or desferrioxamine administered at the time of reperfusion fail to improve postischemic recovery in the isolated rat heart after long-term hypothermic storage

] hlol

(ielI

Cardiol

22, 1211-1220

(1990)

Diltiazem and/or Desferrioxamine Administered at the time of Reperfusion Fail to Im@rove Postischemic Recovery in the Isolated Rat Heart After Long-term Hypothermic Storage Manuel Cardiovascular

Galiiianes

and David

J. Hearse

Research, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK

(Received I4 January 1990, accepted in revised form 4 May 1990) M. GALIGANES AND DAVID J. HEARSE. Diltiazem and/or Desferrioxamine Administered at the time of Reperfusion Fail to Improve Post-ischemic Recovery in the Isolated Rat Heart After Long-term Hypothermic Storage. Journal of Molecular and Cellular Cardiology (1990) 22, 121 I-1220. The ability of diltiazem and/or desferrioxamine to enhance the recovery of cardiac contractile function during reperfusion after prolonged hypothermic storage was assessed. Isolated rat hearts were arrested with St. Thomas’ Hospital Cardioplegic Solution and stored for 10 h at 4°C. Reperfusion in the Langendorff mode was initially carried 01.1’ with crystalloid perfusate with or without added diltiazem (0.5 pmol/l) and/or desferrioxamine (15, 50, 100, 150 or 250 pmol/l). After 15 min the drugs were discontinued and the hearts were perfused for a further 45 min. Diltiazem reduced leakage of creatine (CK) kinase during the first 15 min of reperfusion from 102 + 8 IL/15 min/g dry wt to 67 f 9 IV/ 15 min/g dry wt (P < 0.05). However, during the subsequent period ofdiltiazem-free perfusion, CK leakage was similar to control values (131 f 24 vs 142 f 34 IU/45 min/g dry wt, respectively!. After 1 h of reperfusion there was no significant difference in total CK leakage between the diltiazem and the control groups (198 &- 32 vs 244 + 39 NJ/SO min/g dry wt, respectively). Desferrioxamine had no effect on CK leakage at any of the doses studied. Diltiazem significantly reduced leakage of enzyme during the initial reperfusion phase when combined with desferrioxamine; however, as with diltiazem alone, this protection was losr after the drug was withdrawn. Post-ischemic contents of adenosine triphosphate and creatine phosphate were similar in all groups as was the final recovery of function, as assessed by left ventricular developed pressure at an end-diastolic pressure of 5 mmHg. In conclusion, neither diltiazem nor desferrioxamine nor both together could be shown to confer benefit during reperfusion after long-term storage.

KEY WORDS:

Reperfusion;

Diltiazem;

Desferrioxamine;

Long-term

Introduction The development of procedures for the longterm [ >6 h) preservation of the heart is important if the shortage of donor hearts for transplantation is to be overcome. Two important determinants of the ability to reanimate hearts after extended periods of hypothermic ischemic storage are the condition of the heart at the time of harvest and the extent of injury sustained during the storage. A third possible factor may be “reperfusion-induced in.jury”. Although controversial, it is possible that reperfusion may induce myocardial injury above that sustained as a consequence of the preceding ischemia (Hearse, 1977; Hearse, Please address all correspondence to: Manuel Hospital, London SE1 7EH, UK. 0022-2828/90/l

11211 + 10 $03.0010

Galiiianes,

preservation;

Rat heart

1990). If reperfusion-induced injury does reduce post-ischemic recovery then it should be possible to modify the conditions of reperfusion so as to enhance either the rate or the extent (Hearse, 1988;) of recovery. In this connection it has been proposed (Hearse and Tosaki, 1988; Hearse, 1990; Opie, 1989) that reperfusion-induced calcium overload and/or free radical production might be important factors in determining the outcome of reperfusion. Thus, studies in a variety of species and experimental preparations have shown that transient reduction of calcium (Allen et al., 1986; Follette et al., 1981; Klein et al., 1989; Kuroda et al., 1986), the use of calcium

Cardiovascular

Research,

The Rayne

Institute.

IQ 1990 Academic

St. Thomas’ Press Limited

1212

M. Galihncs

snd D. J. Hearse

antagonists (Allen et al., 1986; Klein et al., 1989) or the use of free radical scavengersor agents that inhibit radical production (Ambrosio et al., 1987; Chambers et al., 1987; Shlafer et al., 1982b) can enhance postischemic recovery. However, some of these studies (as well the entire concept of reperfusion-induced injury) are controversial (Bershon and Shine, 1983; Jennings et al., 1986; Klein et al., 1989; Nejima et al., 1989; Przyklenk and Kloner, 1989). The present studies were therefore designed to determine whether the calcium antagonist diltiazem and/or the iron chelator desferrioxamine, when given at the time of reperfusion, were able to improve the post-ischemic recovery of the rat heart.

Materials

and Methads

Animals and perfusion procedures

Male Wistar rats (300-350 g body wt) were anesthetized (pentobarbital 60 mg/kg, intraperitoneally); sodium heparin (1000 IU/kg) was then injected intravenously and the hearts excised and placed in cold (4°C) perfusion medium until contraction had ceased(approximately 15 s). The aorta was then cannulated and the St. Thomas’ Hospital Cardioplegic Solution (containing in mmol/l: NaCl 110.0, KC1 16.0, MgClz 16.0, CaC12 1.2, NaHCOs 10.0; pH 7.8 at 37”C), gassedwith 100% 02, was infused at a constant perfusion pressure (equivalent to 60 cmHz0) for 1 min at 25°C followed by 2 min at 4°C. Hearts were then removed from the cannula and stored in cold cardioplegia at 4°C. After storage the hearts were again cannulated via the aorta and perfused in the LangendorlI ( 1985) mode at 37°C and a constant perfusion pressure(equivalent to 100 cmHz0) with standard perfusion fluid (containing in mmol/l: glucose 11.1, NaCl 118.5, KC1 4.75, MgS04 1.19, KHzPO‘, 1.18, NaHCOs 25.0, CaClz 1.36), gassedwith 95% 02 plus 50/ COz (pH 7.4). All perfusion fluids were filtered to remove any particulate matter using a 5.0 pm porosity filter. After 10 min of reperfusion a balloon catheter was inserted into the left ventricle via the left atrium and filled with a volume of saline sufficient to produce a constant end-diastolic pressureof 5 mmHg (Curtis et al., 1986). The experiments

were conducted in conformity with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. Experimental

time course

After infusion with St. Thomas’ Hospital Cardioplegic Solution hearts (n = G/group) were immersedin cardioplegic solution at 4°C and stored for 10 h. They were then recannulated and reperfused aerobically for 60 min at 37°C; non-treated hearts were reperfused throughout the 60 min using the standard perfusion fluid whereas the drug treated groups were reperfused for 15 min with diltiazem and/or desferrioxamine included in the perfusion fluid before going over to standard drug-free fluid for the remaining 45 min. Nonischemic hearts (n = 6) perfused for 60 min served as aerobic controls. Coronary flow was measured (by timed collection of coronary effluent) throughout the reperfusion period and coronary effluent was taken for determination of creatine kinase (CK) leakage. At the end of all experiments hearts were freezeclamped (using stainlesssteel tongs cooled to the temperature of liquid nitrogen) and taken for the analysis of adenosine triphosphate (ATP) and creatine phosphate (CP) content (Hearse et al., 1977). Expression of results

CK leakage, expressed as IU/g dry wt, was measuredduring both the first 15 min and the entire 60 min of reperfusion. ATP and CP contents were expressedaspmol/g dry wt, and indices of fuction were expressedin absolute terms. All results are expressed as mean + standard error of the mean. Analysis of variance was used; when a significant F-value was obtained, comparisons between the untreated and each of the treated groups were carried out by the two-tailed Dunnett’s test. A difference was considered statistically significant when P < 0.05. Results Cardiac function

Figures 1, 2 and 3 show the post-ischemic recovery profiles for left ventricular developed

1213

(b)

140 120 ,oo Desferrioxomine

Untreated control

2ob

*I’

0

(c)

,Diltiarem I I 10 15 2025 303540 Time of reperfwon

*p(2) 5

I

(0.5pmol/l) I I I 45 5055 (min)

(15 pmol/l)

*’

2o I 60

14DyP\

0

Desferrloxomlne (15 mol/l) Dtltlozem (0.5Pmol/l) I I I I I I I I IO 15202530354045505560 Time of reperfuslon (mln)

(I ,;+,5

(d)l4D, Desferrloxomlne

0

(e)

140

5 fj

~202530354045505560 Time of reperfuwon

5

10 15 20 25 30 3540 Time of reprefuslon

pmol/l)

45 50 55 6; (mln)

r nine

0

0 (mm)

(100

5

(150

pmol/I)

I20

IO 15 20 25 30 35 4045 50 55 60 Time of reperfuslon (mln)

- Desferrioxamine

0

5

(250

10 15202530354045505560 Time of reperfuslon

~mol/l)

(mln)

FIGURE 1. The effect of diltiazem (0.5 pmol/l) and/or desferrioxamine (15, SO, 100, 150 or 250 pmol/l), administered during the first 15 min of reperfusion, on the recovery of the left ventricular developed pressure (LVDP) following 10 h of hypothermic (4°C) storage. After the first 15 min of reperfusion all hearts were perfused with drug-free medium. Each point represents the mean of six measurements in six hearts and the bars indicate the S.E.M. The open triangles in block (a) represent hearts that were subjected to 1 h of aerobic perfusion. The numbers in parentheses represent the number of hearts remaining for evaluation after exclusion on the grounds of systole or ventricular arrhythmias. (*P < 0.05 from drug-free control hearts.)

pressure (LVDP), heart rate and coronary flow in untreated hearts and hearts reperfused for 15 min with diltiazem and/or various concentrations of desferrioxamine. The results are also compared with those from control hearts which were aerobica!ly perfused for 60 min [open triangles in Figures 1 (a), 2(a) and 3(a)] to give an indication of the extent of functional loss in the groups studied.

(i) Recovery of LVDP LVDP (which, in the absence of ischemia, was in excess of 110 mmHg), recovered to 57 + 8 mmHg

after

10

min

of

reperfusion

in

the

hearts which were not treated with diltiazem or desferrioxamine [open circles in Figure 1 (a)]. This value increased progressively to 71 f 8 mmHg after 60 min of reperfusion.

M. Gdiiiaaes

1214

4oo

Untreoted

and D. 1. Hearse

control

i-1

*

200

0

‘(2) t

150 100 1 50

,

I

Diltiozem ,

5

,

(0.5

~mol/l)

[,

,

,

g

200

2 I

150 loo

I 5

0

of reperfusion

I Desferrloxomine I -..---Dlltlozem (1 1

(15 pmol/l) (0.5 pmol/l)

50

,I

IO 15202530354045505560 Time

\I,;,,

(min)

I I I IO 152025 Time

I

1 I I 3OE40455056

of reperfusion

I

I

I

I) 60

(min)

(d)

Desferrioxomine Diltiozem / 50 /0

5

**I/ I II IO 15202530354045505560 Time of reperfuscon

y

0

5

lo

I

I

I

I

I 0

5

(mm)

IO 152025303540455056 Time of reperfusion

60 (min)

Desferr~oxom~ne(150~mol/l) Diltiozem [0.5~mol/l)

I I I 15 20 25 303540

Time

(50~mol/l) (0.5pmol/I)

of reperfusion

I

I 45

I I I 5055 60

(mm)

0

I I I I I I I 5 IO 15202530354045505560 Time

of reperfusion

I

I

I

I

II

(mm)

FIGURE 2. The effect of diltiazem (0.5 nmol/l) and/or desferrioxamine (15, 50, 100, 150 or 250 pmol/l), administered during the first 15 min of reperfusion, on the recovery of the heart rate (HR) foIlowing 10 h of hypothermic (4°C) storage. After the first 15 min of reperfusion all hearts were perfused with drug-free medium. Each point represents the mean ofsix measurements in six hearts and the bars indicate the S.E.M. The open triangles in block (a) represent hearts that were subjected to 1 h of aerobic perfusion. The numbers in parentheses represent the number of hearts remaining for evaluation after exclusion on the grounds of asystole or ventricular arrhythmias. (*P < 0.05 from drug-free control hearts.)

Inclusion of diltiazem (0.5 pmol/l) during the lowing 30 min of reperfusion the recovery of first 15 min of reperfusion resulted in contrac- function in the two groups was identical, tile arrest during this period. When diltiazem reaching approximately 60 to 70% of mean was removed from the reperfusion solution value in the aerobic control hearts [Fig. 1(a)]. contractile activity recovered to reach a stable Desferrioxamine alone [Fig. 1(b)-(f)] level after 15 min of reperfusion. At this time causeda small reduction in LVDP during the the recovery of LVDP (64 + 10 mmHg) was first 15 min of reperfusion. However, once indistinguishable from that in untreated con- desferrioxamine had been removed from the trol hearts (66 + 7 mmHg). During the fol- perfusate, LVDP recovered to a steady level

Diltiazem,

Dcsferriosamine

and

I I 1 1 1 1 1 ’ ’ ’ 1 ’ 0

5

IO 15 2025 Time

0

30354045505569

of reperfuslon

kb

8 4

0

Desferrloxamine

Time

(50

II II IO 15 2025 Time

I I I 30354045

gmol/l)

of reperfwon

I

II 5055

20

F :<::8 6

t I 5

5 10 15202530354045505560

(mm)

Id)

;

1215

Reperfusion

1 60

0

of reperfusion

DeTrioxamme

Desferrloxamlne Dt ltiazem (0.5

(f)

20

(100

~mol/l)

(IO0 pmol)

pmol/l)

I I I I I I I 5 IO 152025x)354045505560 Time

(mln)

(mln)

of reperfuslon

Desferrloxamlne

I

I

/

II

II

(min)

(250

pmol/l)

(250 pmol)

pmol/l)

-2 .-c s

8-

s

Desferrloxamine Diliazem (0.5

4-

0

I I I I I 5 IO I5 2025 Time

I I I I I I 30 3540 45 5055

of reperfusion

II 60

(min)

0

II I Ill II 5 IO 15202530354045505560 Ttme of reperfusion

I

I

I

I

(mln)

FIGURE 3. The effect of diltiazem (0.5 pmoI/l) and/or desferrioxamine (15, 50, 100, 150 or 250 pmol/l), administered during the first 15 min of reperfusion, on the recovery of the coronary flow (CF) following 10 h of hypothermic (4°C) storage, After the first 15 min of reperfusion all hearts were perfused with drug-free medium. Each point represents the mean ofmeasurement in six hearts and the bars indicate the S.E.M. The open triangles in block !a) represent hearts that were subjected to I h of aerobic perfusion.

indistinguishable from that in untreated controls. Thus, after 60 min of reperfusion LVDP recovered to 71 + 6 mmHg in the untreated control group and to 71 f 6, 70 + 7, 70 + 6, 72 2 10 and 68 + 5 mmHg in the 15,50, 100, 150 and 250 pmol/l desferrioxamine groups. The combination of diltiazem (0.5 pmol/l) with each concentration of desferrioxamine during the first 15 min of reperfusion produced complete or near complete contractile arrest. However, as in the case of diltiazem

alone or desferrioxamine alone, removal of the drugs from the reperfusion solution restored function, which by the end of the reperfusion period was identical to that in the untreated control group. (ii) Heart rate Heart rate in the untreated control group recovered to better than 85% of that in the aerobic control group (Fig. 2); maximal recovery was achieved within the first 10 min of

1216

M. GPliaancs and D. J. Hearse

reperfusion and was maintained at this level for the remainder of the reperfusion period. Treatment with diltiazem alone or in combination with desferrioxamine resulted in a severe reduction in heart rate (to almost zero in most hearts in most groups). However, when diltiazem was discontinued heart rate rapidly recovered to a value similar to that seen in the untreated controls. The use of desferrioxamine alone during the initial reperfusion period resulted in a small reduction in heart rate (not of statistical significance) which recovered to a value indistinguishable from that untreated controls once the desferrioxamine was discontinued. (iii) CoronaryJlow Neither diltiazem nor desferrioxamine nor their combination had any significant effect upon coronary flow whether during the initial reperfusion period or during the subsequent drug-free reperfusion period (Fig. 3). In all instances coronary flow recovered to within 72”/, to 9294 of that measured in the aerobic controls. CK leakage

Neither diltiazem nor desferrioxamine, singly or in combination, had any significant effect upon enzyme leakage when measuredover the entire 60 min period of reperfusion (Fig. 4). However, when measured during the first 15 min of reperfusion enzyme leakage was 20:/, to 4096 lower in hearts which received diltiazem [Fig. 4(B)]. Desferrioxamine alone had no effect in enzyme leakage. Tissue ATP

and CP content

As shown in Figure 5, in comparison with untreated hearts neither diltiazem nor desferrioxamine, singly or in combination, had any beneficial effect upon tissue ATP or CP contents measured at the end of the 60 min period of reperfusion.

c-300

“, 250 s 200 -g 150 B loo B 50 Y 0 Dlltlozem

concentratfon

(0.5 pol/ll

FIGURE 4. The effect ofdiltiazem (0.5 ~mol/l) and/or desferrioxamine (15, 50, 100, 150 or 250 ~mol/l), administered during the first 15 min of reperfusion, on the leakage of creatine kinase (CK) following 10 h of hypothermic (4°C) storage. After the first 15 min of reperfusion all hearts were perfused with drug-free medium. The light hatching indicates the CK leakage during the first 15 min of reperfusion and the dark hatching the leakage during the entire (60 min) period of reperfusion. Each column represents the mean of measurements in six hearts and the bars indicate the S.E.M. (*P < 0.05 from the drug-free control hearts.)

post-ischemic recovery of the heart following an extended (10 h) period of hypothermic storage. On the basisof functional, metabolic and enzymatic criteria no sustained benefit could be demonstrated. A number of aspects of these studies, and their interpretation do, however, warrant discussion. Ischemia- versus reperfusion-induced

injury

While there is no doubt that myocardial ischemia, even in the presenceof cold cardioplegia, can be damaging, the possibility that reperfusionper semight causelethal cell injury (Hearse, 1990) remains controversial. The extent of damage caused by a fixed ischemic Discussion insult can undoubtedly be reduced through The present studies were designed to assess the use of a variety of anti-ischemic agents. whether diltiazem and/or desferrioxamine, Thus, agents, such as calcium antagonists when given at the time of reperfusion (and (Allen et al., 1986; Bershon and Shine, 1983; subsequently discontinued) could enhance the Klein et al., 1989), given either before or

Diltiazem,

Desferrioxamine

ca zo -Z fb 3200 5‘ 150 8” loo ii

500 Untreated control

m

3501,

I5 50 Desferrwxamlne

100 150 250 concentrotlon (pmol/l)

L.\

I

a 5 2ccJ ;g 150 6 100 50 0 Untreated control

0 Dlltlazem

concentrotlon

(05 pnol/lj

FIGURE 5. The effect ofdiltiazem (0.5 pmol/l) and/or desferrioxamine (15, 50, 100, 150 or 250 pmol/l), administered during the first 15 min of reperfusion, on the recovery of the tissue adenosine triphosphate (ATP: light hatched columns) and creatine phosphate (CP: dark hatched columns) contents following 10 h of hypothermic i4”C) storage and 60 min reperfusion. After the first 15 min of reperfusion all hearts were perfused with drug-free medium. Each column represents the mean of measurements in six hearts and the bars indicate the S.E.M.

during a period of myocardial &hernia slow the rate of development of the ischemic injury so that at the time of reperfusion more cells are in a potential salvable state and, thus, postischemic recovery of function can be improved. Numerous agents (Hearse, 1988) have been shown to possess such anti-ischemic properties and many have been incorporated into cardioplegic solutions (Hearse et al., 198 1) . The possibility exists that reperfusion may further injure the tissues and thus limit postischemic recovery. Thus, reperfusion may be associated with potentially lethal ventricular arrhythmias (Golberg et al., 1983; Hearse, 1990; Manning and Hearse, 1984; Tanaka and Hearse, 1988; Tzivoni et al., 1983) and myocardial stunning (Bolli, 1988; Braunwald and Kloner, 1982; Reimer et al., 1981)) and may also hasten the expression of various necrotic processes in cells which had already died before reperfusion was initiated. The

and

Reperfusion

121;

mechanisms underlying these various forms of reperfusion-induced injury are complex and controversial (Bolli, 1988; Hearse and Tosaki, 1988; Opie, 1989), but intracellular calcium overload and/or free radical induced injury are frequently cited as important contributory factors (Ambrosio et al., 1987; Chambers et al., 1987; Chien et al., 1978; Henry et al., 1977; Jennings et al., 1986; Klein et al., 1989; Kuroda el al., 1986; Nejima et al., 1989; Przyklenk and Kloner, 1989; Shen and Jennings. 1972; Shlafer et al., 1982b). It is also possible that reperfusion may actually kill cells that were potentially viable in the moments before reperfusion. The existence of this form of lethal reperfusion-induced injur) is again controversial (Hearse, 1990; Jennings et al., 1986; Reimer et al., 1989) with little definitive evidence for its occurrence, although, once again, free radicals and ralcium have been suggested as important contributory factors. Conclusive proof for the existence of lethal reperfusion injury requires that agents given at the time of reperfusion (not before, so as to exclude anti-ischemit effects) result in a sustained improvement in the recovery ofcontractile function even when the intervention is eventually removed. In the present studies diltiazem and/or desferrioxamine were given at the time of reperfusion and were subsequently withdrawn; sustained benefit was not observed. This observation might question the suitability of these drugs for reducing reperfusion-induced injury or possibly the very existence of reperfusion injury. Rationale for the use of desfeerrioxamine An extensive literature now shows that various agents that inhibit free radical production or promote their elimination once formed. can protect the heart against the adverse conscquences of ischemia and reperfusion i Ambrosio et al., 1987; Chambers e/ al., 1987; Myers et al., 1986; Nejima et al., 1989; Przyklenk and Kloner, 1989; Shlafer et al., 1982a; Shlafer et al., 198213; Stewart et al., 1983;. Conversely, it has been shown that agents that promote radical production can exacerbate injury (Blaustein et al., 1986; Burton et al., 1984; Jackson et al., 1986). The sources of free radicals in the myocardium are man); and

1‘218

M. CaMames

and D. J. Hearst

complex, and include leukocytes, mitochondria, hemoglobin, myoglobin, aldehyde oxidases and catecholamines (Hearse, 1989). Iron, a cofactor in biological systems(Aust el al., 1985), is thought to play a role in free radical production through its involvement in the Fenton reaction which leads to the production of hydroxyl radicals. It has been suggested (Holt et al., 1985) that ischemia and reperfusion may both impair the cellular control of free iron and that iron chelators may therefore protect against free radical-induced injury. Desferrioxamine is capable of chelating iron (Kerbele, 1964)) inhibiting lipid peroxidation (Willis, 1969) and acting as a direct scavenger of free radicals (Halliwell, 1985). It hasalso been shown (Bolli et al., 1987; Menasthe et al., 1986; Reddy et al., 1989) to reduce the injury associatedwith ischemia and reperfusion. However, other studies have failed to demonstrate an effect (Maxwell et al., 1989; Myers et al., 1986). Various factors such as species,dose,model, in vivo vs in vitro, duration of ischemia and temperature of the myocardium might account for these conflicting results. The failure of desferrioxamine to improve recovery in the present studies could be taken as evidence that it doesnot prevent the formation of hydroxyl radicals or that ironmediated hydroxyl radical production plays no role in reperfusion-induced injury in our model. However, it could also be argued that desferrioxamine might have been effective at other concentrations or that our asanguinous preparation is unsuitable for revealing any protective properties. Thus, the absence of leukocytes and hemoglobin from crystalloid perfused hearts eliminates major sources of radicals and iron; however, many other sourcesexist, including the aldehyde oxidases, catecholamines, mitochondria and myoglobin. It could alsobe argued that desferrioxamine, administered at the time of reperfusion, may be confined to the extracellular space or cannot enter the cell rapidly enough to prevent a burst of intracellular radical formation. The possibility also exists that our chosen duration of 15 min exposure to desferrioxamine was too short to allow endogenous defensesto recover fully and that radical damage may arise after the withdrawal of the compound. Studies with longer durations of

treatment would be required to resolve this possibility. Another qualification to be applied to the interpretation of the present studies relates to coronary flow, which during reperfusion (Fig. 3) was approximately 20% lower than the values from the non-ischemic, aerobically perfused hearts. More severe ischemic damage in the subendocardium (possibly including the no-reflow phenomenon) might well have occurred and accounted for this flow deficit. If this were the case,then an impairment of drug delivering might well have accounted, in part at least, for our negative results. Rationale for the use of diltiatem A large body of evidence also suggest that calcium overload might occur during the early moments of reperfusion and limit postischemic recovery (Henry et al., 1977; Shen and Jennings, 1972). In support of this, both calcium antagonists and transient hypocalcemia have been reported to improve the outcome of reperfusion (Allen et al., 1986; Follette et al., 1981; Klein et al., 1989; Kuroda et al., 1986). However, the results of other studies dispute that possibility, particularly when calcium antagonists are administered at the onset of reperfusion (Bourdillon et al., 1982; Chapel et al., 1985; Poole-Wilson et al., 1984).

The failure of diltiazem to afford protection in the present studies might be due to an inappropriate dose. However, since the dose selectedhad a profound effect upon heart rate and contractile function, pharmacological activity can be assumed. It would therefore appear that L-channel blockade offers no protection against reperfusion-induced damage. In this respect our data are consistent with those of others (Grinwald and Brosnahan, 1987; Poole-Wilson and Tones, 1988; Renlund et al., 1984;Tani and Neely, 1989). The ability of diltiazem to reduce CK leakage during the first 15 min of reperfusion could be taken as a sign of efficacy if it were not for the fact that metabolic and functional indices were not improved. The effect of diltiazem on CK leakage is more likely a reflection of the altered contractile state of the heart during the early reperfusion period and the consequent reduction in enzyme clearance (de Leiris and Hearse, 1984).

Dilthzem,

Desferrioxamint

and

Concluding comments

Repmfusion

I?19

Acknowledgements

The present studies in a model of long-term hypothermic global &hernia indicate that diltiazem and desferrioxamine (singly or in combination) when given at the time of reperfusion do not afford any additional protection. Whether this reflects the failure of these drugs to act against reperfusion-induced injury or the existence of such injury itself remains to be resolved. Studies with other drugs and antioxidants will be needed before the concept could be challenged.

This work was supported in part by grants from the National Heart Research Fund, STRUTH and National Heart, Blood and Lung Institute (HL 39457). The valuable discussionof Drs MJ Curtis and MJ Shattock is gratefully acknowledged. We thank Lorex Pharmaceuticals Limited (UK) for donating the diltiazem.

References ALLEN BS, OKAMOTO F, BUCKBERG GD, ACAR C, PART~NGTON M, BUCY H, LEAF J (1986) Studies of controlled rep&&ion after ischemia. IX. Reperfusate composition: benefits of marked hypocalcemia and diltiazem on regional recovery. J Thorac Cardiovasc Surg 92: 564-572. AMBROSIO G, WEISFELDT ML, JACOBUS WE, FLAHERTY JT (1987) Evidence for a reversible oxygen radical-mediated component of reperfusion injury: reduction by recombinant human superoxide dismutase administered at the time of reflow. Circulation 75: 282-291. AUST SD, MOREHAUSE LA, THOMAS CE (1985) Role of metals in oxygen radical reactions. Free Radical Biol Med 1: 3-25. BERSHON MM, SHINE KI (1983) Verapamil protection of ischemic isolated rabbit heart: dependence on pretreatment. J Mol Cell Cardiol 15: 65967 1. BLAUSTEIN AS, SCKINE L, BROOKS WW, FANBURG BL, BING OHL (1986) Influence of exogenously generated oxidant species on myocardial dysfunction. Am J Physiol25lk H595-H599. BOLLI R, PATEL BS, ZHU W, O’Nem PG, HARTLEY CJ, CHARLAT ML, ROBERTS R (1987) The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am J Physiol253: H 1372-H 1380. BOLLI R (1988) Oxygen-derived free radicals and postischemic myocardial dysfunction (“stunned myocardium” J Am Co11 Cardiol12: 239249. BOURDILLON PDV, POOLE-WILSON PA (1982) The effects of verapamil, quiescence, and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res 50: 360-368. BRAUNWALD E, KLONER RA (1982) The stunned myocardium: prolonged post-ischemic ventricular dysfunction. Circulation 66: 11461149. BURTON KP, MCCORD JM, GHAI G (1984) Myocardial alterations due to free-radical generation. Am J Physiol 246: H776-H783. CHAMBERS DJ, BRAIMBRIDGE MV, HEARSE DJ (1987) Free radicals and cardioplegia: free radical scavengers improve post-ischemic function of the rat myocardium. Eur J Cardiothorac Surg 1: 37-45. CHAPEL SP, LEWIS MJ, HENDERSON AH (1985) Myocardial reoxygenation damage: can it be circumvented. Cardiovasc Res 19: 299-305. CHIEN KR, ABRM~S J, SERRONI A, MARTIN JT, FARBER JL (1978) Accelerated phospholipid degradation and associated membrane dysfunction in irreversible, ischemic liver cell injury. J Biol Chem ‘253: 4809. CURTIS MJ, MACLEOD BA, TABRIZCHI R, WALKER MAJ (1986) An improved perfusion apparatus for small animal hearts. J Pharmacol Methods 15: 87-94. FOLLETTE DM, FEY K, BUCKBERG GD, HELLY JJ JR, STEED DL, FOGLIA RP, MALONEY JV JR (1981) Reducing postischemic damage by temporary modification of reperfusate calcium, potassium, pH and osmolarit!. J Thorac Cardiovasc Surg 82: 221-238. GOLDBERG G, GREENSPAN AJ, URBAN PL, MUZA B, BERGER B, WALINSKY P, MAROKO PR (1983) Reperfusion arrhythmia: a marker of restoration of antegrade flow during intracoronary thrombolysis for acute myocardial infarction. Am Heart J 105: 26-32. GRINWALD PM, BROSNAHAN C (1987) Sodium imbalance as a cause of calcium overload in post-hypoxic reoxygenation injury. J Mol Cell Cardiol 19: 487495. HALLIWELL B (1985) Use of desferrioxamine as a probe for iron-dependent formation of. OH. Biochem Pharmacol34: 229233. HEARSE DJ (1977) Reperfusion of the ischemic myocardium. J Mol Cell Cardiol9: 605616. HEARSE DJ, OPIE LH, KATZEFF IE, LUBBE WF, VAN DER WERFF TJ, PEISACH M, BAILLE G (1977) Characterization of the “border zone” in acute or regional ischemia in the dog. Am J Cardiol Uk 7 16726. HEARSE DJ, BRAIMBRIDGE MV, JYNCE P (1981) Protection of the Ischemic Myocardium: Cardiqplegta. New York. Raven Press, pp 341-352.

1220 HEARSEDJ (1988) The protection

M. tZdifbncs

and D. J. Hearse

of the ischemic myocardium: surgical success v clinical failure? Prog Cardiovasc Dis 30: 381402. HEARSE DJ, Tosam A (1988) Free radicals and calcium: simultaneous interacting triggers as determinants of vulnerability to reperfusion-induced arrhythmias in the rate heart. J Mel Cell Cardiol20: 213-223. HEARSE DJ (1989) A possible role for free radicals in tissue injury during myocardial ischemia and reperfusion. Prog Appl Microcirc 13: pp 54-70. HEARSE DJ (1990) Ischemia, reperfusion and the determinants of tissue injury. Cardiovasc Drug Ther (in press). HENRY PD, SCHUCHLEIB R, DAVIS J, WEISS ES, SOBEL BE (1977) Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol233: H677-H684. HOLT S, GUNDERSON KJ, NAYINI NR, cf al (1985) Myocardial tissue iron delotion and evidence for lipid peroxidation after two hours of ischemia (abstr). Ann Emerg Med 14: 499. JACKSON CV, MICKELSON JK, POPE TK, RAO PS, LUCCHESI BR (1986) 0, free radical-mediated myocardial and vascular dysfunction. Am J Physiol 251: H 1225-H 123 1. JENNINGS RB, REIMER KA, STEENBERGER C (1986) Myocardial ischemia revisited: the osmolar load, membrane damage and reperfusion. J Mel Cell Cardiol 18: 769780. KERBELE H (1964) The biochemistry of desferrioxamine and its relation to iron metabolism. Ann NY Acad Sci 119: 758-768. KLEIN HH, PICH S, LINDERT S, NEBENDAHL K, WARNEKE G, KREUZER H (1989) Treatment of reperfusion injury with intracoronary calcium channel and reduced coronary free calcium concentration in regionally ischemic, reperfused porcine hearts. J Am Co11 Cardiol 13: 1395-1401. KURODA H, ISHIGURO S, Mom T (1986) Optimal calcium concentration in the initial reperfusate for post-ischemic myocardial performance (calcium concentration during reperfusion). J Mel Cell Cardiol 18: 625633. LANGENDORFF 0 (1985) Untersuchungen am uberlebenden Saugethierhenen. Pthigers Arch 61: 291-332. DE LEIRIS J, HEARSE DJ (1984) Myocardial enzyme leakage as an indicator of cellular injury: principles and application. In: Methods ofStudying Cardiac Membranes. vol 1, NS Dhalla (Ed.). CRC Press 17: pp 253-277. MANNING AS, HEARSE DJ (1984) Reperfusion-induced arrhythmias: mechanisms and prevention. J Mol Cell Cardiol 16: 497-518. MAXWELL MP, HEARSE DJ, YELLON DM (1989) Inability of desferrioxamine to limit tissue injury in the ischaemic and reperfused rabbit heart. J Cardiovasc Pharmacol 13: 608615. study of free radical scavengers in MENASCHE P, GROU~~ET C, GAUDUEL Y, F%VNICA A (1986) A comparative cardioplegic solutions. J Thorac Cardiovasc Surg 92: 264-27 I. MYERS CL, WEISS SJ, KIRSH MM, SHEPARD BM, SHLAFER M (1986) Effects of supplementing hypothermic crystalloid cardioplegic solution with catalase, superoxide dismutase, allopurinol, or desferrioxamine on functional recovery of globally ischemic and reperfused isolated hearts. J Thorac Cardiovasc Surg 91: 281-289. NEJIMA J, KNIGHT DR, FALLON JT, UEMURA N, MANDERS T, CANFIELD DR, COHEN MV, VATNER SF (1989) Superoxide dismutase reduces reperfusion arrhythmias but fails to salvage regional function or myocardium at risk in conscious dogs. Circulation 79: 143-153. OPIE LH (1989) Repetfusion injury and its pharmacological modification. Circulation 80: 1049-1062. POOLE-WILSON PA, HARDING DP, BOURDILLON PDV, TONES MA ( 1984) Calcium out of control. J Mel Cell Cardiol 16: 175-187. POOLE-WILSON PA, TONES MA (1988) Sodium exchange during hypoxia and on reoxygenation in the isolated rabbit heart. J Mel Cell Cardiol Xl (Suppl II): 15-22. PRZYKLENK K, KLONER RA (1989) “Reperfttsion injury” by oxygen-derived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow. Circ Res 61: 86-96. REDDY BR, KLONER RA, PRZYKLENK K (1989) Early treatment with desferoxamine limits myocardial ischemic/reperfusion injury. Free Radical Biol ,Med 7: 45-52. REIMER KA, MURRY CE, RICHARD VJ (1989) The role of neutrophils and free radicals in the ischemic-reperfused heart: why the confusion and controversy? J Mol Cell Cardiol 21: 1225-1239. RENLUND DG, GERSTENBLITH G, LAKATTA EG, JACOBUS WE, KALLMAN CH, WEISFELDT ML (1984) Pet&sate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart. J Mel Cell Cardiol 16: 795-80 1. SHEN AC, JENNINGS RB (1972) K’ me t its ofcalcium accumulation in acute myocardial injury. Am J Path01 67: 441452. SHLAFER M, KANE PF, KIRSH MM (1982a) Superoxide dismutase plus catalase enhances the efficacy of hypothermic cardioplegia to protect the globally ischemic, reperfused heart. J Thorac Cardiovasc Surg 83: 830-839. SHLAFER M, KANE PF, WIGGINS VY, KIRSH MM (1982b) Possible role for cytotoxic oxygen metabolites in the pathogenesis of ischemic injury. Circulation 66 (Suppl I): 85-92. STEWART JR, BLACKWELL WH, CRUTE SL, LOUGHLIN V, GREENFIELD LJ, HESS ML (1983) Inhibition of surgically induced ischemia/reperfusion injury by oxygen free radical scavengers. J Thorac Cardiovasc Surg 86: 2622272. TANAKA K, HEARSE DJ (1988) Reperfusion-induced arrhythmias on the isolated rabbit heart: characterization of the influence of the duration of regional ischemia and the extracellular potassium concentration. J Mol Cell Cardiol 20: 201-211. TANI M, NEELY J (1989) Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na+ and Nat-Ca2+ exchange. Circ Res 65: 104551056. TZIVONI D, KEREN A, GRANOT H, GO~TLIEB S, BENHORIN J. STERN S (1983) Ventricular fibrillation caused by myocardial reperfusion in Prinzmetal’s angina. Am Heart J 105: 3233325. WILLIS ED (1969) Lipid peroxide formation of microsomes: the role of non-haem iron. Biochem J 113: 325-332.