and cell damage are not well understood. While a number of studies have been performed with perfused hearts and other intact tissue preparations (6-13), the results have often been difficult to interpret in terms of intracellular events.

The aim of the present experiments was to determine the effects of ATP deprivation and free radicals on the function of myocardial muscle cells. Suspensions of isolated cells were used so that specific changes could be induced in the extracellular environment and in the myoplasm. A number of effects of ATP depletion on isolated myocytes have previously been reported (19-22). However, most previous experiments have been performed with quiescent, rather than contracting, cells. In the present studies, electrically stimulated myocytes were employed. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophen- ylhydrazone (CCCP) has been used to decrease intracellular ATP concentrations (22) and a peroxide, H202, has been used to generate free radicals (23,24).

The results indicate that both ATP depletion and the action of H202 markedly impair the function of electrically stimulated myocytes, in part by enhancing Ca2+ inflow across the sarcolemma. The initial stage of ATP depletion was found to be associated with enhanced Ca2’ inflow through voltage-operated Ca2’ channels.

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196 SHEPHERD ET AL.

METHODS

Isolation of myocardial muscle cells and measurement of contraction. The isolation of myocardial muscle cells from rats (25), enrichment of the preparation with rod-shaped cells, stimulation of contraction at 21°C on microscope slides by electrical impulses, and measurement of the number of contracting cells, were conducted as described previously (26). For electrical stimulation on microscope slides, the electrodes were 2.2 cm apart. The applied voltage was 100 V, the duration 10 msec, and the frequency 1 pulse/2 sec. The number of cells counted at any given time point was SO-100 cells. Verapamil was dissolved in dimethyl sulfoxide (final concentration less than 0.5% (v/v)).

Measurement of intracellular free Ca2+ using quin2. Myocytes were loaded with quin2 by incubation with the acetoxymethyl ester of quin2 (27) (50 PM) for 45 min in the presence of 1.3 mM Ca*’ . The final volume was 2.4 ml and the concentration of cells was 6-10 mg protein ml-‘. The incubation buffer, Buffer C, (Haworth et al. (25)) contained (in mM) 118 NaCl, 4.8 KCl, 1.2 MgS04, 1.2 KH2P04, 25 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid (Hepes), and 11 glucose, adjusted to pH 7.4 (25°C) with KOH. The acetoxymethylester of quin2 was dissolved in dimethyl sulfoxide (final concentration, 0.2% (w/v). At the end of the incubation period the cell suspension was centrifuged at 50g for 1.5 min at room temperature, washed once in 5 ml of Buffer C, and finally suspended in 8 ml of Buffer C. The intracellular concentration of quin2, estimated as described previously (26), was 0.6 2 0.1 (n = 3) mM. Control (unloaded) myocytes were treated in a similar manner except that dimethyl sulfoxide was used in place of the acetoxymethylester of quin2. Fluorescence measurements were conducted between 5 and 45 min after final suspension of the quin2-loaded cells in Buffer C.

Fluorescence was measured at 37°C in 1.5-ml volumes of myocytes suspended in Buffer C which contained 50 ,u~ EDTA (cf. Ref. 28). An Aminco-Bowman spectrofluorimeter (SPF 74, American Instrument Company, Silver Spring, MD), equipped with a constant temperature accessory and a magnetic stirrer was used. The wavelengths for excitation and emission were 340 and 490 nm, respectively (27), and the slit width was 0.5 mm. The output from the fluorimeter was channelled to a Brinkman 2543 potentiometric recorder (Brinkman Instruments, Westbury, NY). For calculation of the values of intracellular free Ca2+ concentration the fluorescence of unloaded cells was subtracted from the value obtained for cells loaded with quin2. Estimates of intracellular free Ca2+ concentrations were made as described by Tsien et al. (27) using 25 PM digitonin to lyse the cells and 1.3 mM CaCl, and 0.5 mM MnCl, to estimate maximum and minimum fluorescence, respectively, and a value of 115 IUI for the dissociation constant for the combination of Ca2’ with quin2 (27). Correction for the presence of extracellular quin2 was made using Mnzf as described by Rink and Pozzan (29).

ATP concentrations. ATP was measured by a luminometric method using luciferin and luciferase (from firefly lantern, Photinus pyralis) as described by Wulff and Doppen (30). Photon emission was measured using the tritium channel of an Isocap (Searle Analytic, Inc.) liquid scintillation counter (31). Myocytes

MYOCARDIAL MUSCLE CELLS 197

2 4 6 6 10

Time (min)

FIG. 1. Decrease in the proportion of rod-shaped myocytes induced by incubation with CCCP. Myocytes were mixed with 0.9 mM (circles) or 4.5 mM (triangles) CaClz without (open symbols) or with (closed symbols) 0.25 PM CCCP. The number of rod-shaped cells present was determined at the times indicated after the addition of CCCP and is expressed as a percentage of the total number of cells present. The values are the means ?SEM of eight experiments, or the means of two experiments (0). The values of P, determined using Student’s t test for unpaired samples, for a comparison with the corresponding control value obtained in the absence of CCCP are *P < 0.05.

were incubated in Buffer C in the presence of I .O mM Ca’+ (final volume 1.0 ml) at 37°C and electrical stimulation applied as described previously (26).

Materials. The acetoxymethylester of quin2 was obtained from Amersham Australia Pty. Ltd., Sydney, New South Wales; and digitonin, CCCP, o-luciferin, luciferase (firely lantern), and ATP from the Sigma Chemical Co., St. Louis, Missouri. Verapamil was generously provided by Knoll AG. All other reagents were from the sources described previously (26).

RESULTS

Energy Deprivation

Addition of 0.25 PM CCCP to electrically stimulated myocytes decreased the proportion of rod-shaped cells present during a period of 10 min following addition of the uncoupling agent (Fig. 1). The principal initial effect of CCCP was to cause a marked shortening of rod-shaped cells which was evident as early as 2 min and clearly developed by 4 min. At later times, some rod-shaped shortened cells were converted to round cells (results not shown). The effect of CCCP was more pronounced at 4.5 mM extracellular Ca*+ than at 0.9 mM extracellular Ca’+ (Fig. 1). The absence of electrical stimulation, or the presence of verapamil,

198 SHEPHERD ET AL.

I 2 4 6 8 10

Time (mid

FIG. 2. Effects of electrical stimulation and verapamil on the decrease in the number of rod- shaped myocytes induced by CCCP. Myocytes were incubated in the presence of 0.9 mM CaCI, and subject to electrical stimulation in the absence of further additions (0) or in the presence of 0.25 PM CCCP (0) or 0.25 PM CCCP + verapamil (A) or without electrical stimulation in the presence of 0.25 PM (m) as described in Fig. 1. The values are the means -tSEM of four experiments or the means of two experiments (A).

inhibited the decline in the number of rod-shaped cells induced by CCCP during the first 5 min, but not a later times (Fig. 2).

The time course for the effect of CCCP on the number of cells contracting was similar to that for the effect of the uncoupler on the total number of rod- shaped cells present (results not shown). Addition of verapamil in the absence of CCCP reduced the number of contracting cells to 35% of the rod-shaped cells present. In the presence of the Ca*+ antagonist, the effect of CCCP on the number of cells contracting showed a time course similar to that observed in the absence of verapamil (results not shown). The effects of CCCP on cell contraction and morphology were associated with a marked decrease in cellular ATP concentrations in both quiescent (Fig. 3a) and contracting (Fig. 3b) myocytes.

The effects of the uncoupler on intracellular free Ca*’ concentrations were measured using myocytes loaded with quin2. The results are shown in Fig. 4. In the absence of depolarization, CCCP caused a small increase in intracellular free Ca*+ concentration over a period of. 12 min. This confirms results previously obtained by Cobbold and Bourne (18). However, a much larger increase was observed when the cells were depolarized with by addition of K+ (46 mM total concentration). Increases in free Ca*+ concentration during the first 5 min of exposure of K+-depolarized cells to CCCP were substantially inhibited by verapamil.

MYOCARDIAL MUSCLE CELLS 199

60 t

20.

2 4 5 Time (rnlo)

FIG. 3. The decrease in the concentration of cellular ATP induced by CCCP in myocytes incubated in the absence (a) and presence (b) of electrical stimulation. Myocytes were incubated in the absence or presence of electrical stimulation, applied at t = 0 mitt, and in the absence (0) or presence (0) of 0.25 PM CCCP, added at 30 set (indicated by arrows). and the measurement of ATP conducted as described under Methods. The values obtained at any given time are expressed as a percentage of the value obtained at 0 min. The mean value for the amount of ATP present in control cells at 0 min was 5.7 k 0.9 (n = 3) nmole/mg cellular protein. The results are the means t SEM of three or four experiments.

Effects of H,O,

The addition of H202 to electrically stimulated myocytes decreased the number of rod-shaped cells which contracted (either partially or fully) (Fig. 5). In the presence of low concentrations of H202, the cells were seen to undergo a change in the pattern of contraction upon electrical stimulation. Instead of a normal end- to-end synchronized contraction, a ripple-like effect was observed, producing waves which moved from one end of the cell to the other. In subsequent ex- periments, the effect of Hz02 on the number of rod-shaped cells which showed normal end-to-end synchronized contraction, as distinct from abnormal contraction, was studied.

The number of cells which contracted with normal end-to-end synchronized contraction in the presence of H202 decreased as the concentration of this agent increased (Fig. 6a), or as the time of exposure to a given concentration of H202 increased (Fig. 6b). The decrease in the number of myocytes which contracted normally was more pronounced at increased extracellular Ca2’ concentrations. In the absence of H202, the number of rod-shaped myocytes contracting with

200 SHEPHERD ET AL.

L L 0 0 2 4 6 8 10 12

Time (mm)

FIG. 4. Increases in intracellular free Ca” concentration induced by CCCP. The preparation and incubation of myocytes loaded with quin2 and measurement of intracellular free Ca*+ concentrations were performed as described under Methods. Myocytes loaded with quin2 were incubated in Buffer C in the the presence of I .O mM CaCI,, 50 (XVI EDTA, and verapamil (when present) for 2 min before the addition of saline (0) or 40 mM KC1 (AJ). After a further 2 mitt, 0.25 PM CCCP was added (indicated by the arrow). The closed symbols represent values of intracellular free Ca” concentration obtained in the presence of CCCP at 6 mM K’ (0). 46 mr.r K’ (W), or 46 mM K’ plus IO PM verapamil (A). The open symbols represent the intracellular free Ca” concentrations under the same conditions before addition of CCCP. The values are the means f SEM of four to nine experiments, or the means of two experiments.

2 4 6 8 10

Time (min)

FIG. 5. Decrease in the number of rod-shaped myocytes contracting either partially or fully in the presence of H,O,. Electrically stimufated myocytes were incubated in the presence of 1.0 mM CaCI, in the absence of further additions (A) and in the presence of 1.0 (0) or 5.0 (0) mtu HZOZ. At the times indicated after the addition of H,Oz, the number of myocytes which contracted either fully or partially was determined as described under Methods, and is expressed as a percentage of the total number of rod-shaped cells present. The results are the means -+ SEM of four experiments.

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(a)

0.25 0.50 0.75 1.0

H202 (mM)

I I I 5 10 15 20 25

Time (min)

FIG. 6. Effects of increasing concentrations of H20Z (a) and of increasing time of exposure to HZO, (b) on the number of myocytes contracting with normal end-to-end synchronized contraction. (a) Electrically stimulated myocytes were incubated in the presence of I .O mM CaC& and the indicated concentration of H,02 for 1 min, and the number of cells contracting normally was determined. The results are the means k SEM of 10 experiments. (b) Electrically stimulated myocytes were incubated in the presence of 1.0 mM Ca” in the absence (0) or presence (A) of 0.5 mM HzOz (added at 0 min), and the number of cells contracting normally was determined at the times indicated. The results are the means of two experiments. The number of cells contracting normally is expressed as a percentage of the total number of rod-shaped cells.

normal end-to-end synchronized contraction was 88 + 2, 84 2 7, and 47 2 20% of rod-shaped cells at 0.5, 1.0, and 2.0 mM Ca”, respectively, and in the presence of 0.5 mM H202 the values were 69 k 6, 24 + 8, and 25 + 12% (n = 3) at 0.5, 1.0, and 2.0 mM Ca2+, respectively. The effects of Hz02 were partially reversed by dimethyl sulfoxide (Table 1).

DISCUSSION

The observation that the effects of CCCP on myocyte shape are more pronounced in (a) contracting myocytes than in quiescent cells and (b) in contracting cells incubated at higher rather than lower extracellular Ca*’ concentrations and the partial inhibition by verapamil of the effect of CCCP on myocyte shape indicate that one of the early effects of CCCP is to induce net Ca*+ inflow across the sarcolemma. Consistent with this conclusion is the observation that the increase

202 SHEPHERD ET AL.

TABLE 1 Partial Reversal by Dimethyl S&oxide of the Decrease in the Number of Myocytes Contracting

Normally Caused by H,O*”

Additions

Number of cells contracting normally

(% of rod-shaped cells)

None H,O, (0.5 mu) Dimethyl sulfoxide HZOZ + diemethyl

sulfoxide

86 f 2 32 + 6 78 + 2

52 + 13

’ Electrically stimulated myocytes were incubated in the presence of 1.3 rnM CaCIZ and the additions are indicated. After 15 min, the number of cells contracting with normal end-to-end synchronized contraction and the total number of rod-shaped cells were determined. The results are the means f SEM of three experiments.

in intracellular free Ca*’ concentration caused by CCCP is much larger in cells depolarized with a high concentration of KC1 than in cells incubated at a normal KC1 concentration, and is inhibited by verapamil. While the effects of pulsed electrical stimulation and high K’ on myocyte contraction are different, a common feature is that voltage-operated Ca*’ channels are in the open state for a much longer period of time than those in nonstimulated cells.

The effects of CCCP are likely to be caused, in part, by a decrease in the intracellular ATP concentration (22). However, the observation that there is little difference in the effect of CCCP on cellular ATP concentrations in stimulated and nonstimulated cells indicates that the effects of CCCP on Ca*’ homeostasis are unlikely to be due solely to decreased ATP concentrations. Furthermore, it is also likely that some damage induced by CCCP is caused by mechanisms which do not involve changes in intracellular free Ca*’ (11,12).

The ability of verapamil to provide some protection during the initial, but not at later, times of exposure to CCCP indicates that during this initial period net Ca*’ inflow is catalyzed by voltage-operated Ca*’ channels. The failure of verapamil to provide protection at later times indicates that, under these conditions, net Ca*’ inflow across the sarcolemma is catalyzed by additional processess. The observed effects of verapamil may be due to inhibition of voltage-operated Ca” channels and preservation of intracellular ATP concentrations (IO).

The present results for the effect of treatment with CCCP on intracellular free Ca*’ concentrations confirm those reported previously by Cobbold and Bourne (22) for quiescent myocytes. However, the present study also shows that when myocytes are depolarized, depletion of intracellular ATP causes a much greater increase in the myoplasmic free Ca*+ concentration. K+-depolarized myocytes should reflect the state of myocytes in viva more closely than do quiescent cells.

The results obtained with H202 show that low concentrations of this peroxide induce the abnormal contraction of myocytes by a process dependent on the extracellular Ca*+ concentration. Furthermore, the abnormal behavior of the cells observed in the presence of H202 is similar to that observed by Haworth

MYOCARDIAL MUSCLE CELLS 203

et al. (32) for myocytes incubated at high extracellular Ca2’ concentrations. These observations suggest that the effects of HzOz involve increased net Ca’+ inflow across the sarcolemma. The observations of others that peroxides like Hz02 can generate free radicals (13,24) and the ability of dimethyl sulfoxide, a scavenger of free radicals (33), to partially inhibit the effects of H202, suggest that the effects of H202 observed in the present experiments are mediated by free radicals. Recent studies of Tones and Poole-Wilson (24), using perfused inter-ventricular septa, have shown that H202 and cumene peroxide stimulates Ca*’ uptake by cardiac muscle.

The present results provide evidence which indicates that both depletion of cellular ATP and hydroperoxides induce damage to cardiac myocytes by enhancing net Ca” inflow. Electrically stimulated isolated myocytes should provide an appropriate system for further elucidation of the molecular mechanisms involved in the response of myocytes to ATP depletion and the generation of peroxides.

SUMMARY

The effect of energy deprivation and H202 on the contraction, shape, and intracellular free Ca*+ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhy- drazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mM extracellular Ca*+, CCCP (0.25 PM) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca*’ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mM extracellular K+, CCCP caused a marked increase in the myopiasmic free Ca*+ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K+-induced depolarization.

Exposure of myocytes to H202 (0.5 mM) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H202 were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca”, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H202, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca2’ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.

ACKNOWLEDGMENT This work was supported by a grant from the National Heart Foundation of Australia.

204 SHEPHERD ET AL.

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