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Adenosine versus potassium cardioplegia: Effects on cardiac arrest and postischemic recovery in the isolated rat heart
J THORAC
CARDIOVASC SURG 1989;98: 1057-65
Adenosine cardioplegia Adenosine versus potassium cardioplegia: Effects on cardiac arrest and postischemic recovery in the isolated rat heart Adenosine is a potential cardioplegic agent by virtue of its specific inhibitory properties on nodal tissue. We tested the hypothesis that adenosine could be more effective than potassium in inducing rapid cardiac arrest and enhancing postischemic hemodynamic recovery. Isolated rat hearts were perfused with Krebs-Henseleit buffer or cardioplegic solutions to determine the time to cardiac arrest and the high-energy phosphate levels at the end of cardioplegia. Cardioplegic solutions contained adenosine 10 mmol/L, potassium 20 mmol/L, or adenosine 10 mmol/L + potassium 20 mmol/L and were infused at a rate of 2 ml/min for 3 minutes at 10° C. Both time taken and total number of beats to cardiac arrest during 3 minutes of cardioplegia were reduced by adenosine 10 mmol/L and adenosine 10 mmol/L + potassium 20 mmol/L when compared with potassium 20 mmol/L alone (p < 0.001). Tissue phosphocreatine was conserved by adenosine 10 mmol/L when compared with potassium 20 mmol/L, being 7.1 ± 0.2 (JLmol/gm wet weight (n = 7) and 6.0 ± 0.3 JLmol/gm wet weight (n = 5~ respectively (p < 0.05). Postischemic hemodynamic recovery was tested in isolated working rat hearts. After initial cardiac arrest, the cardioplegic solution was removed with Krebs-Henseleit buffer at a rate of 2 m1/min for 3 minutes at 10° C, and thereafter total ischemia was maintained for 30 or 90 minutes at 10° C before reperfusion. Adenosine 10 mmol/L enhanced recovery of aortic output when compared with potassium 20 mmol/L or adenosine 10 mmol/L + potassium 20 mmoljL, the percentage recovery after 30 minutes of ischemia being 103.0% ± 4.4% (n = ~ 89.0% ± 5.8% (n = 6~ and 86.6% ± 4.3% (n = ~ respectively (p < 0.05 for comparison between adenosine 10 mmoljL and potassium 20 mmol/L), Thus adenosine cardioplegia caused rapid cardiac arrest and improved postischemic recovery when compared with potassium cardioplegia and with a combination of these two agents.
Thorsten Schubert, MD, Herbert Vetter, MD,a Patricia Owen, PhD, Bruno Reichart, MD,a and Lionel H. Opie, MD, PhD, Cape Town, Republic of South Africa
AdenOSine, by its property of antagonizing cardiac calcium channels, inhibits the sinoatrial node, the atrioventricular node, and myocardial contraction, thereby potentially inducing cardiac arrest.l' During ischemia, high-energy phosphates are degraded to adenosine, inosine, hypoxanthine, and uric acid,4-6 and the adenosine
From the Heart Research Unit. Departments of Medicine and Cardiothoracic Surgery! University of Cape Town and Groote Schuur Hospital. Cape Town. Republic of South Africa. Revision received for publication Oct. 31. 1988. Accepted for publication March 2. 1989. Address for reprints: Professor L. H. Opie. Heart Research Unit, University of Cape Town. Medical School. Observatory. 7925, South Africa.
12/1/13098
triphosphate (ATP) levels may be depressed for days after recovery from ischemia.' Earlier studies have shown the benefits of adenosine added to other cardioplegic solutions.t including a solution with a high potassium (K+) concentration." At the same time, there are doubts about the benefits of high K+ cardioplegic solutions. 10-12 We examined the individual and additive potential benefits of adenosine cardioplegia and sought to answer the following questions: I. How rapidly and at what concentration can adenosine arrest the heart when compared with high K+? 2. Was the recovery of adenosine-treated hearts after different ischemic times better than that of K+ -arrested hearts? . 3. Was the combination of adenosine and high K+ concentration beneficial for the heart? 1057
The Journal of
1 0 5 8 Schubert et al.
Thoracic and Cardiovascular Surgery
A.
B.
10 min
3 min
Langendorff Perfusion
cp
10 min
10 min
L-dorff
WH
3'
t Freeze clamp for tissue analysis
3' 30/90 min
I
l
CP WO Ischemia
I
10 min L-dorff
10 min
WH
I
Freeze-clamp for tissue analysis
Fig. 1. Perfusion sequences, perfusion mode, and time division for each perfusion mode. A, Protocol for the effects of different cardioplegic solutions in Langendorff-perfused hearts. B, Protocol for studying hemodynamic recovery in the working heart. L-dorff, Langendorff perfusion; WHo working heart perfusion; CPo cardioplegia at 2 ml/rnin for 3 minutes; WOo washout at 2 ml /min for 3 minutes with KH buffer at 10° C.
Methods Our model was the isolated perfused rat heart, which has also been used in other cardioplegia studies." 9. 13 All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Male Long Evans rats weighing 270 to 330 gm were anesthetized with ether and given 200 units of heparin into the femoral vein. The heart (average fresh weight 1.0 gm) was rapidly removed and plunged into ice-cold perfusion medium before being mounted on an aortic cannula for isolated perfusion. Perfusion techniques. We used two different perfusion techniques: (I) the Langendorff aortic technique for determination of cardiac arrest time and high-energy compounds (after 3 minutes of cardioplegia) and (2) the working heart preparation for studying both the cardiac arrest time and the hemodynamics in the postischemic recovery period (Fig. I). Langendor.lftechnique. The heart was mounted via the aorta on a modified Langendorff perfusion apparatus and was perfused retrogradely with a modified Krebs-Henseleit (KH) buffer containing NaHC0 3 25 mrnol/L, NaCI 118 rnmol/L, KH 2P04 1.2 mrnol/L, KC14.8 mmol/L, MgS0 4 1.2 mmol/L, CaCI 2 1.25 mrnol/L, and glucose 11.1 mrnol/L. The solution was gassed with 95% oxygen and 5% carbon dioxide and maintained at 3]0 C. The heart was perfused at a pressure of 100 em H 20. The apical force displacement technique'" was used to measure contraction with a Grass model 79-D recorder (Grass Instrument Company, Quincy, Mass.), PROTOCOL After a stabilization period of 10 minutes, cardiac arrest was induced by introduction of different cardioplegic solutions through a side arm of the aortic cannula at a rate of 2 ml/rnin for 3 minutes. The cardioplegic solution and the water jacket surrounding the heart during cardioplegia were kept at 10° C by a separate water circuit. The following mechanical parameters were assessed over the 3-minute period of induction of cardioplegia: arrest time (seconds), number of arrest beats,
number of escape beats, and total number of beats. Then the heart was freeze-clamped for determination of phosphocreatine (PCr), creatine (Cr), and adenosine triphosphate (A TP), diphosphate (ADP), and monophosphate (AMP). Working heart technique. The working heart perfusion technique of Neely and associates I 5 allows determination of hemodynamic parameters while the heart is perfused via the left atrium (preload) and ejects the cardiac output against a pressure load (afterload). The aorta was cannulated and the heart was perfused at 100 em H 20 pressure. During the initial Langendorff (aortic) perfusion, the left atrium was cannulated and the pulmonary artery incised. The retrograde perfusion was stopped by supplying perfusate to the left atrium at a pressure of 20 em H 20. The left ventricle then ejected the cardiac output (aortic and coronary flow) against a pressure of 100 em H20. The cardioplegic solutions and the washout (KH) were infused through a side arm of the aortic cannula. The aortic pressure was measured with a Statham P23dB pressure transducer (Spectramed Inc., Critical Care Division, Oxnard, Calif.) on a second side arm of the aortic cannula. An electrocardiographic electrode was attached to the right ventricle. The electrocardiogram and pressure were monitored on a Devices twinchannel recorder (Mx 2P-148, Devices for Science. Inc., Redwood City, Calif.). PROTOCOL After a stabilization period of 10 minutes, the Langendorff perfusion was converted to the working heart model (left atrial perfusion) for another 10 minutes (Fig. 1). Cardiac arrest was then induced by infusing cardioplegic solution at 2 ml/rnin for 3 minutes at 10° C. The cardioplegic solution was washed out of the heart with KH buffer at a rate of 2 rnl/rnin for 3 minutes at lOG C via the same cannula. In two additional groups, KH and adenosine 10 mrnol/L, the cardioplegic solution was not washed out of the heart and remained for the duration of the ischemic period. Total ischemia was then maintained at 10° C for 30 or 90 minutes. During cardioplegia and ischemia the heart was surrounded by a water-jacketed chamber maintained at 10° C. After ischemia the heart was rewarmed by retrograde perfusion of KH buffer at 37° C for 10 minutes. Then the system was switched to a working prepara-
Volume 98 Number 6
Adenosine cardioplegia
December 1989
10 5 9
Table I. Effect of different cardioplegic solutions on cardiac arrest time, number of arrest beats, number of escape beats, and total number of beats during cardioplegia Cardioplegic solution
'0.
Time to cardiac arrest (sec)
A. Langendorff perfused rat hearts K+ 20 mrnol/L Adenosine 10 rnmol/L Adenosine 1 mrnol/L Adenosine 10 mrnol/L + K+ 20 mmol/L Adenosine 1 mrnol/L + K+ 20 mmol/L B. Working rat hearts KH K+ 20 rnrnol/L Adenosine 10 mrnol/L
52 ± 1.6 ± 2.1 ± 1.9 ± 9.0 ±
2.0 0.3* 0.6* 0.2* 5.0*
446 ± 39 63 ± 12 7 ± It
No. of arrest beats
168 ± 7::':: 6± 9± 16 ±
9 1* 1* 2' 8*
640 ± 107 125 ± 27 15 ± 2t
No. of escape beats
Total No. of beats during cardioplegia
0 3 ± 1* 11 ± 2* 4 ± 1* 15 ± 7*
168 ± 8± 17 ± 13 ± 31 ±
38 ::':: 9 37 ± 24 31 ± 7
678±115 162 ± 3 46 ± 5t
9 2* 2* 3' 7*
or experiments = 5 to 7 in each group. KH. Krebs-Hensclcit buffer
'Compared with K + :'0 mmol/ L: f' <0.00 I. modified { test.
+Compared with K + :'0 mmol . L: P <0.05 { test.
tion and mechanical parameters were measured over a 10minute period, at the end of which the heart was freeze-clamped for biochemical analysis. The following parameters were measured: aortic output. coronary flow, heart rate, systolic and diastolic pressure, PCr, ATP, lactate and glycogen. Perfusion solutions. The different cardioplegic solutions contained KH buffer plus: 1. Adenosine 10 mmoljL 2. Adenosine I mmoljL 3. K+ 20 mmoljL 4. Adenosine 10 mmoljL + K+ 20 mmoljL 5. Adenosine I mmoljL + K+ 20 mmoljL 6. Control, standard KH buffer These solutions were tested at 10° C by a single infusion technique. Tissue processing and analysis. Content of high-energy compounds was determined either by high-performance liquid chromatography" or enzymatically.'? Glycogen and lactate were determined cnzyrnatically.P'"? Calculations. The following calculations were made: I. Cardiac arrest time: time (seconds) from the onset of infusion of cardioplegic solution until the heart arrests for 2 seconds 2. Arrest beats: number of heart beats until 2 seconds of arrest 3. Escape beats: beats coming through after initial arrest 4. Total number of beats during 3 minutes of cardioplegic perfusion: number of arrest beats plus escape beats 5. Percentage recovery of aortic output (AO): AO (lor 10 min reperfusion) 00 AO (lor 10 min before ischemia) X I
6. Percentage recovery of systolic pressure X heart rate: (SP 2 X HR 2) (SP I X HR t )
X 100
7. Percentage recovery of pulse pressure X heart rate: ([SP 2 - DP 2] X HR 2 ) ([SP I - DPd X HR I )
X 100
where SP = systolic pressure, DP = diastolic pressure. and HR = heart rate (I = preischemic values, 2 = reperfusion values). Statistics and expansion of data. The results are presented as mean and standard error of the mean. Overall significance of differences between groups was determined by a one-way analysis of variance followed by a modified Student's t test. When only two subgroups were compared, we used the t test with a Bonferroni correction. A p value of less than 0.05 was considered significant. Metabolic data are expressed in terms of wet weight (= dry weight X 5).
Results Effect of adenosine on cardiac arrest time, number of beats to cardiac arrest, and escape beats (Table I, A and B, Fig. 2). In Langendorff-perfused hearts (Table I, A), the time to cardiac arrest, the number of arrest beats, and the total number of beats during the 3-minute cardioplegic period were reduced by all cardioplegic solutions containing adenosine when compared with K+ 20 mmol/L alone. The time to cardiac arrest was reduced from 52 seconds with K+ 20 mmol/L to less than 3 seconds in hearts perfused with solutions containing adenosine 10 mmol/L, adenosine 1 mrnol/L, and adenosine 10 mmol/L + K+ 20 mmol/L and to 9 ± 5 seconds for hearts receiving adenosine I mrnol/L + K+ 20 mrnol/L (p < 0.001 for all comparisons). The number of arrest beats was reduced from 168 ± 9 during K+ 20 mmol/L cardioplegia to less than 16 beats in all groups (p < 0.001 for all comparisons). A difference was observed in the number of escape beats after initial cardiac arrest. Hearts receiving K+ 20 mrnol/L had no escape beats. Solutions containing adenosine 10 mmol/L allowed 3 to 4 escape beats, whereas those containing adenosine 1 rnrnol/L allowed 11 to 15 escape beats (adenosine 10 mmol/L versus adenosine I mmol/L,p < 0.05). Adenosine 10 mmol/ L reduced the total number of beats more than adenosine
The Journal of
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Thoracic and Cardiovascular
Schubert et al.
Surgery
A. POTASSIUM 20mM
IHIIHl\H\\\\P\l:I\\IIHHPlql'\! ~ I j II
1
I
:"~\\\:\\,,~,,,I~~'~':~~~'~~~~~~\\:i~~\:\~,~,~,~~<'~<"~" '~'.', (\,,__.:~'~,~, ~,~,,~,~~.~ ~,~,~~ J.J.:. ~~, . -. . ", ", .:.»:v
cardioplegia onset
escape beat
_
B. ADENOSINE 10mM
o
20
10
40
30
50
60
Time (sec)
Fig. 2. A, Trace of left ventricular contraction measured by apical displacement for. arrest by K+ 20 rnrnol/L. B, Trace for arrest by adenosine 10 mrnol/L.
Table II. High-energy phosphates after 3 minutes of cardioplegia in Langendor./f-perfused hearts Cr
PCr
Cardioplegic solution
Pre-cardioplegia K+ 20 mmoljL Adenosine 10 mmoljL Adenosine I mmoljL Adenosine 10 mmoljL + K+ 20 mmoljL Adenosine I mmoljL + K+ 20 mmoljL
6.6 6.0 7.1 7.5 6.5 5.9
± 0.3 ± 0.3 ±0.2* ± 0.7* ± 0.2 ± 0.1
4.3 5.2 3.6 5.6 4.2 6.1
± ± ± ± ± ±
ATP
0.3 0.3 0.2 1.1 0.4 0.4
4.1 4.2 4.0 4.5 3.8 4.2
± ± ± ± ± ±
0.2 0.1 0.1 0.2 0.1 0.1
ADP
0.9 ± 0.9 ± 0.8 ± 1.1 ± 0.8 ± 1.0 ±
0.05 0.04 0.03
o.n0.02 O.03t
AMP
0.07 0.07 0.08 0.11 0.05 0.09
± ± ± ± ± ±
0.01 0.01 0.01 0.02
am 0.01
PCrjCr ratio
1.6 ± 1.2 ± 2.0 ± 1.6 ± 1.6 ± 1.1 ±
0.2 0.1 0.1* 0.3 0.2 0.2
A TPjADP ratio
4.7 ± 0.3 4.9 ± 0.2 5.0 ± 0.2 4.4 ± 0.3 5.0 ± 0.1 4.2±0.lt
Units: jlmoljgm wet weight. Number of experiments = 5 to 7. •P <0.05, modified t test compared with K+ 20 rnrnol/ L
tp <0.05, modified t test compared with adenosine 10 mmoljL.
1 mmol/L (p < 0.05). In the working heart preparation (Table I, B), adenosine 10 mrnol/L cardioplegia showed similar results, namely a reduced time to cardiac arrest and fewer total beats when compared with K+ 20 mmol/ L cardioplegia (p < 0.05), Thus adenosine was more effective than K+ in inducing electromechanical arrest in both the nonworking and working heart. Effect of adenosine on high-energy phosphate levels after cardioplegia (Table II), At the end of 3 minutes of cardioplegia, tissue PCr was preserved by adenosine 10 mrnol/L and 1 mmol/L (p < 0,05 compared with K+ 20 mmol/L). Replacement of K+ 20 mrnol/L with adenosine 10 mmol/L increased the ratio PCr/Cr. The ATP/ ADP ratio fell with adenosine 1 mmol/L + K+ 20 rnmol/ L when compared with adenosine 10 mmol/L (p <0,05). Effect of adenosine cardioplegia on hemodynamic recovery after 30 and 90 minutes of ischemia (Tables III and IV). We showed that, when the adenosine 10 mmol/L solution remained in the heart during ischemia, the effect was not beneficial because the percentage recovery of the aortic output after 10 minutes of working heart reperfusion was no different from that of KH-
arrested hearts, being 64.6% ± 15.9% and 49.4% ± 14.2%, respectively (Table III), If, however, the cardioplegic solution had been removed from the heart with KH buffer before total ischemia, the percentage recovery of aortic output was increased in the case of adenosine from 65% to 103% (p < 0.01, t test) and for the KHtreated hearts from 49% to 85% (p < 0.01, t test). In hearts subjected to 30 minutes of ischemia, adenosine 10 mmol/L cardioplegia showed improved recovery of aortic output, being 91% at 1 minute and 103% at 10 minutes, in comparison with K+ 20 mrnol/L (p < 0.05 for both comparisons), The combination of adenosine 10 mmol/L + K+ 20 mmol/L improved aortic output after 1 minute but not after 10 minutes. No difference was observed between hearts treated with K+ 20 mmol/L and those receiving KH buffer in recovery of aortic output after 30 minutes of ischemia with washout (Table Ill). Hearts subjected to adenosine 10 mmol/ L cardioplegia and 90 minutes of ischemia showed improved aortic output at 1 minute and at 10 minutes of working heart reperfusion when compared with hearts receiving K+ 20 mmol/L (p < 0.05). The percentage recovery of systolic
Volume 98 Number 6
Adenosine cardioplegia
December 1989
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Table III. Mechanical recovery of working rat hearts after 30 minutes of ischemia -------------------------------------~--~--
Percentage recovery of heart rate X systolic pressure
Percentage recovery of aortic output
Percentage recovery of pulse pressure X heart rate
Time of reperfusion work Cardioplegic solution
KH Adenosine 10 mmoljL KH washout K+ 20 mmoJjL washout Adenosine 10 mmoljL washout Adenosine 10 mmoljL + K+ 20 mmoljL washout
1 min
65.4 ± 50.9± 83.4 ± 80.2 ± 91.1 ± 93.4 ±
10 min
14.6 14.7 2.5* 4.1 4.6*
49.4 ± 64.6 ± 84.6 ± 89.0 ± 103.0 ± 86.6 ±
:nt
10 min
14.2 15.9 3.0* 5.8 4.4* 4.3
72.6 ± 72.7 ± 95.7 ± 97.1 ± 106.8 ± 102.5 ±
18.8 17.1 3.2 6.8 4.4 4.5
10 min
45.0 71.4 87.6 93.5 98.0 96.1
± ± ± ± ± ±
11.2 16.3 5.4 15.5 8.4 9.2
Protocol: Thirty minutes of ischemia at 10 C followed by 10 minutes of LangendortT reperfusion before onset of working heart rcperfusion. :-':umb"r of experiments = 6. Washout = Removal of cardioplegic solution at 2 ml /rnin over 3 minutes at 10' C with Krebs-Henseleit (KH) buffer.
*1' <0.01.
I
test. compared nonwashout with washout.
tp <0.05. modified t test. compared with K+ 20 mrnol/L.
Table IV. Effect of different cardioplegic solutions on the mechanical recovery of isolated working rat hearts after 90 minutes of ischemia Percentage recovery of systolic pressure X heart rate
Percentage recovery of aortic output
Percentage recovery of pulse pressure X heart rate
Time of reperfusion work Cardioplegic solution
KH K+ 20 mmoljL Adenosine 10 mrnol/L Adenosine 10 mmoljL + K+ 20 mmoljL
1 min
58.7 73.5 89.0 76.6
± 4.2 ± 2.8 ± 3.8* ± 4.8
10 min
48.0 75.1 83.9 70.6
± 1.4 ± 2.5 ± 1.3* ± 3.9
10 min
78.3 88.0 92.7 88.7
± 4.2
± 4.4 ± 2.5t ± 3.0
10 min
68.8 84.2 85.5 79.0
± 7.2 ± 6.1
± 4.1t ± 4.7
Protocol: "inety minutes of ischemia at 10 C followed by 10 minutes of Langendorff reperfusion before onset of working heart rcpcrfusion. "umber of experiments = 6. All were done with washout of the cardioplegic solution. KH. Krebs-Henseleit buffer
'r
<0.05 differences with modified t test when compared with K+ 20 rnmol/L,
t p <0.05 modified
t
test compared with KH.
pressure X heart rate was also better when compared with that of KH-treated hearts (p < 0.05). There was no difference between hearts receiving K+ 20 mmol/L and those receiving KH buffer in percentage recovery of systolic pressure X heart rate after 90 minutes of ischemia (Table IV). No other groups of measurements showed improvement when compared with K+ 20 mmol/L. The presence of K+ 20 mmol/L with orwithout adenosine impaired mechanical function in the recovery period (Table IV). Coronary flow during the working recovery period after either 30 or 90 minutes of ischemia showed no difference when compared with preischemic values, even after adenosine had been included in the cardioplegic solution. Examples of precardioplegic and postischemic working heart values for coronary flow after 90 minutes of ischemia are as follows: (1) K + cardioplegia, 19.6 ± 0.7 and 19.4 ± 0.9 ml/gm/rnin (n = 6); (2)
adenosine cardioplegia, 18.2 ± 0.9 and 18.2 ± 0.7 ml/ gm/rnin (n = 6); and (3) adenosine + K+ cardioplegia, 17.8 ± 0.7 and 16.3 ± 1.0 ml/gm/rnin (n = 6) (precardioplegic and postischemic values, respectively). Effect of washout of adenosine on metabolic parameters at the end of the recovery period after 30 minutes of ischemia (Table V). Tissue ATP levels at the end of 10 minutes of working heart reperfusion in hearts subjected to adenosine 10 mmol/L were improved by the additional washout of adenosine from 3.3 ± 0.2 to 4.2 ± 0.1 umol/gm wetweight(p <0.001). Taking hearts subjected to the washout procedure (Table V), the ATP levels at the end of reperfusion were depressed by K+ 20 mrnol/L and adenosine 10 mmol/L + K+ 20 mmol/L when compared with adenosine 10 mmol/L alone. PCr levels were low after K+ 20 mrnol/L and adenosine 10 mmol/L + K+ 20 mmol/L when compared with KH
The Journal of
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Thoracic and Cardiovascular Surgery
Schubert et al.
Table V. Energy status of isolated working rat heart after I D-minute reperfusion ATP
Cardioplegic solution
3.1 3.3 4.2 3.8 4.2 3.3
KH Adenosine 10 mmoljL KH washout K+ 20 mmoljL washout Adenosine 10 mmoljL washout Adenosine 10 mmoljL + K+ 20 mmoljL washout
± ± ± ± ±
0.2 0.2 0.1· 0.2 0.1· ± 0.2:1:
4.1 5.6 6.0 4.6 5.4 4.0
PCr
Glycogen
± ± ± ± ± ±
9.4 ± 0.8 12.0 ± 0.9 8.9±1.1 11.8 ± 2.3 10.2 ± 1.1 9.9 ± 0.9
0.4 0.5 0.4· 0.7t 0.2 0.3t
Lactate 5.3 5.0 4.9 4.1 5.2 4.1
± ± ± ± ± ±
0.7 0.8 0.5 1.1 0.4 0.4
Protocol: Thirty minutes of ischemia at l O" C, followed by 10 minutes of Langendorff reperfusion. followed by 10 minutes of working heart reperfusion. Lnits: I'moljgm wet weight; glycogen units I'mol C6 units/grn wet weight (= dry weight X 5). Number of experiments = 6. Washout = Removal of cardioplcgic solution at 2 ml/rnin over 3 minutes at 10" C with Krebs-Henseleit (KH) buffer. .p <0.05. compared with KH. t P <0.05, compared with control KH
+p <0.05, compared with
+ washout. modified t test. + washout. modified
adenosine 10 mmol/L
t
test.
buffer (p <0.05). There was no difference in the ATP and PCr values between the hearts treated with adenosine 10 mmol/L and those receiving KH solution. We could not show a positive correlation between levels of high-energy phosphates and return of contractile function in the recovery period. Discussion Adenosine cardioplegia. The new observation in this paper is that the addition of adenosine to KH solution is superior to the addition of K+ in achieving cardioplegia. Adenosine 10 mmol/L cardioplegia produced a rapid cardiac arrest and improved PCr levels (Tables I and II, Fig. 1). Postischemic hemodynamic recovery was also better after adenosine 10 mmol/L cardioplegia (Table IV). The mechanisms of rapid cardiac arrest after adenosine cardioplegia are most probably due to (1) a dose-dependent increase in K+ permeability that leads to hyperpolarization of the membranes in the atrial and sinus node tissue, causing inhibition of atrial action potential and atrioventricular block 20• 21 and (2) a reduced calcium influx causing a negative inotropic effect. 1-3 Adenosine 10 mmol/L was more effective than adenosine 1 mmol/L in reducing the total number of beats until cardiac arrest. Therefore, recovery studies were undertaken only with the higher adenosine concentration. We also showed that the washout of the cardioplegic solution by cold KH buffer at 10° C improved mechanical recovery (Tables III and V) and removed the risk that adenosine left in the heart could cause persistent atrioventricular block in the early reperfusion period. Preservation of PCr values by adenosine might be explained by the rapid cardiac arrest, or adenosine could slow net degradation of PCr in the absence of oxygen. Because PCr and Cr are exclusively cytoplasmic and the creatine phosphokinase reaction is generally at near equilibrium with the cytoplasmic [ATP]/[ADP]/[Pi] ratio
(where Pi = inorganic phosphate), these data suggest that it is particularly the cytoplasmic energy status that is maintained by adenosine. Kao and Magovern" showed preservation of ATP levels at the end of 60 minutes of ischemia after adenosine had been added to the cardioplegic solution. The ATP values in our study after 3 minutes of cardioplegia were no different in the various groups, perhaps because the cardioplegic time was too short (Table 11). Postischemic PCr values may be a better parameter for the degree of myocardial protection during ischemia, as suggested in previous studies.P Whether ATP levels correlate with the return of contractile function governing mechanical recovery remains a controversial issue.23-26 Our data suggest that the failure of some reperfused hearts to work is not due to the lack of availability of ATP, PCr, or glycogen during the recovery period (Table V). However, conservation of high-energy phosphates during the ischemic period remains an important aim. n , 28 Improved postischemic hemodynamic function has also been shown after adenosine treatment by Ely and associates." However, such previous studies tested the effects of combined adenosine and high K+ cardioplegia. Higb K+ cardioplegia. K+ 20 rnmol/L produced a slow onset of cardiac arrest and low PCr levels (Tables I and II, Fig. 2); postischemic hemodynamic recovery of function was impaired and PCr levels were depressed. Since 1955, when Melrose and associates-' first used high K+ solutions to induce cardiac arrest, many different solutions, infusions, styles, and additives have been developed and are still currently used. During high K+ cardioplegia, there is diastolic arrest-? caused by depolarization of the membranes and a decrease of sodium influx with a change in the action potential duration. 29-31 The K+ concentration used in cardioplegic solution differs from IS to 40 mmoljL. 29 Higher K+ concentrations result in increased high-energy phosphate utilization be-
Volume 98 Number 6
Adenosine cardioplegia
December 1989
cause of an increase of myocardial wall tension and intracellular Ca 2+ accumulation.F''" whereas low concentrations ofK+ may not depolarize the membranes sufficiently to cause complete cardiac arrest. The harmful effects of the initial Melrose solution could be related to excessive K+ concentration, hyperosmolality, and cytotoxicity." The commonly used combination of high K+ plus hypothermia results in a greater energy conservation.Pf However, hypothermia combined with hyperkalemia is not ideal. First, there is still some low-amplitude atrial electrical activity during ischemic arrest. 10. II Second, some K+ concentrations may decrease the level of A TP during the recovery penod.l ' Third, hypothermia with hyperkalemia produced only a relatively slow onset of cardiac arrest in our study. Thus the combination may not give optimal myocardial protection.!" 11.27 Comparison of adenosine cardioplegia withhigh K+ cardioplegia. The comparison of adenosine 10 mmol/L with K+ 20 mmol/L cardioplegia showed (1) a shorter cardiac arrest time (Table I, Fig. 2), (2) preservation of PCr levels (Table II), and (3) improved mechanical recovery after adenosine cardioplegia (Tables III and IV). Thus, as judged by all these parameters, adenosine was a better cardioplegic agent than high K+. Action of adenosine plus high K+ combination. The action of adenosine in the presence of a high external K+ (20 mrnol/L) is probably due to increased K+ permeability causing an outward K+ current and inhibition of the spontaneous depolarization of the sinus node, even in the presence of depolarization by the high K+ solution.P The combination of K+ and adenosine for cardioplegia was as effective as adenosine alone in inducing a rapid cardiac arrest, but the hemodynamic recovery and biochemical status was not improved by the combination of K+ with adenosine 10 mmol/L (Tables III and IV). Possible reasons might be that the membranes are depolarized by high K+ alone, whereas with adenosine they are hyperpolarized; further electrophysiologic studies are required to determine the effects of the combination (K+ and adenosine) on resting membrane potential. The combination could cause incomplete mechanical arrest during ischemia and depressed A TP values during the recovery period, as previously shown. I 0-12 Thus there is some evidence that adenosine alone may be superior to the combination of adenosine and K+. Possible application and reservations. We used two different protocols for assessing cardiac arrest time and recovery because this procedure is similar to clinical situations. Before the human heart is arrested with cardioplegic solution, the aorta is crossclamped and the heart is not in the normal working mode. Therefore we used the Langendorff nonworking perfusion technique as a model
I063
to determine cardiac arrest time. Before coming off cardiopulmonary bypass, the human myocardium is allowed to rewarm and the cardioplegic solution is washed out. Bypass is discontinued only when a certain cardiac output is reached. Thus in our model we chose to reperfuse by a lO-minute Langendorff nonworking perfusion after the ischemic period before switching to the standard working heart preparation. Nonetheless, it must be emphasized that different protocols or the use of other species could have given varying results, so that further work is required to confirm and extend the present results. Clearly, extrapolation of these results to the clinical situation has to be made with great care, especially as bolus injections of high doses of adenosine can cause pain and discomfort. 39. 4o However, the data here presented suggest that adenosine may warrant consideration for testing as a cardioplegic agent, especially because it is an agent already available in intravenous form for the therapy of supraventricular tachycardias."! We emphasize that (1) further testing is required in large animal models, particularly primates, before trials of the use of adenosine in patients become warranted and that (2) this study is limited to a comparison between adenosine and K+ cardioplegia. Additional studies now under way will show whether the addition of adenosine can improve the efficacy of established cardioplegic solutions, such as the St. Thomas' Hospital solution. We gratefully acknowledge the technical assistance of Dr. Mathias Karck, Dr. Dieter Boehm, Christof Schmid, Valerie Tate, and Sedick Isaacs, MSc (statistician), and the secretarial assistanceof June Chambers. REFERENCES 1. Schrader J, Rubio R, Berne RM. Inhibitionof slow action
2. 3.
4.
5. 6.
potentialsof guinea-pigatrial muscle by adenosine: a possibleeffecton Ca2+influx. J Mol Cell Cardiol 1975;7:42733. Stafford A. Potentiationof adenosineand the adenine nucleotides by dipyridamole. Br J Pharmacal Chemother 1966;28:218-27. Drury AN, Szent-Gyorgyi A. The physiological activityof adenine compounds with especial reference to their action upon the mammalian heart. J Physiol (Lond) 1929/ 1930;68:213-37. Jennings RB, Hawkins HK, Lowe LE, Hill MC, Klotman S, Reimer KA. Relation between high-energy phosphate and lethal injury in myocardial ischemiain the dog. Am J PathoI1978;92:187-214. BerneRM, Rubio R. Adeninenucleotide metabolismin the heart. Circ Res 1974;35(suppl 3):109-20. Jennings RB, Steenbergen C Jr. Nucleotide metabolism and cellular damage in myocardial ischemia. Ann Rev Physiol 1985;47:727-49.
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7. Ward HB, Kriett JB, Einzig S, Bianco R W, Anderson R W, Foker JE. Adenine nucleotides and cardiac function following global myocardial ischemia. Surg Forum 1983; 34:264-6. 8. Kao RL, Magovern GJ. Prevention of reperfusional damage from ischemic myocardium. J THORAC CARDIOVASC SURG ;1986;91: 106-14. 9. Ely SW, Mentzer RM, Lasley RD, Lee BK, Berne RM. Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J THORAC CARDIOVASC SURG 1985;90:549-56. 10. Landymore RW, Marble AE, Trillo A, MacAulay M, Faulkner G, Cameron C. Effect of small amplitude electrical activity on myocardial preservation in cold potassiumarrested heart. J THORAC CARDIOVASC SURG 1986;91 :684-
Surgery
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9. 11. Ferguson TB, Smith PK, Lofland G K, Holman WL, Helms MA, Cox JL. The effects of cardioplegic potassium concentration and myocardial temperature on electrical activity in the heart during elective cardioplegic arrest. J THORAC CARDIOVASC SURG 1986;92:755-65. 12. Engelman RM, Dobbs W A, Roussou JH, Lemeshow S. The optimal potassium concentration in a cardioplegic solution. In: Proceedings of a symposium. Royal Society, London. Cardioplegia: the first quarter century. London: 1980:89-90. 13. Hearse DJ, Stewart DA, Chain EB. Recovery from cardiac bypass and elective cardiac arrest: metabolic consequences of various cardioplegic procedures in the isolated rat heart. Circ Res 1974;35:448-57. 14. Bricknell 0, Opie LH. Effects of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion. Circ Res 1978;43: 102-15. 15. Neely JR, Liebermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967;212:804-14. 16. Sellevold OFM, Jynge P, Aarstad K. High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine nucleotides in myocardial tissue. J Mol Cell Cardiol 1986; 18:51727. 17. Lamprecht W, Trautschold I. Adenosine-5' -triphosphate: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer H, ed. Methods of enzymatic analysis. Vo14. London: Academic Press, 1974:210110. 18. Good CA, Kramer H, Somogyi M. The determination of glycogen. J Bioi Chem 1933; I00:485-91. 19. Gutmann I, Wahlefeld A W. L-( +)-Iactate: determination with lactate dehydrogenase and NAD. In: Bergmeyer H, ed. Methods of enzymatic analysis. Vol 3. London: Academic Press, 1974:1464-8. 20. Belardinelli L, West GA. Cardiac electrophysiological effects of adenosine. In: de Jong JW, ed. Cardiac energy metabolism. Dordrecht: Nijhoff, 1988:93-104. 21. Belardinelli L, Giles WR, West A. Ionic mechanisms of
26.
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adenosine actions in pacemaker cells from rabbit heart. J Physiol (Lond) 1988;405:615-33. Rosenkranz ER, Okamoto F, Buckberg GD, et al. Studies of controlled reperfusion after ischemia. II. Biochemical studies: Failure of tissue adenosine triphosphate levels to predict recovery of contractile function after controlled reperfusion. J THORAC CARDIOVASC SURG 1986;92:488501. Melrose DG, Dreyer B, Bentall HH, Baker JBE. Elective cardiac arrest. Lancet 1955;2:21-3. Vary TC, Angelakos ET, Schaffer SW. Relationship between adenine nucleotide metabolism and irreversible ischemic tissue damage in isolated perfused rat heart. Cire Res 1979;45:218-25. Foker JE, Einzig S, Wang T, Anderson RW. Adenosine metabolism and myocardial preservation: consequences of adenosine catabolism on myocardial high-energy compounds and tissue blood flow. J THORAC CARDIOVASC SURG 1980;80:506-16. Taegtmeyer H, Roberts AFC, Raine AEG. Energy metabolism in reperfused heart muscle: metabolic correlates to return of function. J Am Coil Cardiol 1985;6:864-70. Hearse DJ, Braimbridge MV, Jynge P. Protection of the ischemic myocardium: cardioplegia. New York: Raven Press, 1981:209-99. Longmore DB. Towards safer cardiac surgery, ed. I. Boston: G K Hall, 1981. Lell WA, Buttner E. Myocardial preservation during cardiopulmonary bypass. In: Kaplan JA, ed. Cardiac anesthesia. Vol 2. Cardiovascular pharmacology. New York: Grune & Stratton, 1983:525-50. Ettinger PO, Regan TJ, Oldewurtel HA. Hyperkalemia, cardiac conduction and the electrocardiogram: a review. Am Heart J 1974;88 :360-71. Rosen MR, Wit AL, Hoffman BF. Electrophysiology and pharmacology of cardiac arrhythmias. I. Cellular electrophysiology of the mammalian heart. Am Heart J 1974;88:380-5. Hearse DJ, Stewart DA, Braimbridge MV. Hypothermic arrest and potassium arrest: metabolic and myocardial protection during elective cardiac arrest. Circ Res 1975;36:481-9. Isselhard W, Merguet H, Aengenvoort J, et al. Vergleich des Herzstoffwechsels bei verschiedenen Methoden des kunstlichen Herzstillstandes. Pflugers Arch 1965;286:33671. Rich TL, Brady AJ. Potassium contracture and utilization of high-energy phosphates in rabbit heart. Am J Physiol 1974;226:105-13. Tyers GF, Todd GJ, Niebauer 1M, et al. The mechanism of myocardial damage following potassium citrate (Melrose) cardioplegia. Surgery 1975;78:45-53. Flaherty JT, Weisfeldt ML, Hollis DP, et al. Mass spectrometry and phosphorus-31 nuclear magnetic resonance demonstrate additive myocardial protection by potassium cardioplegia and hypothermia during global ischemia. Adv Myocardiol 1980;2:487-99.
Volume 98 Number 6 December 1989
37. Hearse OJ, Stewart DA, Braimbridge MY. The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat. J THORAC CARDIOVASC SURG 1980;79:39-43. 38. Rosenfeldt FL, Hearse OJ, Darracott-Cankovic S, et al. The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. J THORAC CARDIOVASC SURG 1980;79:29-38. 39. Sylven C, Beermann B, Jonzon B, Brandt R. Angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers. Br Med J 1986;293:227-30.
Adenosine cardioplegia 1 0 6 5
40. Sylven C, Jonzon B, Fredholm BB, Kaijser L. Adenosine injection into the brachial artery produces ischaemia like pain or discomfort in the forearm. Cardiovasc Res 1988;22:674-8. 41. DiMarco JP, Sellers TO, Berne RM, West GA, Belardinelli L. Adenosine: electrophysiologic effects and therapeutic use for terminating paroxysmal supraventricular tachycardia. Circulation 1983;68: 1254-63.
Bound volumes available to subscribers Bound volumes of THE JOURl'AL OFTHORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) for the 1989 issues from the Publisher, at a cost of$55.00 ($72.00 international) for Vol. 97 (January-June) and Vol. 98 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue of the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C.V. Mosby Company, Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
CARDIOVASC SURG 1989;98: 1057-65
Adenosine cardioplegia Adenosine versus potassium cardioplegia: Effects on cardiac arrest and postischemic recovery in the isolated rat heart Adenosine is a potential cardioplegic agent by virtue of its specific inhibitory properties on nodal tissue. We tested the hypothesis that adenosine could be more effective than potassium in inducing rapid cardiac arrest and enhancing postischemic hemodynamic recovery. Isolated rat hearts were perfused with Krebs-Henseleit buffer or cardioplegic solutions to determine the time to cardiac arrest and the high-energy phosphate levels at the end of cardioplegia. Cardioplegic solutions contained adenosine 10 mmol/L, potassium 20 mmol/L, or adenosine 10 mmol/L + potassium 20 mmol/L and were infused at a rate of 2 ml/min for 3 minutes at 10° C. Both time taken and total number of beats to cardiac arrest during 3 minutes of cardioplegia were reduced by adenosine 10 mmol/L and adenosine 10 mmol/L + potassium 20 mmol/L when compared with potassium 20 mmol/L alone (p < 0.001). Tissue phosphocreatine was conserved by adenosine 10 mmol/L when compared with potassium 20 mmol/L, being 7.1 ± 0.2 (JLmol/gm wet weight (n = 7) and 6.0 ± 0.3 JLmol/gm wet weight (n = 5~ respectively (p < 0.05). Postischemic hemodynamic recovery was tested in isolated working rat hearts. After initial cardiac arrest, the cardioplegic solution was removed with Krebs-Henseleit buffer at a rate of 2 m1/min for 3 minutes at 10° C, and thereafter total ischemia was maintained for 30 or 90 minutes at 10° C before reperfusion. Adenosine 10 mmol/L enhanced recovery of aortic output when compared with potassium 20 mmol/L or adenosine 10 mmol/L + potassium 20 mmoljL, the percentage recovery after 30 minutes of ischemia being 103.0% ± 4.4% (n = ~ 89.0% ± 5.8% (n = 6~ and 86.6% ± 4.3% (n = ~ respectively (p < 0.05 for comparison between adenosine 10 mmoljL and potassium 20 mmol/L), Thus adenosine cardioplegia caused rapid cardiac arrest and improved postischemic recovery when compared with potassium cardioplegia and with a combination of these two agents.
Thorsten Schubert, MD, Herbert Vetter, MD,a Patricia Owen, PhD, Bruno Reichart, MD,a and Lionel H. Opie, MD, PhD, Cape Town, Republic of South Africa
AdenOSine, by its property of antagonizing cardiac calcium channels, inhibits the sinoatrial node, the atrioventricular node, and myocardial contraction, thereby potentially inducing cardiac arrest.l' During ischemia, high-energy phosphates are degraded to adenosine, inosine, hypoxanthine, and uric acid,4-6 and the adenosine
From the Heart Research Unit. Departments of Medicine and Cardiothoracic Surgery! University of Cape Town and Groote Schuur Hospital. Cape Town. Republic of South Africa. Revision received for publication Oct. 31. 1988. Accepted for publication March 2. 1989. Address for reprints: Professor L. H. Opie. Heart Research Unit, University of Cape Town. Medical School. Observatory. 7925, South Africa.
12/1/13098
triphosphate (ATP) levels may be depressed for days after recovery from ischemia.' Earlier studies have shown the benefits of adenosine added to other cardioplegic solutions.t including a solution with a high potassium (K+) concentration." At the same time, there are doubts about the benefits of high K+ cardioplegic solutions. 10-12 We examined the individual and additive potential benefits of adenosine cardioplegia and sought to answer the following questions: I. How rapidly and at what concentration can adenosine arrest the heart when compared with high K+? 2. Was the recovery of adenosine-treated hearts after different ischemic times better than that of K+ -arrested hearts? . 3. Was the combination of adenosine and high K+ concentration beneficial for the heart? 1057
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A.
B.
10 min
3 min
Langendorff Perfusion
cp
10 min
10 min
L-dorff
WH
3'
t Freeze clamp for tissue analysis
3' 30/90 min
I
l
CP WO Ischemia
I
10 min L-dorff
10 min
WH
I
Freeze-clamp for tissue analysis
Fig. 1. Perfusion sequences, perfusion mode, and time division for each perfusion mode. A, Protocol for the effects of different cardioplegic solutions in Langendorff-perfused hearts. B, Protocol for studying hemodynamic recovery in the working heart. L-dorff, Langendorff perfusion; WHo working heart perfusion; CPo cardioplegia at 2 ml/rnin for 3 minutes; WOo washout at 2 ml /min for 3 minutes with KH buffer at 10° C.
Methods Our model was the isolated perfused rat heart, which has also been used in other cardioplegia studies." 9. 13 All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Male Long Evans rats weighing 270 to 330 gm were anesthetized with ether and given 200 units of heparin into the femoral vein. The heart (average fresh weight 1.0 gm) was rapidly removed and plunged into ice-cold perfusion medium before being mounted on an aortic cannula for isolated perfusion. Perfusion techniques. We used two different perfusion techniques: (I) the Langendorff aortic technique for determination of cardiac arrest time and high-energy compounds (after 3 minutes of cardioplegia) and (2) the working heart preparation for studying both the cardiac arrest time and the hemodynamics in the postischemic recovery period (Fig. I). Langendor.lftechnique. The heart was mounted via the aorta on a modified Langendorff perfusion apparatus and was perfused retrogradely with a modified Krebs-Henseleit (KH) buffer containing NaHC0 3 25 mrnol/L, NaCI 118 rnmol/L, KH 2P04 1.2 mrnol/L, KC14.8 mmol/L, MgS0 4 1.2 mmol/L, CaCI 2 1.25 mrnol/L, and glucose 11.1 mrnol/L. The solution was gassed with 95% oxygen and 5% carbon dioxide and maintained at 3]0 C. The heart was perfused at a pressure of 100 em H 20. The apical force displacement technique'" was used to measure contraction with a Grass model 79-D recorder (Grass Instrument Company, Quincy, Mass.), PROTOCOL After a stabilization period of 10 minutes, cardiac arrest was induced by introduction of different cardioplegic solutions through a side arm of the aortic cannula at a rate of 2 ml/rnin for 3 minutes. The cardioplegic solution and the water jacket surrounding the heart during cardioplegia were kept at 10° C by a separate water circuit. The following mechanical parameters were assessed over the 3-minute period of induction of cardioplegia: arrest time (seconds), number of arrest beats,
number of escape beats, and total number of beats. Then the heart was freeze-clamped for determination of phosphocreatine (PCr), creatine (Cr), and adenosine triphosphate (A TP), diphosphate (ADP), and monophosphate (AMP). Working heart technique. The working heart perfusion technique of Neely and associates I 5 allows determination of hemodynamic parameters while the heart is perfused via the left atrium (preload) and ejects the cardiac output against a pressure load (afterload). The aorta was cannulated and the heart was perfused at 100 em H 20 pressure. During the initial Langendorff (aortic) perfusion, the left atrium was cannulated and the pulmonary artery incised. The retrograde perfusion was stopped by supplying perfusate to the left atrium at a pressure of 20 em H 20. The left ventricle then ejected the cardiac output (aortic and coronary flow) against a pressure of 100 em H20. The cardioplegic solutions and the washout (KH) were infused through a side arm of the aortic cannula. The aortic pressure was measured with a Statham P23dB pressure transducer (Spectramed Inc., Critical Care Division, Oxnard, Calif.) on a second side arm of the aortic cannula. An electrocardiographic electrode was attached to the right ventricle. The electrocardiogram and pressure were monitored on a Devices twinchannel recorder (Mx 2P-148, Devices for Science. Inc., Redwood City, Calif.). PROTOCOL After a stabilization period of 10 minutes, the Langendorff perfusion was converted to the working heart model (left atrial perfusion) for another 10 minutes (Fig. 1). Cardiac arrest was then induced by infusing cardioplegic solution at 2 ml/rnin for 3 minutes at 10° C. The cardioplegic solution was washed out of the heart with KH buffer at a rate of 2 rnl/rnin for 3 minutes at lOG C via the same cannula. In two additional groups, KH and adenosine 10 mrnol/L, the cardioplegic solution was not washed out of the heart and remained for the duration of the ischemic period. Total ischemia was then maintained at 10° C for 30 or 90 minutes. During cardioplegia and ischemia the heart was surrounded by a water-jacketed chamber maintained at 10° C. After ischemia the heart was rewarmed by retrograde perfusion of KH buffer at 37° C for 10 minutes. Then the system was switched to a working prepara-
Volume 98 Number 6
Adenosine cardioplegia
December 1989
10 5 9
Table I. Effect of different cardioplegic solutions on cardiac arrest time, number of arrest beats, number of escape beats, and total number of beats during cardioplegia Cardioplegic solution
'0.
Time to cardiac arrest (sec)
A. Langendorff perfused rat hearts K+ 20 mrnol/L Adenosine 10 rnmol/L Adenosine 1 mrnol/L Adenosine 10 mrnol/L + K+ 20 mmol/L Adenosine 1 mrnol/L + K+ 20 mmol/L B. Working rat hearts KH K+ 20 rnrnol/L Adenosine 10 mrnol/L
52 ± 1.6 ± 2.1 ± 1.9 ± 9.0 ±
2.0 0.3* 0.6* 0.2* 5.0*
446 ± 39 63 ± 12 7 ± It
No. of arrest beats
168 ± 7::':: 6± 9± 16 ±
9 1* 1* 2' 8*
640 ± 107 125 ± 27 15 ± 2t
No. of escape beats
Total No. of beats during cardioplegia
0 3 ± 1* 11 ± 2* 4 ± 1* 15 ± 7*
168 ± 8± 17 ± 13 ± 31 ±
38 ::':: 9 37 ± 24 31 ± 7
678±115 162 ± 3 46 ± 5t
9 2* 2* 3' 7*
or experiments = 5 to 7 in each group. KH. Krebs-Hensclcit buffer
'Compared with K + :'0 mmol/ L: f' <0.00 I. modified { test.
+Compared with K + :'0 mmol . L: P <0.05 { test.
tion and mechanical parameters were measured over a 10minute period, at the end of which the heart was freeze-clamped for biochemical analysis. The following parameters were measured: aortic output. coronary flow, heart rate, systolic and diastolic pressure, PCr, ATP, lactate and glycogen. Perfusion solutions. The different cardioplegic solutions contained KH buffer plus: 1. Adenosine 10 mmoljL 2. Adenosine I mmoljL 3. K+ 20 mmoljL 4. Adenosine 10 mmoljL + K+ 20 mmoljL 5. Adenosine I mmoljL + K+ 20 mmoljL 6. Control, standard KH buffer These solutions were tested at 10° C by a single infusion technique. Tissue processing and analysis. Content of high-energy compounds was determined either by high-performance liquid chromatography" or enzymatically.'? Glycogen and lactate were determined cnzyrnatically.P'"? Calculations. The following calculations were made: I. Cardiac arrest time: time (seconds) from the onset of infusion of cardioplegic solution until the heart arrests for 2 seconds 2. Arrest beats: number of heart beats until 2 seconds of arrest 3. Escape beats: beats coming through after initial arrest 4. Total number of beats during 3 minutes of cardioplegic perfusion: number of arrest beats plus escape beats 5. Percentage recovery of aortic output (AO): AO (lor 10 min reperfusion) 00 AO (lor 10 min before ischemia) X I
6. Percentage recovery of systolic pressure X heart rate: (SP 2 X HR 2) (SP I X HR t )
X 100
7. Percentage recovery of pulse pressure X heart rate: ([SP 2 - DP 2] X HR 2 ) ([SP I - DPd X HR I )
X 100
where SP = systolic pressure, DP = diastolic pressure. and HR = heart rate (I = preischemic values, 2 = reperfusion values). Statistics and expansion of data. The results are presented as mean and standard error of the mean. Overall significance of differences between groups was determined by a one-way analysis of variance followed by a modified Student's t test. When only two subgroups were compared, we used the t test with a Bonferroni correction. A p value of less than 0.05 was considered significant. Metabolic data are expressed in terms of wet weight (= dry weight X 5).
Results Effect of adenosine on cardiac arrest time, number of beats to cardiac arrest, and escape beats (Table I, A and B, Fig. 2). In Langendorff-perfused hearts (Table I, A), the time to cardiac arrest, the number of arrest beats, and the total number of beats during the 3-minute cardioplegic period were reduced by all cardioplegic solutions containing adenosine when compared with K+ 20 mmol/L alone. The time to cardiac arrest was reduced from 52 seconds with K+ 20 mmol/L to less than 3 seconds in hearts perfused with solutions containing adenosine 10 mmol/L, adenosine 1 mrnol/L, and adenosine 10 mmol/L + K+ 20 mmol/L and to 9 ± 5 seconds for hearts receiving adenosine I mrnol/L + K+ 20 mrnol/L (p < 0.001 for all comparisons). The number of arrest beats was reduced from 168 ± 9 during K+ 20 mmol/L cardioplegia to less than 16 beats in all groups (p < 0.001 for all comparisons). A difference was observed in the number of escape beats after initial cardiac arrest. Hearts receiving K+ 20 mrnol/L had no escape beats. Solutions containing adenosine 10 mmol/L allowed 3 to 4 escape beats, whereas those containing adenosine 1 rnrnol/L allowed 11 to 15 escape beats (adenosine 10 mmol/L versus adenosine I mmol/L,p < 0.05). Adenosine 10 mmol/ L reduced the total number of beats more than adenosine
The Journal of
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Thoracic and Cardiovascular
Schubert et al.
Surgery
A. POTASSIUM 20mM
IHIIHl\H\\\\P\l:I\\IIHHPlql'\! ~ I j II
1
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:"~\\\:\\,,~,,,I~~'~':~~~'~~~~~~\\:i~~\:\~,~,~,~~<'~<"~" '~'.', (\,,__.:~'~,~, ~,~,,~,~~.~ ~,~,~~ J.J.:. ~~, . -. . ", ", .:.»:v
cardioplegia onset
escape beat
_
B. ADENOSINE 10mM
o
20
10
40
30
50
60
Time (sec)
Fig. 2. A, Trace of left ventricular contraction measured by apical displacement for. arrest by K+ 20 rnrnol/L. B, Trace for arrest by adenosine 10 mrnol/L.
Table II. High-energy phosphates after 3 minutes of cardioplegia in Langendor./f-perfused hearts Cr
PCr
Cardioplegic solution
Pre-cardioplegia K+ 20 mmoljL Adenosine 10 mmoljL Adenosine I mmoljL Adenosine 10 mmoljL + K+ 20 mmoljL Adenosine I mmoljL + K+ 20 mmoljL
6.6 6.0 7.1 7.5 6.5 5.9
± 0.3 ± 0.3 ±0.2* ± 0.7* ± 0.2 ± 0.1
4.3 5.2 3.6 5.6 4.2 6.1
± ± ± ± ± ±
ATP
0.3 0.3 0.2 1.1 0.4 0.4
4.1 4.2 4.0 4.5 3.8 4.2
± ± ± ± ± ±
0.2 0.1 0.1 0.2 0.1 0.1
ADP
0.9 ± 0.9 ± 0.8 ± 1.1 ± 0.8 ± 1.0 ±
0.05 0.04 0.03
o.n0.02 O.03t
AMP
0.07 0.07 0.08 0.11 0.05 0.09
± ± ± ± ± ±
0.01 0.01 0.01 0.02
am 0.01
PCrjCr ratio
1.6 ± 1.2 ± 2.0 ± 1.6 ± 1.6 ± 1.1 ±
0.2 0.1 0.1* 0.3 0.2 0.2
A TPjADP ratio
4.7 ± 0.3 4.9 ± 0.2 5.0 ± 0.2 4.4 ± 0.3 5.0 ± 0.1 4.2±0.lt
Units: jlmoljgm wet weight. Number of experiments = 5 to 7. •P <0.05, modified t test compared with K+ 20 rnrnol/ L
tp <0.05, modified t test compared with adenosine 10 mmoljL.
1 mmol/L (p < 0.05). In the working heart preparation (Table I, B), adenosine 10 mrnol/L cardioplegia showed similar results, namely a reduced time to cardiac arrest and fewer total beats when compared with K+ 20 mmol/ L cardioplegia (p < 0.05), Thus adenosine was more effective than K+ in inducing electromechanical arrest in both the nonworking and working heart. Effect of adenosine on high-energy phosphate levels after cardioplegia (Table II), At the end of 3 minutes of cardioplegia, tissue PCr was preserved by adenosine 10 mrnol/L and 1 mmol/L (p < 0,05 compared with K+ 20 mmol/L). Replacement of K+ 20 mrnol/L with adenosine 10 mmol/L increased the ratio PCr/Cr. The ATP/ ADP ratio fell with adenosine 1 mmol/L + K+ 20 rnmol/ L when compared with adenosine 10 mmol/L (p <0,05). Effect of adenosine cardioplegia on hemodynamic recovery after 30 and 90 minutes of ischemia (Tables III and IV). We showed that, when the adenosine 10 mmol/L solution remained in the heart during ischemia, the effect was not beneficial because the percentage recovery of the aortic output after 10 minutes of working heart reperfusion was no different from that of KH-
arrested hearts, being 64.6% ± 15.9% and 49.4% ± 14.2%, respectively (Table III), If, however, the cardioplegic solution had been removed from the heart with KH buffer before total ischemia, the percentage recovery of aortic output was increased in the case of adenosine from 65% to 103% (p < 0.01, t test) and for the KHtreated hearts from 49% to 85% (p < 0.01, t test). In hearts subjected to 30 minutes of ischemia, adenosine 10 mmol/L cardioplegia showed improved recovery of aortic output, being 91% at 1 minute and 103% at 10 minutes, in comparison with K+ 20 mrnol/L (p < 0.05 for both comparisons), The combination of adenosine 10 mmol/L + K+ 20 mmol/L improved aortic output after 1 minute but not after 10 minutes. No difference was observed between hearts treated with K+ 20 mmol/L and those receiving KH buffer in recovery of aortic output after 30 minutes of ischemia with washout (Table Ill). Hearts subjected to adenosine 10 mmol/ L cardioplegia and 90 minutes of ischemia showed improved aortic output at 1 minute and at 10 minutes of working heart reperfusion when compared with hearts receiving K+ 20 mmol/L (p < 0.05). The percentage recovery of systolic
Volume 98 Number 6
Adenosine cardioplegia
December 1989
1 06 1
Table III. Mechanical recovery of working rat hearts after 30 minutes of ischemia -------------------------------------~--~--
Percentage recovery of heart rate X systolic pressure
Percentage recovery of aortic output
Percentage recovery of pulse pressure X heart rate
Time of reperfusion work Cardioplegic solution
KH Adenosine 10 mmoljL KH washout K+ 20 mmoJjL washout Adenosine 10 mmoljL washout Adenosine 10 mmoljL + K+ 20 mmoljL washout
1 min
65.4 ± 50.9± 83.4 ± 80.2 ± 91.1 ± 93.4 ±
10 min
14.6 14.7 2.5* 4.1 4.6*
49.4 ± 64.6 ± 84.6 ± 89.0 ± 103.0 ± 86.6 ±
:nt
10 min
14.2 15.9 3.0* 5.8 4.4* 4.3
72.6 ± 72.7 ± 95.7 ± 97.1 ± 106.8 ± 102.5 ±
18.8 17.1 3.2 6.8 4.4 4.5
10 min
45.0 71.4 87.6 93.5 98.0 96.1
± ± ± ± ± ±
11.2 16.3 5.4 15.5 8.4 9.2
Protocol: Thirty minutes of ischemia at 10 C followed by 10 minutes of LangendortT reperfusion before onset of working heart rcperfusion. :-':umb"r of experiments = 6. Washout = Removal of cardioplegic solution at 2 ml /rnin over 3 minutes at 10' C with Krebs-Henseleit (KH) buffer.
*1' <0.01.
I
test. compared nonwashout with washout.
tp <0.05. modified t test. compared with K+ 20 mrnol/L.
Table IV. Effect of different cardioplegic solutions on the mechanical recovery of isolated working rat hearts after 90 minutes of ischemia Percentage recovery of systolic pressure X heart rate
Percentage recovery of aortic output
Percentage recovery of pulse pressure X heart rate
Time of reperfusion work Cardioplegic solution
KH K+ 20 mmoljL Adenosine 10 mrnol/L Adenosine 10 mmoljL + K+ 20 mmoljL
1 min
58.7 73.5 89.0 76.6
± 4.2 ± 2.8 ± 3.8* ± 4.8
10 min
48.0 75.1 83.9 70.6
± 1.4 ± 2.5 ± 1.3* ± 3.9
10 min
78.3 88.0 92.7 88.7
± 4.2
± 4.4 ± 2.5t ± 3.0
10 min
68.8 84.2 85.5 79.0
± 7.2 ± 6.1
± 4.1t ± 4.7
Protocol: "inety minutes of ischemia at 10 C followed by 10 minutes of Langendorff reperfusion before onset of working heart rcpcrfusion. "umber of experiments = 6. All were done with washout of the cardioplegic solution. KH. Krebs-Henseleit buffer
'r
<0.05 differences with modified t test when compared with K+ 20 rnmol/L,
t p <0.05 modified
t
test compared with KH.
pressure X heart rate was also better when compared with that of KH-treated hearts (p < 0.05). There was no difference between hearts receiving K+ 20 mmol/L and those receiving KH buffer in percentage recovery of systolic pressure X heart rate after 90 minutes of ischemia (Table IV). No other groups of measurements showed improvement when compared with K+ 20 mmol/L. The presence of K+ 20 mmol/L with orwithout adenosine impaired mechanical function in the recovery period (Table IV). Coronary flow during the working recovery period after either 30 or 90 minutes of ischemia showed no difference when compared with preischemic values, even after adenosine had been included in the cardioplegic solution. Examples of precardioplegic and postischemic working heart values for coronary flow after 90 minutes of ischemia are as follows: (1) K + cardioplegia, 19.6 ± 0.7 and 19.4 ± 0.9 ml/gm/rnin (n = 6); (2)
adenosine cardioplegia, 18.2 ± 0.9 and 18.2 ± 0.7 ml/ gm/rnin (n = 6); and (3) adenosine + K+ cardioplegia, 17.8 ± 0.7 and 16.3 ± 1.0 ml/gm/rnin (n = 6) (precardioplegic and postischemic values, respectively). Effect of washout of adenosine on metabolic parameters at the end of the recovery period after 30 minutes of ischemia (Table V). Tissue ATP levels at the end of 10 minutes of working heart reperfusion in hearts subjected to adenosine 10 mmol/L were improved by the additional washout of adenosine from 3.3 ± 0.2 to 4.2 ± 0.1 umol/gm wetweight(p <0.001). Taking hearts subjected to the washout procedure (Table V), the ATP levels at the end of reperfusion were depressed by K+ 20 mrnol/L and adenosine 10 mmol/L + K+ 20 mmol/L when compared with adenosine 10 mmol/L alone. PCr levels were low after K+ 20 mrnol/L and adenosine 10 mmol/L + K+ 20 mmol/L when compared with KH
The Journal of
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Thoracic and Cardiovascular Surgery
Schubert et al.
Table V. Energy status of isolated working rat heart after I D-minute reperfusion ATP
Cardioplegic solution
3.1 3.3 4.2 3.8 4.2 3.3
KH Adenosine 10 mmoljL KH washout K+ 20 mmoljL washout Adenosine 10 mmoljL washout Adenosine 10 mmoljL + K+ 20 mmoljL washout
± ± ± ± ±
0.2 0.2 0.1· 0.2 0.1· ± 0.2:1:
4.1 5.6 6.0 4.6 5.4 4.0
PCr
Glycogen
± ± ± ± ± ±
9.4 ± 0.8 12.0 ± 0.9 8.9±1.1 11.8 ± 2.3 10.2 ± 1.1 9.9 ± 0.9
0.4 0.5 0.4· 0.7t 0.2 0.3t
Lactate 5.3 5.0 4.9 4.1 5.2 4.1
± ± ± ± ± ±
0.7 0.8 0.5 1.1 0.4 0.4
Protocol: Thirty minutes of ischemia at l O" C, followed by 10 minutes of Langendorff reperfusion. followed by 10 minutes of working heart reperfusion. Lnits: I'moljgm wet weight; glycogen units I'mol C6 units/grn wet weight (= dry weight X 5). Number of experiments = 6. Washout = Removal of cardioplcgic solution at 2 ml/rnin over 3 minutes at 10" C with Krebs-Henseleit (KH) buffer. .p <0.05. compared with KH. t P <0.05, compared with control KH
+p <0.05, compared with
+ washout. modified t test. + washout. modified
adenosine 10 mmol/L
t
test.
buffer (p <0.05). There was no difference in the ATP and PCr values between the hearts treated with adenosine 10 mmol/L and those receiving KH solution. We could not show a positive correlation between levels of high-energy phosphates and return of contractile function in the recovery period. Discussion Adenosine cardioplegia. The new observation in this paper is that the addition of adenosine to KH solution is superior to the addition of K+ in achieving cardioplegia. Adenosine 10 mmol/L cardioplegia produced a rapid cardiac arrest and improved PCr levels (Tables I and II, Fig. 1). Postischemic hemodynamic recovery was also better after adenosine 10 mmol/L cardioplegia (Table IV). The mechanisms of rapid cardiac arrest after adenosine cardioplegia are most probably due to (1) a dose-dependent increase in K+ permeability that leads to hyperpolarization of the membranes in the atrial and sinus node tissue, causing inhibition of atrial action potential and atrioventricular block 20• 21 and (2) a reduced calcium influx causing a negative inotropic effect. 1-3 Adenosine 10 mmol/L was more effective than adenosine 1 mmol/L in reducing the total number of beats until cardiac arrest. Therefore, recovery studies were undertaken only with the higher adenosine concentration. We also showed that the washout of the cardioplegic solution by cold KH buffer at 10° C improved mechanical recovery (Tables III and V) and removed the risk that adenosine left in the heart could cause persistent atrioventricular block in the early reperfusion period. Preservation of PCr values by adenosine might be explained by the rapid cardiac arrest, or adenosine could slow net degradation of PCr in the absence of oxygen. Because PCr and Cr are exclusively cytoplasmic and the creatine phosphokinase reaction is generally at near equilibrium with the cytoplasmic [ATP]/[ADP]/[Pi] ratio
(where Pi = inorganic phosphate), these data suggest that it is particularly the cytoplasmic energy status that is maintained by adenosine. Kao and Magovern" showed preservation of ATP levels at the end of 60 minutes of ischemia after adenosine had been added to the cardioplegic solution. The ATP values in our study after 3 minutes of cardioplegia were no different in the various groups, perhaps because the cardioplegic time was too short (Table 11). Postischemic PCr values may be a better parameter for the degree of myocardial protection during ischemia, as suggested in previous studies.P Whether ATP levels correlate with the return of contractile function governing mechanical recovery remains a controversial issue.23-26 Our data suggest that the failure of some reperfused hearts to work is not due to the lack of availability of ATP, PCr, or glycogen during the recovery period (Table V). However, conservation of high-energy phosphates during the ischemic period remains an important aim. n , 28 Improved postischemic hemodynamic function has also been shown after adenosine treatment by Ely and associates." However, such previous studies tested the effects of combined adenosine and high K+ cardioplegia. Higb K+ cardioplegia. K+ 20 rnmol/L produced a slow onset of cardiac arrest and low PCr levels (Tables I and II, Fig. 2); postischemic hemodynamic recovery of function was impaired and PCr levels were depressed. Since 1955, when Melrose and associates-' first used high K+ solutions to induce cardiac arrest, many different solutions, infusions, styles, and additives have been developed and are still currently used. During high K+ cardioplegia, there is diastolic arrest-? caused by depolarization of the membranes and a decrease of sodium influx with a change in the action potential duration. 29-31 The K+ concentration used in cardioplegic solution differs from IS to 40 mmoljL. 29 Higher K+ concentrations result in increased high-energy phosphate utilization be-
Volume 98 Number 6
Adenosine cardioplegia
December 1989
cause of an increase of myocardial wall tension and intracellular Ca 2+ accumulation.F''" whereas low concentrations ofK+ may not depolarize the membranes sufficiently to cause complete cardiac arrest. The harmful effects of the initial Melrose solution could be related to excessive K+ concentration, hyperosmolality, and cytotoxicity." The commonly used combination of high K+ plus hypothermia results in a greater energy conservation.Pf However, hypothermia combined with hyperkalemia is not ideal. First, there is still some low-amplitude atrial electrical activity during ischemic arrest. 10. II Second, some K+ concentrations may decrease the level of A TP during the recovery penod.l ' Third, hypothermia with hyperkalemia produced only a relatively slow onset of cardiac arrest in our study. Thus the combination may not give optimal myocardial protection.!" 11.27 Comparison of adenosine cardioplegia withhigh K+ cardioplegia. The comparison of adenosine 10 mmol/L with K+ 20 mmol/L cardioplegia showed (1) a shorter cardiac arrest time (Table I, Fig. 2), (2) preservation of PCr levels (Table II), and (3) improved mechanical recovery after adenosine cardioplegia (Tables III and IV). Thus, as judged by all these parameters, adenosine was a better cardioplegic agent than high K+. Action of adenosine plus high K+ combination. The action of adenosine in the presence of a high external K+ (20 mrnol/L) is probably due to increased K+ permeability causing an outward K+ current and inhibition of the spontaneous depolarization of the sinus node, even in the presence of depolarization by the high K+ solution.P The combination of K+ and adenosine for cardioplegia was as effective as adenosine alone in inducing a rapid cardiac arrest, but the hemodynamic recovery and biochemical status was not improved by the combination of K+ with adenosine 10 mmol/L (Tables III and IV). Possible reasons might be that the membranes are depolarized by high K+ alone, whereas with adenosine they are hyperpolarized; further electrophysiologic studies are required to determine the effects of the combination (K+ and adenosine) on resting membrane potential. The combination could cause incomplete mechanical arrest during ischemia and depressed A TP values during the recovery period, as previously shown. I 0-12 Thus there is some evidence that adenosine alone may be superior to the combination of adenosine and K+. Possible application and reservations. We used two different protocols for assessing cardiac arrest time and recovery because this procedure is similar to clinical situations. Before the human heart is arrested with cardioplegic solution, the aorta is crossclamped and the heart is not in the normal working mode. Therefore we used the Langendorff nonworking perfusion technique as a model
I063
to determine cardiac arrest time. Before coming off cardiopulmonary bypass, the human myocardium is allowed to rewarm and the cardioplegic solution is washed out. Bypass is discontinued only when a certain cardiac output is reached. Thus in our model we chose to reperfuse by a lO-minute Langendorff nonworking perfusion after the ischemic period before switching to the standard working heart preparation. Nonetheless, it must be emphasized that different protocols or the use of other species could have given varying results, so that further work is required to confirm and extend the present results. Clearly, extrapolation of these results to the clinical situation has to be made with great care, especially as bolus injections of high doses of adenosine can cause pain and discomfort. 39. 4o However, the data here presented suggest that adenosine may warrant consideration for testing as a cardioplegic agent, especially because it is an agent already available in intravenous form for the therapy of supraventricular tachycardias."! We emphasize that (1) further testing is required in large animal models, particularly primates, before trials of the use of adenosine in patients become warranted and that (2) this study is limited to a comparison between adenosine and K+ cardioplegia. Additional studies now under way will show whether the addition of adenosine can improve the efficacy of established cardioplegic solutions, such as the St. Thomas' Hospital solution. We gratefully acknowledge the technical assistance of Dr. Mathias Karck, Dr. Dieter Boehm, Christof Schmid, Valerie Tate, and Sedick Isaacs, MSc (statistician), and the secretarial assistanceof June Chambers. REFERENCES 1. Schrader J, Rubio R, Berne RM. Inhibitionof slow action
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potentialsof guinea-pigatrial muscle by adenosine: a possibleeffecton Ca2+influx. J Mol Cell Cardiol 1975;7:42733. Stafford A. Potentiationof adenosineand the adenine nucleotides by dipyridamole. Br J Pharmacal Chemother 1966;28:218-27. Drury AN, Szent-Gyorgyi A. The physiological activityof adenine compounds with especial reference to their action upon the mammalian heart. J Physiol (Lond) 1929/ 1930;68:213-37. Jennings RB, Hawkins HK, Lowe LE, Hill MC, Klotman S, Reimer KA. Relation between high-energy phosphate and lethal injury in myocardial ischemiain the dog. Am J PathoI1978;92:187-214. BerneRM, Rubio R. Adeninenucleotide metabolismin the heart. Circ Res 1974;35(suppl 3):109-20. Jennings RB, Steenbergen C Jr. Nucleotide metabolism and cellular damage in myocardial ischemia. Ann Rev Physiol 1985;47:727-49.
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7. Ward HB, Kriett JB, Einzig S, Bianco R W, Anderson R W, Foker JE. Adenine nucleotides and cardiac function following global myocardial ischemia. Surg Forum 1983; 34:264-6. 8. Kao RL, Magovern GJ. Prevention of reperfusional damage from ischemic myocardium. J THORAC CARDIOVASC SURG ;1986;91: 106-14. 9. Ely SW, Mentzer RM, Lasley RD, Lee BK, Berne RM. Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J THORAC CARDIOVASC SURG 1985;90:549-56. 10. Landymore RW, Marble AE, Trillo A, MacAulay M, Faulkner G, Cameron C. Effect of small amplitude electrical activity on myocardial preservation in cold potassiumarrested heart. J THORAC CARDIOVASC SURG 1986;91 :684-
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9. 11. Ferguson TB, Smith PK, Lofland G K, Holman WL, Helms MA, Cox JL. The effects of cardioplegic potassium concentration and myocardial temperature on electrical activity in the heart during elective cardioplegic arrest. J THORAC CARDIOVASC SURG 1986;92:755-65. 12. Engelman RM, Dobbs W A, Roussou JH, Lemeshow S. The optimal potassium concentration in a cardioplegic solution. In: Proceedings of a symposium. Royal Society, London. Cardioplegia: the first quarter century. London: 1980:89-90. 13. Hearse DJ, Stewart DA, Chain EB. Recovery from cardiac bypass and elective cardiac arrest: metabolic consequences of various cardioplegic procedures in the isolated rat heart. Circ Res 1974;35:448-57. 14. Bricknell 0, Opie LH. Effects of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion. Circ Res 1978;43: 102-15. 15. Neely JR, Liebermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967;212:804-14. 16. Sellevold OFM, Jynge P, Aarstad K. High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine nucleotides in myocardial tissue. J Mol Cell Cardiol 1986; 18:51727. 17. Lamprecht W, Trautschold I. Adenosine-5' -triphosphate: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer H, ed. Methods of enzymatic analysis. Vo14. London: Academic Press, 1974:210110. 18. Good CA, Kramer H, Somogyi M. The determination of glycogen. J Bioi Chem 1933; I00:485-91. 19. Gutmann I, Wahlefeld A W. L-( +)-Iactate: determination with lactate dehydrogenase and NAD. In: Bergmeyer H, ed. Methods of enzymatic analysis. Vol 3. London: Academic Press, 1974:1464-8. 20. Belardinelli L, West GA. Cardiac electrophysiological effects of adenosine. In: de Jong JW, ed. Cardiac energy metabolism. Dordrecht: Nijhoff, 1988:93-104. 21. Belardinelli L, Giles WR, West A. Ionic mechanisms of
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37. Hearse OJ, Stewart DA, Braimbridge MY. The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the rat. J THORAC CARDIOVASC SURG 1980;79:39-43. 38. Rosenfeldt FL, Hearse OJ, Darracott-Cankovic S, et al. The additive protective effects of hypothermia and chemical cardioplegia during ischemic cardiac arrest in the dog. J THORAC CARDIOVASC SURG 1980;79:29-38. 39. Sylven C, Beermann B, Jonzon B, Brandt R. Angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers. Br Med J 1986;293:227-30.
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40. Sylven C, Jonzon B, Fredholm BB, Kaijser L. Adenosine injection into the brachial artery produces ischaemia like pain or discomfort in the forearm. Cardiovasc Res 1988;22:674-8. 41. DiMarco JP, Sellers TO, Berne RM, West GA, Belardinelli L. Adenosine: electrophysiologic effects and therapeutic use for terminating paroxysmal supraventricular tachycardia. Circulation 1983;68: 1254-63.
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