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J Thorac Cardiovasc Surg 2000;119:842-848
© 2000 The American Association for Thoracic Surgery

CARDIOPULMONARY SUPPORT AND PHYSIOLOGY

ADENOSINE TRIPHOSPHATE–DEPENDENT POTASSIUM CHANNEL MODULATION AND CARDIOPLEGIA-INDUCED PROTECTION OF HUMAN ATRIAL MUSCLE IN AN IN VITRO MODEL OF MYOCARDIAL STUNNING

From the Laboratory of Cardiovascular Pharmacology, Department of Cardiac Surgery and Second Section of Cardiology, University of Rome "La Sapienza," Rome, Italy.

Supported in part by Ministero dell’Università e della Ricerca Scientifica, Rome (Ricerche di Ateneo 145/1997) and by Cardioricerca, Rome, Italy. Dr Wagner from E. Merck, Darmstadt, Germany, provided a gift of bimakalim used in this study.

Address for reprints: Paolo Emilio Puddu, MD, FESC, FACC, Istituto di Chirurgia del Cuore e Grossi Vasi, II Cattedra di Cardiologia, Università degli Studi di Roma "La Sapienza," Viale del Policlinico, 155, Rome 00161, Italy (E-mail: puddu.pe{at}iol.it ).

	Abstract

Top Abstract Introduction Patients and methods Results Discussion References

 
Objectives: Although adenosine triphosphate–dependent potassium channel openers have been shown to enhance cardioplegic protection in animal myocardium, there is a lack of data on human cardiac tissues. We aimed at determining, on human atrial muscle, whether adenosine triphosphate– dependent potassium channels are involved in protection caused by high-potassium cardioplegia and whether adenosine triphosphate–dependent potassium channel activation might improve cardioplegic protection in an in vitro model of myocardial stunning.
Methods: Human atrial trabeculae were obtained from adult patients undergoing cardiac operations. In an organ bath at 37°C, the preparations were subjected to 60 minutes of hypoxia at a high stimulation rate either in Tyrode solution (control, n = 17) or in St Thomas’ Hospital solution without additives (n = 6) or associated with 100 nmol/L bimakalim (n = 7) or 1 µmol/L glibenclamide (n = 7), followed by 60 minutes of reoxygenation and 15 minutes of positive inotropic stimulation with 1 µmol/L dobutamine.
Results: Atrial developed tension was reduced by hypoxia to 27% ± 5% of baseline and incompletely recovered after reoxygenation to 38% ± 7%, whereas dobutamine restored contractility to 74% ± 7% of basal values. St Thomas’ Hospital solution with or without bimakalim improved developed tension after reoxygenation and dobutamine (P < .0001 vs control), whereas glibenclamide inhibited these protective effects of cardioplegic arrest (P = .001 vs St Thomas’ Hospital solution). After reoxygenation, the protective effect of bimakalim disappeared at a high pacing rate (400- and 300-ms cycle length) but recovered during dobutamine superfusion.
Conclusions: Adenosine triphosphate–dependent potassium channels are likely involved in the cardioprotective effects of cardioplegia in human atrial trabeculae and adenosine triphosphate–dependent potassium channel activation with bimakalim used as an additive to cardioplegia enhanced protection.

	Introduction

Top Abstract Introduction Patients and methods Results Discussion References

 
Cardioplegic arrest provides essential cardioprotection during cardiac operations, and advantages of continuous warm blood cardioplegia have been advanced for minimizing ischemia and avoiding risks related to hypothermia. However, postoperative ventricular dysfunction and injury caused by prolonged cardiac arrest led to the development of additional cardioprotective strategies. ^1,2 Adenosine triphosphate–dependent potassium (K_ATP) channel openers have been shown to limit contractile dysfunction, ^3-5 infarct size, ⁶ and cellular energy depletion ^7,8 caused by ischemia-reperfusion. Moreover, experimental studies carried out on the rabbit, ⁹ pig, ¹⁰ guinea pig, ^11,12 and rat ¹³ have demonstrated benefits of K_ATP channel activators to improve myocardial preservation provided by both cold and warm cardioplegia.

The susceptibility to ischemic injury and responsiveness to myocardial protection, however, differ among mammals, ¹⁴ and the efficacy of a K_ATP channel opener on cardiac muscle may differ between human and other species, as we demonstrated in a previous work. ¹⁵ Considering that the goal of experimental studies on cardioplegia is use in the human myocardium and determination of the potential benefits that patients might find in strategies aimed at reducing postoperative myocardial stunning, confirmation on human cardiac muscle of protective action of K_ATP channel openers as additives to cardioplegic solution is of interest.

We aimed at determining whether K_ATP channels are implicated in the cardioprotective effects of cardioplegic arrest with St Thomas’ Hospital solution (STHS) superfusion on human atrial trabeculae and whether K_ATP channel activation may enhance the benefits of cardioplegia against myocardial stunning induced by hypoxia-reoxygenation. For this, we used the selective K_ATP channel blocker glibenclamide and the K_ATP channel opener bimakalim as additives to the cardioplegic solution during the hypoxic phase. Normothermic instead of cold cardioplegia was selected to prevent complicating factors related to temperature shift in the analysis of contractility changes among experimental groups. ¹²

	Patients and methods

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Preparation
Informed consent was obtained from 41 adult patients (68% male and 32% female subjects) undergoing elective cardiac operations, whose ages ranged from 23 to 79 years (57 ± 11 years) and who had coronary artery disease (59%), rheumatic valve disease (34%), or congenital lesions (7%). Patients were routinely taking cardioactive drugs according to standard prescriptions. Ethical considerations prevented us from stopping these drugs more than 24 hours before the operations. Exclusion criteria were arrhythmia, right ventricular failure, and antiarrhythmic or oral hypoglycemic medication. Premedication, given 1 hour before induction of anesthesia, usually consisted of diazepam, antibiotic (cephalotine), and aprotinin. General anesthesia was then induced with diazepam, fentanyl, pancuronium, and sometimes nitrous oxide.

A sample of the right atrial appendage (approximately 1 cm², 500-1000 mg) was removed and immersed in preoxygenated modified Tyrode solution (NaCl, 120 mmol/L; KCl, 4 mmol/L; CaCl₂, 2.7 mmol/L; MgCl₂, 1.1 mmol/L; NaHCO₃, 25.7 mmol/L; NaH₂PO₄, 1.8 mmol/L [pH 7.4]; and glucose, 11 mmol/L) at room temperature. ^5,15-18 Free-running trabeculae were isolated and fixed to a precalibrated force transducer (TRN001; Kent Scientific Corporation) in an organ bath, where the preparation was superfused at 5 mL/min with Tyrode solution gassed with 95% oxygen and 5% carbon dioxide (PO ₂, 500 mm Hg measured at 755 mm Hg of barometric pressure) and gradually warmed up (circulating thermostat-regulated bath: Polystat 86602, Bioblock Scientific) to 37°C.

Stimulation and recordings
The muscle was stimulated at 1600 ms of basal cycle length (CL) by means of a bipolar Teflon-coated 99.99% silver wire electrode (AG-15T, 0.375 mm in diameter; PHYMEP sarl). Square pulses of 1-ms duration and 4-mA intensity were delivered by an orthorhythmic stimulator (Explorer 1000, VPA Medical). The trabecula was lengthened to the top of its length-tension curve. Isometric force development was monitored on a digital memory oscilloscope (Tektronix 2230, Tektronix Inc), and data were digitized at a sampling frequency of 8 KHz (Datapac 13.2, Biologic). The software automatically measured developed tension (DT) and time to peak tension. After the experiment, the contracting trabecular portion was dried and weighed on a precise balance (Sartorius BA 110S, Sartorius AG).

Experimental protocol (Fig 1)
In a time-related control group (TRC group, n = 17), we assessed the time-dependent contractility loss of trabecular strips stimulated at 1600-ms CL. Thereafter the Paradise test ¹⁹ was performed in 10 preparations included in the latter group, changing 95% oxygen and 5% carbon dioxide in Tyrode solution to 80% oxygen, 5% carbon dioxide, and 15% nitrogen for 15 minutes. Another 17 trabeculae were subjected to 60 minutes of hypoxia at 400-ms CL by superfusion with nonoxygenated Tyrode solution (PO ₂ 180 mm Hg) and then returned to normal for 60 minutes (reoxygenation phase), after which they were subjected to 1 µmol/L dobutamine for 15 minutes. The same protocol was achieved for another 20 preparations undergoing the hypoxic phase at a high pacing rate under normothermic cardioplegia by superfusion with nonoxygenated hyperkalemic STHS, ²⁰ either without additive (STHS, n = 6) or in the presence of 100 nmol/L bimakalim (E. Merck Laboratories; n = 7) or 1 µmol/L glibenclamide prediluted in dimethylsulfoxide 0.01% (Sigma Chemical Co; n = 7). At 0.01%, dimethylsulfoxide 0.0% had no significant effect on human atrial contractility. The composition of STHS was as follows: NaCl, 110 mmol/L; KCl, 16 mmol/L; CaCl₂, 1.2 mmol/L; MgCl₂, 16 mmol/L; and NaHCO₃, 10 mmol/L (pH 7.8). High stimulation rate (400-ms CL) was used during the hypoxic phase to raise the severity of hypoxia, leading to stunned human atrial muscle. ^5,16,17 Stimulation was continued during cardioplegia to maintain the experimental conditions similarly to the other groups (TRC and control groups). A previous study showed that the in vitro contractile protection afforded by STHS was independent of pacing during cardioplegic arrest. ¹² As previously discussed, ^5,17 in this model the stunning phenomenon was disclosed by 1 µmol/L dobutamine challenge, testing the reversibility of the hypoxia/reoxygenation–induced contractile dysfunction. After each experimental phase, the force-frequency (FF) relationship was studied by decreasing stimulation CL from 1600 ms to 1200, 1000, 800, 400, and 300 ms. ^12,17

View larger version (34K): [in this window] [in a new window]   Fig. 1. Experimental protocol for investigation of protective effects of normothermic (37°C) cardioplegia (STHS), either alone or associated with KATP channel modulators, on human atrial trabeculae in an in vitro model of myocardial stunning. FF relationships were studied after each different experimental phase. Hypoxia was performed at a high pacing rate (400-ms CL).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effects of hypoxia-reoxygenation and dobutamine challenge on human atrial contractility. DT variations are expressed as means ± SEM. P values refer to ANOVA for repeated measures performed for the 4 hypoxia-related groups: control, STHS, STHS plus bimakalim, and STHS plus glibenclamide. TRCs are included for visual comparative purposes. Note highly significant P values for group (G), time (T) and group · time (G*T) factors. Note marked positive inotropic action of dobutamine in preparations subjected to cardioplegic arrest, either alone or in the presence of 100 nmol/L bimakalim, whereas 1 µmol/L glibenclamide inhibited protective effects of cardioplegia after hypoxia.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Changes of velocity of tension developed by human atrial trabeculae after hypoxia-reoxygenation and dobutamine challenge. Values of log[velocity of DT] are expressed as means ± SEM. For comparison, 95% confidence intervals of basal values for all 54 preparations included in the randomized study are shown in gray. A, *P < .05 and **P < .01 versus control, Student t test with Bonferroni correction. Note that 100 nmol/L bimakalim added to cardioplegic solution significantly improved the velocity of DT after hypoxia-reoxygenation and dobutamine challenge. B, *P < .05 and **P < .005 versus base, Student t test for paired data. Note that velocity of DT was decreased after dobutamine challenge when 1 µmol/L glibenclamide was associated with cardioplegia, and conversely it was improved in the STHS plus bimakalim group (P < .05, Fisher exact test).

View larger version (18K): [in this window] [in a new window]   Fig. 4. Changes in FF relationships of human atrial trabeculae after reoxygenation (A) and dobutamine challenge (B) in control and STHS-related groups. Developed tension variation (%DT) of DT measured during basal FF at corresponding cycle length is expressed as means ± SEM. P values refer to ANOVA for repeated measures performed for the 4 groups. Note higher FF-related developed tension variation after cardioplegic treatment, either alone or in the presence of 100 nmol/L bimakalim, than in the control and STHS plus glibenclamide groups, which fits with improved human atrial contractility, as illustrated in Fig 2. Note that the protective effect of STHS plus bimakalim after reoxygenation disappeared at the high pacing rate (A) , whereas after dobutamine treatment (B) , there was no such interference.

	Results

Top Abstract Introduction Patients and methods Results Discussion References

DT of atrial preparations exposed to oxygenated Tyrode solution (TRC group) decreased to 91% ± 5% after 60 minutes and to 73% ± 7% after 135 minutes (Fig 2), corresponding to an attrition rate of 9% during the first hour and 15% per hour thereafter. After 15 minutes of the Paradise test, trabecular DT was reduced to 74% ± 4% of DT values measured before the test.

Effects of cardioplegia and K_ATP channel modulators on human trabecular contractility during hypoxia-reoxygenation and dobutamine challenge
After 60 minutes of hypoxia at a high pacing rate (Fig 2, control), DT was lessened to 27% ± 5% of basal values and recovered slightly only after reoxygenation to 38% ± 7% before reaching 74% ± 7% of basal DT after dobutamine, and this was similar to values measured in the TRC group. After cardioplegic arrest, during reoxygenation and dobutamine challenge, DT was significantly improved in the STHS and STHS plus bimakalim groups (both P < .0001 versus control), whereas the percentage of DT observed in the STHS plus glibenclamide group was similar to that found in the control group (P = .12), demonstrating that glibenclamide inhibited the protective effect of cardioplegic arrest (P = .001 vs STHS). Also, on velocity of DT, bimakalim lessened the decrease induced by hypoxia-reoxygenation (Fig 3, A ) and enhanced velocity of DT after dobutamine (Fig 3, A and B ), whereas velocity of DT was reduced in the control and STHS plus glibenclamide groups compared with basal values (Fig 3, B ).

FF relationships
Before hypoxia, human atrial trabeculae exhibited a slight positive FF relationship as CL decreased until 800 ms, and then depressed DT was seen at the high pacing rate. At low frequency, the FF relationship became negative after hypoxia (P = .004 vs baseline) and returned to positive after reoxygenation. After dobutamine, no DT reduction occurred at the highest stimulation frequencies (P < .005 vs baseline).

After reoxygenation (Fig 4, A ), the improved DT observed at the low pacing rate in the STHS plus bimakalim group (P < .005 vs control) disappeared at rapid pacing (400- and 300-ms CL). After 1 µmol/L dobutamine (Fig 4, B ), at short CL, all groups showed DT values higher than DT values obtained during basal FF at corresponding CL (P < .0001 for time factor), and the enhanced DT induced by STHS plus bimakalim treatment remained during the high pacing rate also.

	Discussion

Top Abstract Introduction Patients and methods Results Discussion References

 
This in vitro study shows that (1) K_ATP channels are likely involved in the cardioprotective effects of cardioplegic arrest in stunned human atrial muscle because the contractile protection was inhibited by glibenclamide and (2) K_ATP channel activation by bimakalim, used as an additive of STHS, may enhance the human atrial protection caused by cardioplegic arrest. This latter additional benefit was, however, abolished by the high pacing rate after hypoxia-reoxygenation but was present after dobutamine challenge, even at rapid pacing. Changes in FF relationship were superposable to variations reported previously, ¹⁷ showing that the negative inotropic action of a high pacing rate (400- and 300-ms CL), likely because of dramatic shortening of human atrial action potential duration, ^17,18 consists of a normal physiologic adaptation of human atrial contractile function to rapid pacing. More interesting were the negative FF relationships induced by hypoxia and then reversed after reoxygenation and the abolition of the negative inotropic action of the high pacing rate by dobutamine, as observed in the rabbit heart. ²¹ Considering that our findings in human cardiac tissue arise from a model of normothermic continuous STHS delivery, it will be important to confirm the additional protective effects of bimakalim during other cardioplegic methods, such as cold cardioplegia, because it has been reported extensively, although only in animal preparations. ^9-12

Although data suggest that K_ATP channels are likely involved in cardioprotective mechanisms related to normothermic cardioplegia in human atrial myocardium, it is unclear whether sarcolemmal K_ATP channels alone are implicated or whether the mitochondrial channels also may play a role. A recent animal study showed that diazoxide, a specific mitochondrial K_ATP opener, may exert cardioprotective effects, whereas 5-OH-decanoate, a mitochondrial K_ATP blocker, may block cardioprotection. ²² Animal studies already reported benefits with other K_ATP channel openers as additives of cardioplegic solutions ^9,10 and particularly advantages of nicorandil. ^11-13,23 We chose bimakalim, which exerts selective K_ATP channel activating action, as opposed to nicorandil, which also possesses nitrate-related properties. In addition, we used a relatively low concentration (100 nmol/L) of bimakalim on the basis of the previous studies demonstrating protective effects of bimakalim on human atrial contractile function ⁵ at concentrations (10-100 nmol/L) devoid of negative inotropic action and depressant effect on human atrial action potential duration. ^5,15 Indeed, it is important that additive cardioprotection may be obtained in the absence of action potential changes, thus ruling out potential proarrhythmic risks related to K_ATP channel activators. ^24,25 Moreover, the bimakalim concentration used here may be considered relevant for the clinical setting. Senior and associates ²⁶ reported bimakalim plasmatic concentrations of about 50 nmol/L in normal volunteers. Further studies are now needed to clarify the mechanisms involved in the bimakalim-induced protection during cardioplegia. In view of the strong pharmacologic evidence that K_ATP channels are involved, mitochondrial K_ATP channel is a strong candidate for the putative end effector of bimakalim-induced benefits ²⁷ because additive protection was obtained during cardioplegic arrest and therefore in absence of any contractile and electrical activity.

The additional cardioprotective effects of bimakalim disappeared during the high pacing rate just after hypoxia-reoxygenation, whereas positive inotropic stimulation with dobutamine restored these effects. These findings may suggest that additional benefits of K_ATP channel activation might be overshadowed in the presence of high heart rate, arrhythmic events, or both after hypoxia-reoxygenation where contractile function is about to recover. Although caution is needed when extrapolating in vitro findings to clinical situations, these data support the usefulness of FF relationship studies when investigating the effects of K_ATP channel openers. Otherwise, the restoration of bimakalim’s protective effects by dobutamine at high pacing may be due to the positive inotropic stimulation within cardiac cells because dobutamine was able to revert to positive the negative part of the basal FF relationship in accordance with data previously obtained from terminally failing human ventricular myocardium. ²⁸ Further studies are now needed to clarify the mechanisms involved in the bimakalim-dobutamine interaction.

Limits of in vitro experimental models have to be considered. First, mechanical abnormalities, as seen in this model, may be different from contractile dysfunction caused by regional ischemia in in vivo models, which use the recovery of segmental wall function as an index of stunning. However, the reversibility of contractility loss in human atrial trabeculae under dobutamine challenge fulfills the definition of stunned myocardium. ^5,17 Second, although atrial and ventricular tissues have shown similar responses to hypoxia ¹² and to positive inotropic stimulation, ²⁹ some tissue-related specificity may account for cardiac cell responses to action of K_ATP channel activators. ^15,30 Clinical extrapolation of our findings must therefore await further studies in human ventricular muscle.

	References

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