Deleterious effects of digitalis on newborn rabbit myocardium after simulated cardiac surgery

J THORAC

CARDIOVASC SURG

1991;101:337-41

Deleterious effects of digitalis on newborn rabbit myocardium after simulated cardiac surgery We studied the effect of the digitalis gJycoside ouabain on isoJated blood-pel'f'.-d neonatal (4- to 6-day-old) rabbit hearts during 60 minutes of hypothermic/ischemic arrest (at 15° C), simulating conditions during cardiac surgery. Compared with a control (no ouabain) group, both low- and high-dose ouabain groups had increased left ventricular diast06c chamber stiffness ("contracture") during and after the arrest period. The high-dose ouabain group also showed less recovery of contractile function than did the control group. We conclude that digitalis gJycosides have the potential to impair recovery of myocardial function in the newborn infant ·after hypothermic/ischemic arrest; myocardial reJaxation and diast06c function appear more sensitive to this manifestation of digita6s toxicity than does contractile function.

Takashi Konishi, MD, and Carl S. Apstein, MD, Boston, Mass.

Cardiac surgery in neonates is associated with a relativelyhigh risk of morbidity and death, and protection of immature inyocardiurnhas been recognizedas high priority by the National Heart, Lung, and Blood Institute. Inadequate myocardial preservation during surgical cardiac arrest has been implicated as a major factor in this high risk,I despite numerous reports that newborn myocardium is more resistant to hypoxicand ischemicinjury than adult myocardium.l? We reasoned that this documented poor recovery from cardiac surgery may be due in part to deleterious effects of pharmacologic agents. Many neonates undergoing cardiac surgery have heart failure and may be treated with digitalis glycosides. Recent studies of adult myocardium from our laboratory have demonstrated that digitalis (ouabain) increases myocardialdysfunctionduring and after a brief period of

From the Cardiac Muscle Research Laboratory, the Whitaker Cardiovascular Institute, Boston University School of Medicine, and the Cardiology Section, Thorndike Memorial Laboratory, Boston City Hospital, Boston, Mass. Supported by the United Stated Public Health Service Grant JL35675. Dr. Konishi was supported by a research fellowship (13-437-856) from the Massachusetts Affiliate of the American Heart Association. Received for publication July 13, 1989. Accepted for publication Jan. 30, 1990. Address for reprints: Carl S. Apstein, MD, Cardiac Muscle Research Laboratory, Boston University School of Medicine, 80 E. Concord St., R217, Boston, MA 02118.

12/1/19925

ischemia" or hypoxia," We performed the current study to determine whether newborn hearts, when treated with ouabain and subjectedto the hypothermicischemicarrest conditionsassociatedwith cardiac surgery, would exhibit impaired cardiac function similar to that of the adult hearts we previously studied. Methods Isolated heart preparation.We used an isolated isovolumic (balloon-in-left ventricle) rabbit-heart model perfused with fresh whole blood, a system that we have recently developed.' In this system heparinized blood is pumped from a venous reservoir, through an oxygenator, into a pressurized arterial reservoir, through a 40 J.Lm filter (SQ40; Pall Corp., Biomedical Products Division, Glen Cove, N.Y.), and into an aortic cannula. Coronary venous blood collects in the venous reservoir and is recirculated through the system. A heparinized (500 units/ kg) and anesthetized (sodium pentobarbital, 50 mg/kg) 2 to 3 kg male New Zealand white rabbit served as a blood donor, contributing 70 to 80 ml fresh whole blood from the carotid artery. Gentamicin (6 mg/IOO ml) was added to the blood to prevent bacterial growth. The hematocrit of the blood perfusate was 29% to 33%, blood glucose was maintained between 80 and 120 mg/dl, oxygen tension (P02) was 70 to 150 mm Hg, and pH was 7.35 to 7.45. A neonatal albino New Zealand rabbit (4 to 6 days old, male or female) was heparinized (200 units) and anesthetized with sodium pentobarbital (50 mg). The thorax was rapidly opened and the heart and proximal great vessels excised and placed in iced saline. The aortic stump was perfused by use of the isolated heart system described above. The time from thoracotomy to institution of coronary perfusion on the isolated heart apparatus was approximately I to 2 minutes. Coronary perfusion pressure was adjusted to 60 mm Hg and coronary flow was determined by coronary autoregulation. A coronary perfusion

337

The Journal of Thoracic and Cardiovascular Surgery

3 3 8 Konishi and Apstein

Table I. Effect ofouabain on cardiac function with hypothermic/ischemic arrest Before ouabain Control (n = 9) Low-dose ouabain (n = 8) High-dose ouabain (n = 6)

DP(mm Hg) EDP (mm Hg) CF (ml/m/gm) DP(mmHg) EDP (mm Hg) CF (ml/m/gm) DP(mm Hg) EDP(mmHg) CF ml/rn/gm)

81 ± II ± 2.89 ± 97 ± II ± 1.9 ± 90 ± II ± 2.14 ±

5 0.3 2.31 10 0.5 0.44 9 0.4 0.61

After ouabain 74 ± 9± 2.88 ± 103 ± II ± 2.23 ± 80 ± 15 ± 4.10 ±

5 0.5 2.31 12 0.6 0.47 9* 1* 1.52

End

End

ischemia

reperfusion

% Recovery

0 10 ± 2 0 0 18 ± 3* 0 0 '57.0 ± 11** 0

63 ± 4 II ± I 2.58 ± 1.59 77±1O 17 ± 2* 3.06 ± 1.13 57 ± 5 17 ± 2* 4.19 ± 1.27

78 ± 100 ± 104 ± 79 ± 151 ± 130 ± b63 ± 150 ± 229 ±

2 II 19 7 17 84 4 17 119

Isolated isovolumic (balloon-in-left ventricle) newborn rabbit hearts were perfused at a coronary perfusion pressure of 60 mm Hg during a 15 to 20-minute preouabain stabilization period. The two ouabain groups received ouabain as described in the text. The control group did not receive ouabain; the values for the controls reported in the columns titled "Before ouabain" and "After ouabain" indicate measurements made during the baseline preischemic period at time intervals comparable to the preinfusion and postinfusion measurements in the ouabain groups. Then all groups underwent hypothermic/ischemic arrest (at 15° C) for 60 minutes and reperfusion for 30 minutes. Values are mean ± standard error of the mean. DP, Developed pressure; EDP, end-diastolic pressure; CF, coronary flow; % Recovery, percent recovery relative to preischemic, preouabain values. Compared to value before ouabain, 'p < 0.05, "p < 0.001. 'p < 0.05 compared to low-dose ouabain group. bp < 0.05 compared to control group.

pressure of 60 mm Hg was maintained during the preischemic and recovery periods, since we have shown that this pressure results in optimal performance of the neonatal heart.? Coronary perfusion pressure was monitored from a side arm of the aortic perfusion cannula connected to a pressure transducer (Statham P23Db; Spectramed Inc., Critical Care Division, Oxnard, Calif.). A polyethylene cannula was passed across the left ventricular apex to decompress the left ventricle of thebesian drainage. Another cannula was inserted into the right ventricle by way of the pulmonary artery for complete collection of coronary sinus effluent and maintenance of a decompressed right ventricle throughout the experiment. A pacing wire was introduced into the right ventricle through the right atrium.To measure left ventricular pressures, a collapsed latex balloon was placed in the left ventricle through the left atrium. After its placement in the left ventricle, the balloon was filled with saline to acheive a physiological left ventricular end-diastolic pressure (LVEDP) of 7 to 15 mm Hg. Left ventricular pressure was monitored through a short, rigid, fluid-filled polyethylene tube (PE 240; Intra-Medic, Clay Adams, Division of Becton Dickinson and Company, Parsippany, N.J.) attached to a pressure transducer (Statham P23Db). The heart was then suspended in warm saline at 37° C; Bath temperature was monitored throughout the experiment and maintained by adjusting the temperature of the water circulating through the water-jacketed chamber. During a 15 to 2D-minute preischemic period, the heart was allowed to stabilize at a temperature of 37° C, a paced heart rate of 240 beats/min, a coronary perfusion pressure of 60 mm Hg, and a left ventricular balloon volume producing an LVEDP of 7 to 15 mm Hg. Hearts were discarded if they failed to achieve a left ventricular developed pressure of at least 60 mm Hg during the preischemia stabilization period; approximately 5% of the experimental preparations were rejected under this criterion. Coronary flow rate was measured by collecting timed samples of the coronary sinus effluent through the pulmonary artery cannula. Ouabain dose. Our goal was to use a clinically relevant, nontoxic level of ouabain. Two doses of ouabain were used. The

ouabain solution was infused into the aortic cannula by a microinfusion pump, and the dose was titrated by monitoring left ventricular contractile function and cardiac rhythm. During the ouabain infusion the coronary venous effluent was diverted from the venous reservoir and discarded to prevent ouabain accumulation in the recirculating perfusate. High-dose ouabain group. In the high-dose ouabain group (final concentration, 400 nmol/L; n = 6) ouabain was infused until definite toxicity was indicated by arrhythmia, deterioration of systolic and diastolic function, or both. After this "upper limit" of ouabain tolerance was determined, the ouabain infusion was terminated, and the heart was perfused with ouabainfree blood until any arrhythmia resolved. The ouabain infusion was not reinstituted during reperfusion in this group. Low-dose ouabain group. In the low-dose ouabain group (final concentration, 70 nmol/L; n = 8) ouabain was titrated until a minimal detectable positive inotropic effect was indicated by an increase in left ventricular developed pressure and rate of pressure rise. During reperfusion the ouabain infusion was reinstituted at the same rate as during the preischemic period. Control group. A control group (n = 9) not receivingouabain underwent the same experimental protocol as did the two ouabain groups. Hypothermic/ischemic arrest protocol. After ouabain loading and measurement of preischemic function, the pacemaker was turned off and coronary perfusion was stopped. The hearts were then rapidly cooled to 15° C by submersing them in cold saline and maintained at that temperature during a 60minute arrest period. After the 60-minute hypothermic/ ischemic arrest period, reperfusion was initiated with room-temperature blood (25° C) at a coronary perfusion pressure of 60 mm Hg. During the first 15 minutes of reperfusion, the heart was gradually rewarmed to 37° C, and then pacing was reinstituted during an additional 15 minutes of reperfusion. The studies involving experimental animals herein described conformed to the guiding principles of the American Physiological Society. Statistical analysis.The results are reported as the mean value ± standard error of the mean. Statistical significance for comparisons within a group was assessed by Wilcoxon matched-

Volume 101 Number 2 February 1991

pair signed-rank test or Student's paired t test. Student's unpaired t test wasusedfor comparisons amonggroups,and the Bonferroni correction was used for multiple comparisons.

Results The results are summarized in Table I. There were no significant differences among groups before the addition of ouabain with regard to left ventricular developed pressure, end-diastolic pressure, or coronary flow rates. In the low-dose ouabain group after the addition of ouabain, a modest inotropic effect was manifested by a slight (6%) increase in left ventricular developed pressure from 97 ± 10 to 103 ± 12 mm Hg (p = 0.1) and a 22% increase in rate of pressure rise from 1810 ± 690 mm Hg/sec before application of ouabain to 2210 ± 960 mm Hg/sec after application of ouabain (p < 0.02). There was no significant effect of the low-dose ouabain on LVEDP. In the high-dose ouabain group after loading to the point of toxicity and then terminating the infusion of ouabain, a mild negative inotropic effect was indicated by a slight (11 %) decrease in developed pressure from 90 ± 9 to 80 ± 9 mm Hg (p < 0.05) and a slight (2%) decrease in rate of pressure rise (p = NS*). Myocardial diastolic function was slightly impaired, as manifested by the increase in LVEDPfrom 11 ± 0.4 to 15 ± 1 mmHg (p < 0.05). During the ischemic/hypothermic arrest all hearts became asystolic. The intraventricular balloon was maintained at constant volume and intraventricular (enddiastolic) pressure was monitored as an index of chamber stiffness or "contracture." At the end of ischemia in the nonouabain control group, there was no alteration of chamber stiffness from the initial diastolic pressure value (11 ± 0.3 versus 10 ± 2 mm Hg). In the low-dose ouabain group at the end of ischemia, left ventricular pressure had increased to 18 ± 3 mm Hg from an initial left ventricu1ardiastolicpressureofll ± O.5mmHg(p = 0.02), indicating a significant increase in chamber stiffness or "contracture" of the arrested ventricle. In the high-dose ouabain group at the end of ischemia, intracavitary pressure had increased more than fivefold, to 57 ± 11 mm Hg, from the initial LVEDP value of 11 ± 0.4 mm Hg (p < 0.001), indicating a marked increase in chamber stiffness or contracture in the high-dose ouabain group. This increase in end-ischemic end-diastolic pressure in the high-dose ouabain group was significantly greater (p < 0.05) than that of the low-dose ouabain group. After 30 minutes of reperfusion, the control group recovered 78% ± 2% of developed pressure and mani*NS

= Not significant.

Digitalis and recovery from ischemic arrest 3 3 9

fested no significant change of LVEDP or coronary flow relative to preischemic values. The low-dose ouabain group had a recovery of developed pressure that was comparable to that in the control group (79% ± 7%), but LVEDP in the low-dose ouabain group was significantly increased relative to the preischemic value (11 ± 0.5 versus 17 ± 2 mm Hg, p < 0.05). This indicates a persistent impairment of diastolic relaxation and an increase in diastolic chamber stiffness in the low-dose ouabain group during the reperfusion period. The high-dose ouabain group recovered developed pressure to only 63% ± 4% of the preischemic control level, a value significantly less (p < 0.05) than that of the control group. Diastolic function was also impaired in the high-dose ouabain group during the reperfusion period, as manifested by the increased end-diastolic pressure relative to the preischemic value (11 ± 0.4 versus 17 ± 2 mm Hg, p < 0.05). Discussion

Our results demonstrate that the digitalis glycoside ouabain has the potential to impair functional recovery of newborn myocardium after 1 hour of hypothermic/ ischemic arrest such as that occurring during cardiac surgery. The good recovery of the control group in these experiments is consistent with the high tolerance of neonatal myocardium to hypothermic/ischemic arrest previously reported by ourselves and others. 2-5 In the high-dose ouabain group, both systolic and diastolic cardiac function were reduced in the recovery period, but in the lowdose ouabain group, only diastolic function was significantly impaired. Our results may help explain an apparent paradox regarding cardiac surgery in neonates. Most investigators have found that neonatal myocardium is more tolerant to hypoxia and ischemia than adult myocardium.i" yet inadequate myocardial preservation during cardiac surgery has been implicated as a major cause of postoperative morbidity and death.' Our results suggest that the presence of digitalis glycosides can decrease the tolerance of neonatal myocardium to ischemic arrest, resulting in postoperative dysfunction. This could be interpreted in a clinical setting as "inadequate myocardial preservation." The effect of ouabain was to impair diastolic function more than systolic function. Distinction between systolic and diastolic dysfunction can be made readily in an isovolurnic isolated heart apparatus, but clinical distinction between systolic and diastolic dysfunction in the immediate postoperative period can be difficult; both conditions can cause elevated ventricular filling pressures (either right- or left-sided) and a decreased stroke volume and cardiac output as a result of reduced diastolic filling. When postoperative failure is primarily caused by dias-

The Journal 01 Thoracic and Cardiovascular Surgery

3 40 Konishi and Apstein

tolic dysfunction, the use of digitalis glycosides is not indicated and may be contraindicated. In general, postischemic diastolicdysfunction appears to be a more sensitive index of myocardial injury than does systolic dysfunction, as illustrated by the low-dose ouabain group in these experiments. Similarly, in both experimental and clinical studies of adult hearts, myocardial diastolicrelaxationhas beenimpairedduring and after reversible hypoxic and ischemic injury,whilesystolic function has occasionally remained relatively preserved, especially in studies of hypertrophied hearts.8- IO An increase in myocardial cytosolic calcium is common to digitalis glycoside treatment.U'P myocardial hypertrophy,14-16 and lschemia.l": 18 The impairment of myocardial relaxation in the presenceof ouabain during and after ischemia may be mediated by a cytosolic calcium overload, which prevents the complete abatement of active tension during diastole. Such a mechanism has been invoked to explain the impairment of myocardial relaxation in adult hearts treated with ouabain and subjected to either low-flow ischemia or hypoxias-? and a similar mechanism is likely responsible for the impairment of relaxationin the ouabaingroupsduring and after ischemic arrest in the neonatalheartsin our currentstudy. Limitations of this study. Our investigation suffers from intrinsicexperimentallimitations. We tried to simulate experimentally the myocardial conditions during cardiac surgery and to measure the effect of a clinically relevant dose of digitalis glycoside on the myocardium. One limitation is the relatively brief experimental recovery periodof 30 minutes.Our observations are limitedto the immediate "postoperative" period, and we cannot determine whether the impaired recovery observed in the ouabain groups was reversible with further reperfusion. Our conclusions are also limited by the ouabain levels we used. We attempted to titrate the dose of ouabain as might be done under clinicalcircumstances. Thus, in the high-dose ouabain group, ouabain was administered to the point of toxicityand then stopped. Despitethe cessation of the ouabain infusion, the high-dose ouabaingroup probably had an excess ouabain level at the end of the preischemic period, as evidenced by a lower developed pressure and a higher LVEDP in the postouabain measurements. Thus the high-dose ouabain group probably represents a level that borders on clinicaldigitalis toxicity. On the other hand, the low-dose ouabain group represents a lowor therapeutic level of ouabain,sincethe postouabain, preischemia measurements demonstrated a slightinotropiceffectwith no increasein LVEDP and no arrhythmias. Thus the reduced recovery of diastolic

function (i.e., the persistent postischemic increase in LVEDP) in this group cannot be attributed to an excessiveexperimental dose of ouabain. Our study is limited by its experimental nature, and any clinical extrapolation should be made with great caution. Nonetheless, our results suggesta potentialdeleteriousinteractionbetween preoperative digitalis therapy and subsequent cardiac surgery in newborn infants. Since cardiac surgery in the neonatal periodis generally performedonlyin criticallyillinfants,digitalis glycosides may be used in heart failure therapy beforesurgery. Our resultssuggestthat useofdigitalis in this settingwarrants further clinicalinvestigation. An alternativepreoperative inotropic agent may be a better choice than digitalis. In adult hearts, low-dose isoproterenol did not impair myocardial relaxation during brief periods of ischemia, in contrast to the effect of ouabain," This observation suggests that a l3-adrenoreceptor agonist may be preferable to digitalisfor preoperative inotropic supportin neonates. However, such a conclusion requires clinical confirmation and should not be made on the basis of our experimental observations alone. We are grateful to William Grice for his assistance in performing these experiments and to Maryanne Mills for her expert editing and manuscript preparation. REFERENCES 1. Bull C, Cooper J, Stark J. Cardioplegic protection of the child's heart. J THORAC CARDIOVASC SURG 1984;88:28793. 2. Grice WN, Konishi T, Apstein CS. Resistance of neonatal myocardium to injury during normothermic and hypothermic ischemic arrest and reperfusion. Circulation 1987; 76(Pt 2):VI50-5. 3. Nishioka K, Jarmakani JM. Effect of ischemia on mechanical function and high-energy phosphates in rabbit myocardium. Am J Physiol 1982;242(Heart Circ Physiol 11):HlO77-83. 4. Coles JG, Watanabe T, Wilson GJ, et al. Age-related differences in the response to myocardial ischemic stress. J THORAC CARDIOVASC SURG 1987;94:526-34. 5. Yano Y, Braimbridge MV, Hearse DJ. Protection of the pediatric myocardium. J THORAC CARDIOVASC SURG 1987;94:887-96. 6. Lorell BH, Isoyama S, Grice WN, Weinberg EO, Apstein CS. Effect of ouabain and isoproterenol on left ventricular diastolic function during low flow ischemia in isolated, blood-perfused rabbit hearts. Circ Res 1988;63:457-67. 7. Cunningham MJ, Apstein CS, Weinberg EO, Lorell BH. Deleterious effect of ouabain on myocardial function during hypoxia. Am J Physiol 1989;256(Heart Circ Physiol 25):H681-7.

Volume 101 Number 2

Digitalis and recovery from ischemic arrest 3 4 1

February 1991

8. Apstein CS, Grossman W. Opposite initial effects of supply and demand ischemia on left ventricular diastolic compliance: the ischemia-diastolic paradox. J Mol Cell Cardiol 1987;19:119-28. 9. Lorell BH, Grossman W. Cardiac hypertrophy: the consequences for diastole. J Am Coli Cardiol 1987;9:1189-93. 10. Menasche P, Grousset C, Apstein CS, Marotte F, Mouas C, Piwnica A. Increased injury of hypertrophied myocardium with ischemic arrest: preservation with hypothermia and cardioplegia. Am Heart J 1985;110:1204-9. II. Cleeman L, Pizano G, Monrad M. Optical measurements of extracellular calcium depletion during a single heart beat. Science 1984;226:174-7. 12. Barry WH, Hasin Y, Smith TW. Sodium pump inhibition, enhanced calcium influx via sodium-calcium exchange, and positive inotropic response in cultured heart cells. Circ Res 1985;56:231-41. 13. Biedert S, Barry WH, Smith TW. Inotropic effects and changes in sodium and calcium contents associated with

14.

15.

16.

17.

18.

inhibition of monovalent cation active transport by ouabain in cultured myocardial cells. J Gen PhysioI1979;74:479-94. Keung EC, Berg R, Katzung BG. Calcium current is increased in single myocytes from hypertrophied rat myocardium [Abstract]. Circulation 1987;76(Pt 2):IV329. Andrawis NS, Kuo TH, Giacomelli F, Wiener J. Altered calcium regulation in the cardiac plasma membrane in experimental renal hypertension. J Mol Cell Cardiol 1988;20:625-34. Gwathmey JK, Morgan JP. Altered calcium handling in experimental pressure-overload hypertrophy in the ferret. Circ Res 1985;57:836-43. Lee H, Smith N, Mohabir R, Clusin WT. Cytosolic calciurn transients from the beating mammalian heart. Proc Nat! Acad Sci USA 1987;84:7793-7. Steenbergen C, Murphy E, Levy L, London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 1987;60:700-7.