Electrophysiological and biochemical changes in ... - Clinical Science

Clinical Science (2001) 101, 367–376 (Printed in Great Britain)

Electrophysiological and biochemical changes in rabbit hearts stored at 4 mC for 6 or 24 h Oluwole S. FAGBEMI, Kieran BRACK, Surjit GOLAR, David CRISP and Apollo ECONOMIDES School of Natural and Environmental Sciences, Coventry University, Coventry CV1 5FB, U.K.

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This study examines the electrophysiological and metabolic changes that occur in rabbit hearts during hypothermic storage in vitro. Hearts were microperfused at 4 mC for 6 or 24 h with either normal Krebs–Henseleit buffer (KHB) or KHB containing 2,3-butanedione monoxime (BDM). After hypothermic storage, hearts were rewarmed to 37 mC with KHB. Cardiac function was then assessed in Langendorff perfusion mode. Electrophysiological changes were also assessed from the ventricular paced-evoked responses. After storage, mitochondria were isolated from the hearts and their respiratory control ratio, rate of ATP synthesis and outer membrane intactness were assessed. Compared with values from fresh non-stored hearts, hearts stored hypothermically for 24 h showed significant decreases in both left ventricular developed pressure and coronary flow when reperfused in Langendorff mode. On the other hand, the decrease in left ventricular developed pressure in hearts that were stored for only 6 h (with or without BDM) was not significant. Compared with values obtained from fresh non-stored hearts, hypothermic storage significantly decreased the R-wave amplitude, and both the R–E and ST–E intervals of paced-evoked responses. This was true for hearts microperfused for 6 h (with or without BDM) and for hearts microperfused with buffer containing BDM for 24 h. The ST–R intervals in hearts microperfused hypothermically for 6 h were prolonged, but this change was not statistically significant compared with those obtained from unstored hearts. In hearts microperfused with KHB containing BDM for 24 h, the ST–R interval was significantly prolonged. Hypothermic microperfusion for 24 h significantly decreased both the mitochondrial coupling ratio and the rate of ATP synthesis. In hearts microperfused with BDM for 6 h, mitochondrial coupling ratios and the rate of ATP synthesis were not significantly different from those in fresh hearts. In conclusion, the present study has shown that long-term hypothermic storage significantly impaired both paced-evoked responses and mitochondrial function. Inclusion of BDM in the perfusion buffer during storage significantly ameliorated some of these changes.

INTRODUCTION Hypothermic cardioplegia is widely used as a means of myocardial preservation. However, cold ischaemic storage and reperfusion of donor hearts often results in significant contractile dysfunction [1,2]. Previous studies

have shown that the inclusion of 2,3-butanedione monoxime (BDM) in storage solutions greatly improves myocardial function on resumption of normothermic perfusion [3,4]. BDM is a nucleophilic oxime that has been shown to be an effective, quick-acting and reversible inhibitor of cardiac contractility. It protects against

Key words: 2,3-butanedione monoxime, hypothermic preservation, paced-evoked response, rabbit heart. Abbreviations: BDM, 2,3-butanedione monoxime ; CF, coronary flow ; KHB, Krebs–Henseleit buffer ; LVDP, left ventricular developed pressure ; LVEDP, left ventricular end-diastolic pressure ; PER, paced-evoked response ; RCR, respiratory control ratio. Correspondence: Dr Oluwole S. Fagbemi, at present address Department of Biological Sciences, University of Lincolnshire and Humberside, Brayford Pool, Lincoln LN6 7TS, U.K. (e-mail sfagbemi!lincoln.ac.uk).

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hypoxia- and calcium paradox-induced myocardial damage [5,6] by inhibiting actin–myosin cross-bridge formation. It has been shown that BDM protects the ischaemic myocardium from calcium-stimulated ATP loss and from the damaging effects of ischaemic contracture. In a previous study, we showed that rat hearts stored for 16 h, even in a buffer containing BDM and staurosporine as protectants, attained only approx. 60 % of normal systolic contractile function and rate after normothermic reperfusion [3]. Among the potential mechanisms behind this residual depression of both cardiac contractility and rate are various abnormalities of calcium homoeostasis mediated by the sarcoplasmic reticulum [7], depletion of stores of ATP [8], functional interruption of cardiac sympathetic nerves [9], inhibition of the phosphocreatine shuttle [10], and perhaps regional electrophysiological abnormalities [11]. Previous studies have shown that electrical pacing (to the pre-ischaemic heart rate) during rewarming after a period of hypothermic ischaemia improves the functional recovery of stored hearts. This improvement has been variously attributed to an increase in sodium and calcium influx [12,13] and to an enhancement of oxidative phosphorylation [14]. However, relatively few previous studies have investigated the electrophysiological changes induced by hypothermic storage. In the present study, we have investigated the effects of hypothermic storage on the cardiac action potential using ventricular paced-evoked responses (PERs). The PER has been found to be a useful tool for assessing the effects of physiological or pharmacological interventions on cardiac electrophysiology [15]. We have now also investigated the effects of hypothermic storage on the functional capability of mitochondria isolated from these stored hearts, and whether BDM has a protective effect on the mitochondria.

METHODS Isolated heart preparation New Zealand White rabbits (1.2–1.8 kg) were rendered fully unconscious by cervical dislocation. The heart was removed rapidly through a medial sternotomy and placed in ice-cold Krebs–Henseleit buffer (KHB) to induce cardiac arrest. Within 1 min of excision, the aorta was dissected free of extraneous tissue, cannulated and then connected rapidly to a Langendorff perfusion apparatus. Hearts were perfused thereafter in Langendorff mode with KHB containing (in mM) : NaCl 118.5, KCl 4.75, MgSO 1.2, CaCl 2.0, NaHCO 25, KH PO 1.18, % # $ # % glucose 11.1 and sodium aspartate 2.0, as well as 5 units\l insulin. The solution was maintained at 37 mC and gassed with 95 % O \5 % CO to give a pH of 7.4. Hearts were # # perfused retrogradely with KHB through the aorta at a constant perfusion pressure of 100 mmHg. After 5 min # 2001 The Biochemical Society and the Medical Research Society

a latex balloon connected to a polyethylene catheter was inserted through the left atrium into the left ventricle for measurement of left ventricular developed pressure (LVDP). The balloon was expanded with distilled water to give a left ventricular end-diastolic pressure (LVEDP) of 5 mmHg on a Bell and Howell pressure transducer (type 4327L). A unipolar wedgeless tip platinum electrode (Telectronics) was placed into the right ventricle for pacing the heart and measuring its electrical activity. All hearts were paced during normothermic perfusion, by delivering 0.6 ms pulses of 5 V at 4.1 Hz. The electrical signal recorded from the electrode is known as the PER. The usefulness and sensitivity of this technique have been discussed in an earlier paper [15]. The PER was displayed continuously on a digital storage oscilloscope and also fed through an analogue-to-digital converter into a Picolog computer program on a 286 Viglan computer. LVDP and its differential (at a frequency response of 115 Hz) were recorded using a Graphtec Thermal Arraycorder (WK7700). Coronary flow (CF) was measured by timed collections of coronary effluent. All hearts were allowed a stabilization period of 15 min before acquisition of data began, which continued thereafter for a further 60 min. Hearts with a CF greater than 20 ml\min or a heart rate of less than 180 beats\min at the end of the initial 5 min of Langendorff perfusion were rejected from the study.

Hypothermic preservation Hearts that were to be stored were first perfused in Langendorff mode with the above solution for 5 min. Then the aortic cannula was connected to a peristaltic pump and the organ was microperfused slowly at 0.3 ml\min for 6 or 24 h at 4 mC, as described by Wincomb and Collins [16]. The term ‘ microperfusion ’ is used to describe the very low rate of perfusion used during hypothermic preservation, and should be distinguished from the conventional continuous normothermic constant-pressure perfusion. The composition of the storage solution was as follows (in mM) : NaCl 110, KCl 6.48, MgSO 6, CaCl 0.05, NaHCO 25, KH PO % # $ # % 1.18, glucose 11.1 and sodium pyruvate 2.0, as well as 1 % poly(ethylene glycol) 2000 and 5 units\l insulin. The pH was adjusted to 7.4 and the osmolarity of the medium was 320 mOsm\l. To this solution was added 5 mM BDM for some groups of hearts.

Rewarming At the end of the 6 or 24 h period of hypothermic storage, each heart was rewarmed to 37 mC using KHB, and was reperfused in Langendorff mode for 60 min. A latex balloon for measuring pressure was inserted into the left ventricle via the left atrium after 10 min of rewarming, and the LVEDP was readjusted to 5 mmHg. Aortic pressures were raised progressively from 30 to

Electrophysiological changes in rabbit heart

100 mmHg over a 15 min period. Data were collected thereafter for 60 min. A total of six groups were studied for changes in contractile and electrical functions. Group I (n l 10) consisted of fresh unstored hearts perfused normothermically with KHB for 60 min. This group served as the main control or reference group. Group II hearts were unstored hearts treated with BDM and perfused for 60 min. Hearts in groups III and IV (n l 6 in each group) were stored hypothermically for 6 and 24 h respectively and were then reperfused with KHB. Hearts in groups V and VI were stored in a buffer containing BDM for 6 and 24 h respectively, but were both reperfused with KHB.

Mitochondrial isolation and measurement of function Seven experimental groups were used for this series of experiments. Mitochondria were isolated immediately from freshly harvested (reference) hearts (group I), from hearts perfused for 60 min without hypothermic storage with and without BDM (groups II and VII respectively) and from hearts stored for 6 or 24 h with (groups V and VI respectively) or without (groups III and IV respectively) BDM. We used a slightly modified technique compared with that described previously by Rickwood et al. [17]. Ventricular tissue was trimmed, weighed and finely chopped. It was then suspended in an equal volume of enzymic digestion medium consisting of 300 mM sucrose, 1 mM EGTA, 1 mM CaCl , 5 mM Hepes, 5 mM # KH PO and 0.1 % BSA, adjusted to pH 7.4 with KOH. # % Finally, 3.5 mg\50 ml collagenase (Type IV ; Sigma) was added. After 40 min of incubation at 0–4 mC, more EGTA was added (to a final concentration of 2 mM) to terminate collagenase activity. The digestion medium was then drained from the tissue. The tissue was disaggregated using a glass rod in 3 vol. of ice-cold homogenization medium consisting of 300 mM sucrose, 1 mM EGTA, 5 mM Hepes, 5 mM KH PO and 0.1 % BSA, adjusted to # % pH 7.4 with KOH. The tissue was then homogenized in a further 6 vol. of the same medium with five upand-down strokes using a loose-fitting (0.25–0.33 mm clearance) motor-driven glass\Teflon homogenizer. The homogenate was centrifuged at 1800 g for 5 min at 4 mC. The carefully decanted supernatant was then recentrifuged at 18 000 g for 4 min at 4 mC. The resultant pellet was gently resuspended by hand homogenization and centrifuged once again at 18 000 g for 4 min at 4 mC. The final mitochondrial pellet was resuspended in a small volume of ice-cold 300 mM sucrose to a protein concentration of 10 mg\ml. A Clark-type oxygen electrode incubation chamber (Rank Brothers, Cambridge, U.K.) was used to measure mitochondrial State 3 (after ADP addition) and State 4h (after ADP depletion) respiratory rates at 25 mC. Respiratory control ratios (RCRs) were calculated as the

ratio of State 3 to State 4h respiration rates. ADP\O ratios and rates of oxidative phosphorylation (State 3 respiration rateiP\O ratio) were estimated and used to calculate the rate of ATP synthesis, as described by Andres et al. [18]. A 100 µl aliquot of the mitochondrial suspension was added to 2.85 ml of incubation medium consisting of 8 mM glutamic acid, 8 mM malic acid, 225 mM sucrose, 5 mM KH PO , 1 mM EGTA and # % 20 mM Tris\HCl at pH 7.4. After 3 min, 0.05 ml of 12 mM ADP solution was added to briefly stimulate State 3 respiration, followed by a return to State 4 respiration. In some experiments, 8 µM cytochrome c was added to the incubation chamber at the end of State 3 respiration, to test for the intactness of the mitochondrial outer membrane.

Statistics Data are presented as meanspS.E.M. All comparisons of those parameters measured over time were performed using a two-way repeated-measures ANOVA in order to test for group differences. If significance was detected (P 0.05 for the associated F value), further comparisons among groups at each individual time point were performed to determine at which time points differences existed. If more than two groups were compared with a single control group, analysis was performed using a Dunnett’s t test for the appropriate number of comparisons. In comparisons involving only two groups, an unpaired Student’s t test was used. Comparisons of parameters not determined over time were performed using a factorial ANOVA, followed by a Dunnett’s t test if more than two groups were compared. Statistical analyses were carried out on a Macintosh computer using Stat-View SEj Graphics (Abacus Concepts, Berkeley, CA, U.S.A.) and Multistat software (Biosoft, Cambridge, U.K.). A P value of 0.05 was considered to be statistically significant.

Chemicals All chemicals used were purchased from Sigma (Poole, Dorset, U.K.).

RESULTS Of all hearts harvested, 2 % failed to meet the criteria for inclusion in the study.

Left ventricular pressure Figure 1(a) shows the LVDP values of freshly isolated hearts (reference or control group), and of those hearts stored at 4 mC for 6 or 24 h before being reperfused at 37 mC for 60 min in Langendorff mode. In freshly isolated hearts, LVDP reached a value of 137.2p 3.1 mmHg within 20 min of the start of normothermic perfusion. In unstored hearts, BDM (5 mM) reduced LVDP by approx. 18 % when added to the perfusion # 2001 The Biochemical Society and the Medical Research Society

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Figure 1 (a) LVDP and (b) CF in reference (unstored) hearts and in hearts preserved for 6 or 24 h in either untreated buffer or buffer containing BDM and then reperfused in the Langendorff mode for 60 min

Group I, reference hearts ; group III, hearts stored in untreated buffer alone for 6 h ; group IV, hearts stored in untreated buffer alone for 24 h ; group V, hearts stored for 6 h with buffer containing BDM ; group VI, hearts stored for 24 h with buffer containing BDM. *Significantly different from reference hearts at all time points (P 0.05). Error bars are visible when they are larger than the point symbols. buffer during normothermic perfusion. The LVDP values of hearts that were stored in untreated buffer for either 6 h or 24 h and then rewarmed (groups III and IV) were significantly lower than those of non-stored reference hearts at all time points studied. Hearts preserved for 24 h in a buffer containing BDM had significantly higher LVDP values during normothermic reperfusion than those hearts stored for 24 h in an untreated buffer at all corresponding time points. Hearts microperfused hypothermically with untreated buffer for 6 h and those hearts microperfused with a buffer containing BDM (5 mM) for 6 h and then reperfused normothermically with untreated buffer (groups III and V) had significantly higher LVDP values than hearts stored in untreated buffer for 24 h. LVDP values in these latter two groups of hearts were slightly (but not significantly) lower than those of reference hearts. LVEDP in reference hearts was 5.2p0.8 mmHg after the equilibration period. During the 60 min of normothermic perfusion, this rose to 8.6p1.4 mmHg. LVEDP values in hearts microperfused for 6 or 24 h in all of the solutions tested were between 6.2p2.6 and 20.7p2.1 mmHg. No significant difference was found in LVEDP values between group I (reference) hearts and those hearts microperfused hypothermically for 6 h (groups III and V) or those hearts perfused for 24 h with a buffer containing BDM (group VI). However, as normothermic perfusion progressed, the LVEDP values # 2001 The Biochemical Society and the Medical Research Society

of even those hearts stored with BDM for 24 h increased significantly, to a value of 12.3p2.6 mmHg at approx. 20 min after the start of normothermic reperfusion. LVEDP was highest in those hearts that had been microperfused in normal buffer for 24 h. This high LVEDP was also reflected in the very low LVDP values that were attained by this group of hearts.

CF values Figure 1(b) shows the CF values in reference hearts (18.3p1.8 ml\min) and in those hearts microperfused hypothermically for 6 or 24 h at 4 mC before being reperfused at normothermia in Langendorff mode for 60 min. In the reference hearts, BDM (5 mM) had no significant effect on CF. Hearts microperfused for 6 or 24 h and then reperfused normothermically with untreated buffer had CF values reduced by between 18 % and 83 % compared with the reference hearts. The decrease in CF was most pronounced in hearts that were microperfused for 24 h. CF values in hearts microperfused hypothermically for 6 h with a buffer containing BDM were lower than those of the reference hearts after 30 min of normothermic perfusion, but this difference was not significant.

Changes in electrical activity of the heart Changes in electrical activity of both the reference hearts and hearts microperfused hypothermically for either 6 or


from, those of the reference hearts. However, in hearts microperfused with BDM for 24 h the ST–R intervals were prolonged significantly when compared with values obtained from reference hearts at all time points investigated. In hearts microperfused with untreated buffer for 24 h, the PER values were too small to be measured accurately.

Effects of storage on mitochondrial function The functional competence of isolated mitochondria was evaluated by determining the RCR, the rate of ATP synthesis and the intactness of the outer membrane. Table 1 summarizes the effects of hypothermic storage on the RCR and the rate of ATP synthesis in reference hearts and in hearts microperfused hypothermically for 6 or 24 h with or without BDM in the perfusion buffer. Figure 2 Typical ventricular PER recorded from an isolated perfused rabbit heart stimulated at 4.1 Hz

The PER shown is an average of 10 paced signals recorded by the computer acquisition system. The point of stimulation (St) is followed by the stimulus artifact. R represents the peak of the R wave, and E represent the end of the response. 24 h and then reperfused in Langendorff mode for 60 min were monitored using the PER technique. The PER obtained from reference hearts (Figure 2) at the end of the stabilization period displayed a negative R wave (mean amplitude 5.53p1.15 mV). The ST–R, R–E and ST–E intervals had mean durations of 24.7p2.3, 143.7p3.3 and 169.5p4.2 ms respectively. These values decreased only slightly during 60 min of normothermic perfusion. Inclusion of BDM (5 mM) in the perfusion buffer significantly increased the R-wave amplitudes of the unstored hearts (Figure 3d). BDM also shortened the ST–R interval values slightly, while having little or no effect on either the R–E or ST–E intervals of this group of hearts (Figures 3a–3d). Figures 3(a)–3(d) also summarize the changes in the R-wave amplitude, and in the ST–R, R–E and ST–E intervals, of hearts that were microperfused hypothermically either with untreated buffer or with a buffer containing BDM for 6 or 24 h. The comparison group in each case was the group of unstored hearts with no BDM. The R-wave amplitude, and the R–E and ST–E intervals, were all decreased significantly in hearts microperfused with untreated buffer for 6 h, and in hearts microperfused with a buffer containing BDM for 24 h. The R-wave amplitude, and the ST–E and R–E intervals, obtained from hearts microperfused with a buffer containing BDM for 6 h were lower than values obtained from reference hearts at corresponding time points, but these changes were not significant. The ST–R intervals in hearts microperfused for 6 h either with untreated buffer or with a buffer containing BDM were longer than, but not significantly different

RCR The RCR was used as an indicator of coupling between oxidative phosphorylation and the mitochondrial electron transport chain. Mitochondria obtained from fresh hearts (reference group) had an RCR of 4.36p0.14. Normothermic perfusion for 60 min had no significant effect on the RCR value (4.28p0.22). Similarly, perfusion of freshly isolated hearts with a buffer containing BDM (5 mM) had no significant effect on the measured RCR value. RCR values of hearts microperfused at 4 mC were all significantly decreased, except in those hearts microperfused for 6 h with a buffer containing BDM (5 mM). RCR values in this group of hearts were slightly (but not significantly) lower than those of reference hearts. The decrease in RCR was most pronounced in those hearts that were microperfused for 24 h with untreated buffer. Mitochondria obtained from this latter group of hearts were highly uncoupled. BDM significantly protected the RCR during 6 h of storage of the heart at 4 mC. The slight protection during 24 h of storage was not significant.

Rate of ATP synthesis The rate of ATP synthesis by mitochondria from freshly harvested hearts was 373p18 nmol of ATP:min−":mg−". This rate was unaffected by 60 min of perfusion with untreated buffer (354p16 nmol:min−":mg−") or with a buffer containing BDM. In hearts microperfused with buffer at 4 mC for 24 h, the rate of ATP synthesis was significantly decreased to approx. 50 % of the control rate. The rates of ATP synthesis in groups IV and VI were significantly differently from the rate in mitochondria obtained from hearts microperfused for only 6 h (Table 1). In hearts microperfused for 6 h with either untreated buffer or a buffer containing BDM (groups III and V respectively), the rates of ATP synthesis were lower than in controls, but these differences were not significant. The rate of ATP synthesis in mitochondria from hearts microperfused with buffer containing BDM # 2001 The Biochemical Society and the Medical Research Society

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Figure 3 (a) ST–R interval, (b) R–E interval, (c) ST–E interval and (d) R-wave amplitude in reference (unstored) hearts and in hearts preserved for 6 or 24 h in either untreated buffer or buffer containing BDM and then reperfused in Langendorff mode for 60 min

, Reference hearts ; , control unstored hearts treated with BDM ; >, hearts stored for 24 h with buffer containing BDM ; #, hearts stored for 6 h with buffer containing BDM ; =, hearts stored for 6 h in untreated buffer. Significance of differences : *P 0.05 compared with reference hearts ; §P 0.05 compared with hearts stored for 6 h in untreated buffer. Standard error bars are visible where they are larger than the point symbols.

for 6 h was higher than that from hearts microperfused with untreated buffer for 6 h, but again these differences were not significant.

Test for membrane intactness ATP synthesis can occur only when the mitochondrial outer membrane is intact. In mitochondria with an intact outer membrane, adequate endogenous cytochrome # 2001 The Biochemical Society and the Medical Research Society

c stays in the intermembrane space, and addition of exogenous cytochrome c will have no effect on State 4h respiration. However, if the outer membrane has been damaged, endogenous cytochrome c is lost from the intermembrane space, and addition of exogenous cytochrome c will then stimulate State 4h respiration. Addition of cytochrome c had no observable effect on the State 4h respiration of mitochondria that were obtained from


Table 1

Effects of hypothermic storage on the RCR and the rate of ATP synthesis

Values are expressed as meanspS.E.M. Significance of differences : *P 0.05 compared with mitochondria from unstored reference hearts ; †P 0.05 compared with mitochondria from hearts microperfused for 6 h with or without BDM ; ‡P 0.05 compared with mitochondria from hearts microperfused with buffer alone for 6 h. Group

RCR

ATP synthesis (nmol:min−1:mg−1 mitochondrial protein)

I : unstored reference hearts II : unstored heartsjBDM perfused for 60 min III : hearts microperfused with buffer alone for 6 h IV : hearts microperfused with buffer alone for 24 h V : hearts microperfused with bufferjBDM for 6 h VI : hearts stored in bufferjBDM for 24 h VII : unstored hearts perfused for 60 min

4.36p0.14‡ 4.31p0.36‡ 2.53p0.07 1.42p0.26*† 3.81p0.17‡ 1.70p0.21*† 4.28p0.36‡

373p18 362p12 284p13 154p9* 298p6 172p9* 354p16

control hearts or from hearts microperfused for 6 h with or without BDM. On the other hand, addition of exogenous cytochrome c to mitochondria that had been obtained from hearts microperfused for 24 h (with or without BDM) resulted in a stimulated State 4h respiration rate, suggesting that the mitochondrial membranes had become damaged by prolonged hypothermic perfusion.

DISCUSSION The most widely used method of preserving the integrity of a potential donor heart during transportation is to arrest it with a cold cardioplegic solution, then store it in a plastic bag containing iced electrolyte solution [19,20]. This allows the heart to be stored for approx. 4–6 h, within which time transplantation must take place. Hearts stored beyond this time limit usually suffer from irreversible damage during subsequent reperfusion. Although various new preservation solutions and associated techniques have been reported to improve viability [4,21], the stored heart is yet to attain the longevity that is already shown by several other organs. The most obvious effect of prolonged hypothermic storage on the heart is a depression of mechanical function. Decreases in both contractility and rate during normothermic reperfusion of the stored heart were seen in the present study and have been reported previously by us and others [16,22,23], and are usually attributed to changes in both the intracellular and extracellular ionic milieu. Increases in intracellular sodium and calcium concentrations may be involved, in addition to an increase in extracellular potassium as well as shrinkage of the extracellular space and associated cell swelling. An increase in the extracellular potassium concentration will depolarize the resting membrane potential. This decreases the maximum rate of rise of the action potential upstroke (Vmax), while lowering action potential amplitude and the plateau potential, thereby shortening the duration of the

action potential [24–27]. Other factors, such as a decrease in the intracellular and extracellular pH and the intracellular accumulation of sodium, calcium and fatty acid metabolites, may all contribute significantly to the observed changes in electrical conduction and contractility seen in ischaemic hearts [28–31]. During normothermic reperfusion, the generation of oxygen free radicals may aggravate these changes further [32]. In this present study, the ST–R interval, which represents the latency of activation, was prolonged by storage. This was especially so in hearts that had been stored for more than 6 h before being reperfused. The R–E and ST–E intervals (representing the duration of repolarization and overall action potential respectively) were significantly decreased in stored hearts. The electrophysiological changes that we observed are analogous to the altered action potentials that have been recorded from hypoxic and ischaemic myocardial fibres in previous studies [33,34]. In particular, previous studies have shown that ischaemia causes a large depolarization of the cell membrane, which is attributable to an increase in extracelluar potassium [26,28], and which in turn inactivates the voltage-dependent INa (Na current) [25]. Recovery from this inactivation is normally very fast (1–10 ms), but, after long depolarizations (as may occur during hypothermic storage), recovery may take seconds [35–37]. Another effect of slowed recovery from inactivation of the ion channels is a prolongation of the effective refractory period to beyond the end of the action potential [35]. These changes will have the net effect of reducing excitability, and may also account for the decreased heart rate of hypothermically stored hearts. In the present study, BDM greatly increased R-wave amplitudes, while having little or no effect on the ST–R, R–E or ST–E intervals, of normothermically perfused unstored hearts (Figures 3a–3d). Speeding up of the recovery from inactivation of sodium channels may be involved in these effects. Previous studies have shown that BDM can reversibly shorten and depress the plateau phase of cardiac action # 2001 The Biochemical Society and the Medical Research Society

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potentials in embryonic chick hearts [38], in cat ventricular myocardium [39] and in dog Purkinje fibres [40]. These effects of BDM have been attributed to an ability to decrease calcium entry into the cell via less than normal opening of the slow calcium channels, possibly due to dephosphorylation of the channels through a chemical phosphatase activity [41]. BDM also inhibits the delayed rectifier current [42], it can block ATP-sensitive potassium channels [43] and it can enhance the normal inactivation of the cardiac L-type calcium channels during an action potential [44,45]. Nevertheless, in hearts stored with BDM (5 mM) in the present study, the observed electrophysiological changes that were not due to storage alone were not pronounced. Although BDM exerted a mild negative inotropic effect on unstored hearts in this as in previous studies [22,23], hearts stored in a buffer containing BDM for 6 or 24 h had significantly higher LVDPs when compared with hearts stored in buffer alone for 24 h. A similar protective effect of BDM has been reported previously in rat [3], guinea-pig [23] and rabbit [4] hearts. This protective effect of BDM is likely to be multifactorial in origin. BDM has been shown to inhibit reperfusioninduced contraction band formation, as well as mitochondrial swelling and sarcolemmal damage in myocardial cells [46,47]. It also decreases myofibrillar Ca#+ sensitivity [48–50]. The observation that hearts microperfused with BDM did not show a significant elevation of LVEDP on reperfusion may indicate protection against cytoplasmic Ca#+ overload in these hearts. Although BDM may improve cardiac function in this way, other effects of BDM may be involved, and deserve further study. During cardioplegic storage, mitochondrial disruption and profound depletion of intracellular high-energy phosphates are among the well-documented and fundamental changes that may contribute to the depressed left ventricular functional recovery seen during reperfusion. ATP depletion leads to disruption of cellular ionic homoeostasis, with consequent and possibly irreversible cell injury. Preservation of levels of high-energy phosphates before, during and after cardioplegic arrest, therefore, has become a major goal for those involved in the long-term preservation of hearts. In a previous study, we reported that the ATP content of hearts stored with BDM was well preserved during reperfusion [22]. We showed in the present study that the rate of ATP synthesis in hearts stored in a buffer containing BDM for 6 h was not significantly different from values obtained in unstored hearts, and was slightly higher than those in hearts stored in untreated buffer. Mitochondria from this BDM-treated group of hearts showed significantly higher RCR values (and were therefore more coupled) than those obtained from hearts stored in untreated buffer. The observation that exogenous cytochrome c stimulated State 4h respiration in mitochondria obtained from hearts # 2001 The Biochemical Society and the Medical Research Society

that were stored for 24 h with or without BDM in the buffer indicates that the outer membrane of some of the mitochondria had been damaged, and would therefore be less capable of synthesizing ATP. One of the major limitations to the extended hypothermic preservation of donor hearts for transplantation is the structural damage caused by progressive myocardial contracture resulting from falling levels of ATP. Although storage solutions such as University of Wisconsin solution and St. Thomas ’ solution have proved useful for storing organs such as kidney, liver and pancreas, they are less effective for prolonged heart preservation. In our present study, as in studies using these solutions, the level of ATP was decreased in all stored hearts, indicating that, during storage, the rate of ATP utilization far exceeds the rate of its generation. However, in the present study, the decrease in ATP levels was more pronounced in hearts stored in buffer without BDM. Since the functional recovery of any stored organ depends on the cellular integrity of its energy production apparatus, the observation that BDM protects the mitochondria from hypothermic ischaemic damage suggests that BDM may be a useful addition to cardioplegic solution. In conclusion, the results of the present study suggest that BDM improves cardiac function in hypothermic stored hearts by opposing electrical derangement and the loss of mitochondrial function.

ACKNOWLEDGMENTS We thank Professor B. J. Northover for his constructive suggestions while preparing the manuscript.

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