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Cardiac function and myocardial energy level after prolonged hypothermic storage
Cardiac Function and Myocardial Energy Level After Prolonged Hypothermic Storage K. Ku and H. Oku
N
UMEROUS preservation solutions have been proposed and experimentally evaluated in response to the need for extending the safe limit of organ preservation. UW solution has been demonstrated to provide significant improvement in organ function after the extended preservation of kidney, liver and pancreas.1–3 Several studies have found UW solution superior to other preservation solutions for the heart;4,5 however the clinically safe time limit remains only 4 to 6 hours.6 HTK solution has been reported to be effective in kidney and liver transplantation.7–9 However, it has not been as extensively tested for the donor heart. Hendry et al.10 have reported better recovery of isometric contraction of the human right atrial trabeculae in vitro after preservation with HTK solution, compared with UW solution. We also have reported that HTK solution provided excellent protection after 8 hours of cold storage.11 This study was designed to test the efficacy of HTK solution after prolonged cold storage as compared with the conventional glucose–insulin–potassium (GIK) and UW solutions in experimental heart preservation. GIK solution was chosen as control to mimic current clinical regimens. Variables of cardiac function, myocardial tissue water, and adenine nucleotide pool metabolites were employed to assess prolonged myocardial preservation in the isolated rat heart model. MATERIALS AND METHODS The investigation conformed with the guidelines set out in the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. Male Wistar rats (250 to 350 g) were anesthetized with sodium pentobarbital (65 mg/kg IP) and systemically heparinized (500 IU, IP). The hearts were divided into three groups (N 5 6 per group) according to each preservation solution used: group 1, GIK solution; group 2, UW solution; and group 3, HTK solution. Experiments were performed in vitro using isolated hearts from each group in parallel. The heart was excised and immediately immersed in Krebs–Henseleit bicarbonate buffer (KHB) solution, consisting of following compounds (concentration in mmol/L): NaCl (118), KCl (4.7), MgSO4 (1.2), KH2PO4 (1.2), CaCl2 (2.5), NaHCO3 (25.0), and glucose (11.0) at 37°C. Then, it was mounted on a Langendorff apparatus (IPH-W, Labo Support, Osaka, Japan) via the aorta, and perfused at a constant pressure of 60 mmHg in a nonrecirculating Langendorff mode (L-mode) for 10 minutes. Perfusion was performed with KHB solution which was filtered
(0.22 mm), equilibrated with 95% O2 and 5% CO2, and maintained at 37°C. Following measurement of pre-preservative baseline cardiac function, the hearts were stored in each preservation solution at 4°C for 6, 8, and 12 hr. The recovery of cardiac function of the stored hearts was then evaluated. Myocardial samples frozen in liquid nitrogen before storage in each group were analyzed for adenylate content. All results are expressed as the mean 6 SEM. Statistical evaluation of the data was performed using one-way ANOVA. A P value of less than .05 was considered statistically significant.
RESULTS Cardiac Function
The mean values of baseline cardiac function were: 46.3 6 2.3 mL/min of AF, 14.6 6 0.5 mL/min of CF, 60.4 6 2.3 mL/min of CO, 235 6 12 beats/min of HR, 109 6 6 mmHg of SP, 25600 6 400 of RPP, and 2340 6 140 mmHg/sec. The data in Table 1 show the recovery of hemodynamic function after 6, 8, and 12 hours of preservation in each group. Although the hearts stored in HTK solution (group 3) up to 8 hr showed maintenance of cardiac function almost to 80% of pre-preservative baseline function, a progressive decline of cardiac function occurred at 12 hr. Recovery data of AF and CO of the hearts stored in UW solution (group 2) revealed an initial loss of the function at 6 hr almost to 60% and declined to 50% at 8 hr. Recovery data of AF and CO of the hearts stored in GIK solution (group 1) revealed a progressive loss of the function at 6 hr almost to 50% and declined to 30% at 8 hr. Although there was a dramatic decline in cardiac function between 8 and 12 hrs of storage in each group, the recovery values of AF and CO after 12 hrs of storage in group 3 were significantly higher than in groups 1 and 2. In addition, recovery of CF after 12 hrs of storage in group 3 was significantly higher than in group 1.
From the Department of Cardiovascular Surgery, Kinki University School of Medicine, Osaka, Japan. This work was supported by Grant-in-Aid 06671348 from the Ministry of Education. Address reprint requests to Kwansong Ku, MD, Department of Cardiovascular Surgery, Kinki University School of Medicine, 377-2, Ohno-Higashi, Osaka-Sayama, Osaka 589, Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)01050-1
Transplantation Proceedings, 30, 3331–3333 (1998)
3331
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KU AND OKU Table 1. Recovery of Cardiac Function and Myocardial Energy Level After Cold Storage
Data are expressed as mean 6 SEM. *P , .05. AF, aortic flow; CF, coronary flow; CO, cardiac output. Percent value (%), post-preservative cardiac function/pre-preservative baseline cardiac function 3 100. Adenylate energy charge (EC) 5 (0.5 ADP1ATP)/(ATP1ADP1AMP).
Actual Myocardial Adenylate Contents After Storage in Each Group
The values of ATP/ADP ratio and energy charge (EC) in the myocardium before storage in each group were 3.46 6 0.27 and 0.87 6 0.05. After 8 hrs of storage in HTK solution, the ATP/ADP ratio was sustained at 50% and the value of EC was still maintained at 70% of the prepreservation level. However, in UW and GIK solutions, the ATP/ADP ratios declined to below 15% and the values of EC declined to 30% of the prepreservation levels after 8 hrs of storage. After 12 hrs of storage even in HTK solution, the ATP/ADP ratio also declined to 10% and the value of EC declined to below 30% of the prepreservative level and there were no significant differences in these values after 12 hrs of storage among the groups. DISCUSSION
The efficacy of HTK solution is attributed to the high buffering capacity provided by histidine, which suppresses ischemia-induced tissue acidosis.12 In addition, it has a low concentration of potassium and magnesium. Tryptophane and a-ketoglutarate have been added because of their membrane-protecting effect. In UW solution, Ca21 is also absent; however the potassium concentration is very high. During cold storage in the presence of a low intracellular pH, a high potassium concentration has been shown to
cause myocardial and endothelial damage.13,14 Therefore, UW solution, which has a high potassium concentration, may not be suitable for cardiac preservation compared to HTK solution. In addition, in the present study, the recovery of cardiac function of the hearts stored in GIK solution was impaired more than the recovery in UW solution. Intracellular solutions such as HTK and UW solutions have the potential advantage over extracellular solutions such as GIK solution of minimizing the reequilibration of normal ion gradients that are required during reperfusion.10 Cold storage causes energy exhaustion and leads to ischemic contracture of myocardium.15 In addition, a heart, which needs to perform mechanical work immediately, has a much greater energy demand upon reperfusion than other organs. In the present study, the myocardial ATP/ADP ratio stored in HTK solution was significantly higher than the ratios found in UW and GIK solutions and the recovery of cardiac function was well maintained at 70% to 80% of pre-preservative baseline function until 8 hrs of storage. The myocardial EC values of the hearts stored in HTK solution were also sufficiently maintained until 8 hrs of storage, whereas those values in UW and GIK solutions declined to below 30% at 8 hrs of storage. The suppression of ischemia-induced tissue acidosis and sustenance of cytosolic ATP pools by the high histidine buffering capacity might also lead to the better recovery of function of the
PROLONGED HYPOTHERMIC STORAGE
hearts stored in HTK solution.13 In the present study, even after 12 hrs of storage, the recovery of cardiac function stored in HTK solution was also significantly higher than the hearts stored in UW and GIK solutions; however, there was a remarkable energy deprivation between 8 and 12 hrs of storage and the recovery of cardiac function between 8 and 12 hrs of storage declined dramatically. Successful cold storage of the heart may thus be highly energy-dependent. In conclusion, HTK solution is much more effective than UW and GIK solutions for isolated rat heart preservation; however, successful cold storage of the heart is highly energy-dependent, and a dramatic breakdown of myocardial energy level which causes crucial decline of cardiac function occurs between 8 and 12 hrs of storage. REFERENCES 1. Plog RJ, Goossens D, McAnulty JF, et al: Transplantation 46:191, 1988 2. Jamieson NV, Sundberg R, Lindell S, et al: Transplantation 46:517, 1988 3. Wahlberg JA, Love R, Landegaard L, et al: Transplantation 43:5, 1987
3333 4. Fremes SE, Li RK, Weisel RD, et al: J Thorac Cardiovasc Surg 102:666, 1991 5. Gott JP, Pan-Chih, Dorsey LMA, et al: Ann Thorac Surg 50:348, 1990 6. Furukawa H, Todo S, Imventarsa O, et al: Transplant Proc 23:1550, 1991 7. Holscher M, Groenwoud AF: Transplant Proc 23:2334, 1991 8. Groenwoud AF, Buchholz B, Gubernatis F, et al: Transplant Proc 22:2212, 1990 9. Van Gulik TM, Reinders ME, Nio R, et al: Transplantation 57:167, 1994 10. Hendry PJ, Labow RS, Keon WJ: J Thorac Cardiovasc Surg 105:667, 1993 11. Gu K, Kin S, Saitoh Y, et al: Transplantation 61:1572, 1996 12. Kallerhof M, Blech M, Kehrer G: Urol Res 14:271, 1986 13. del Nido PJ, Wilson GJ, Mickle DAG, et al: J Thorac Cardiovasc Surg 89:689, 1985 14. Mankad PS, Chester AH, Yacoub MH: Ann Thorac Surg 51:89, 1991 15. Stringham JC, Southard JH, Hegge J, et al: Transplantation 53:287, 1992
N
UMEROUS preservation solutions have been proposed and experimentally evaluated in response to the need for extending the safe limit of organ preservation. UW solution has been demonstrated to provide significant improvement in organ function after the extended preservation of kidney, liver and pancreas.1–3 Several studies have found UW solution superior to other preservation solutions for the heart;4,5 however the clinically safe time limit remains only 4 to 6 hours.6 HTK solution has been reported to be effective in kidney and liver transplantation.7–9 However, it has not been as extensively tested for the donor heart. Hendry et al.10 have reported better recovery of isometric contraction of the human right atrial trabeculae in vitro after preservation with HTK solution, compared with UW solution. We also have reported that HTK solution provided excellent protection after 8 hours of cold storage.11 This study was designed to test the efficacy of HTK solution after prolonged cold storage as compared with the conventional glucose–insulin–potassium (GIK) and UW solutions in experimental heart preservation. GIK solution was chosen as control to mimic current clinical regimens. Variables of cardiac function, myocardial tissue water, and adenine nucleotide pool metabolites were employed to assess prolonged myocardial preservation in the isolated rat heart model. MATERIALS AND METHODS The investigation conformed with the guidelines set out in the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. Male Wistar rats (250 to 350 g) were anesthetized with sodium pentobarbital (65 mg/kg IP) and systemically heparinized (500 IU, IP). The hearts were divided into three groups (N 5 6 per group) according to each preservation solution used: group 1, GIK solution; group 2, UW solution; and group 3, HTK solution. Experiments were performed in vitro using isolated hearts from each group in parallel. The heart was excised and immediately immersed in Krebs–Henseleit bicarbonate buffer (KHB) solution, consisting of following compounds (concentration in mmol/L): NaCl (118), KCl (4.7), MgSO4 (1.2), KH2PO4 (1.2), CaCl2 (2.5), NaHCO3 (25.0), and glucose (11.0) at 37°C. Then, it was mounted on a Langendorff apparatus (IPH-W, Labo Support, Osaka, Japan) via the aorta, and perfused at a constant pressure of 60 mmHg in a nonrecirculating Langendorff mode (L-mode) for 10 minutes. Perfusion was performed with KHB solution which was filtered
(0.22 mm), equilibrated with 95% O2 and 5% CO2, and maintained at 37°C. Following measurement of pre-preservative baseline cardiac function, the hearts were stored in each preservation solution at 4°C for 6, 8, and 12 hr. The recovery of cardiac function of the stored hearts was then evaluated. Myocardial samples frozen in liquid nitrogen before storage in each group were analyzed for adenylate content. All results are expressed as the mean 6 SEM. Statistical evaluation of the data was performed using one-way ANOVA. A P value of less than .05 was considered statistically significant.
RESULTS Cardiac Function
The mean values of baseline cardiac function were: 46.3 6 2.3 mL/min of AF, 14.6 6 0.5 mL/min of CF, 60.4 6 2.3 mL/min of CO, 235 6 12 beats/min of HR, 109 6 6 mmHg of SP, 25600 6 400 of RPP, and 2340 6 140 mmHg/sec. The data in Table 1 show the recovery of hemodynamic function after 6, 8, and 12 hours of preservation in each group. Although the hearts stored in HTK solution (group 3) up to 8 hr showed maintenance of cardiac function almost to 80% of pre-preservative baseline function, a progressive decline of cardiac function occurred at 12 hr. Recovery data of AF and CO of the hearts stored in UW solution (group 2) revealed an initial loss of the function at 6 hr almost to 60% and declined to 50% at 8 hr. Recovery data of AF and CO of the hearts stored in GIK solution (group 1) revealed a progressive loss of the function at 6 hr almost to 50% and declined to 30% at 8 hr. Although there was a dramatic decline in cardiac function between 8 and 12 hrs of storage in each group, the recovery values of AF and CO after 12 hrs of storage in group 3 were significantly higher than in groups 1 and 2. In addition, recovery of CF after 12 hrs of storage in group 3 was significantly higher than in group 1.
From the Department of Cardiovascular Surgery, Kinki University School of Medicine, Osaka, Japan. This work was supported by Grant-in-Aid 06671348 from the Ministry of Education. Address reprint requests to Kwansong Ku, MD, Department of Cardiovascular Surgery, Kinki University School of Medicine, 377-2, Ohno-Higashi, Osaka-Sayama, Osaka 589, Japan.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)01050-1
Transplantation Proceedings, 30, 3331–3333 (1998)
3331
3332
KU AND OKU Table 1. Recovery of Cardiac Function and Myocardial Energy Level After Cold Storage
Data are expressed as mean 6 SEM. *P , .05. AF, aortic flow; CF, coronary flow; CO, cardiac output. Percent value (%), post-preservative cardiac function/pre-preservative baseline cardiac function 3 100. Adenylate energy charge (EC) 5 (0.5 ADP1ATP)/(ATP1ADP1AMP).
Actual Myocardial Adenylate Contents After Storage in Each Group
The values of ATP/ADP ratio and energy charge (EC) in the myocardium before storage in each group were 3.46 6 0.27 and 0.87 6 0.05. After 8 hrs of storage in HTK solution, the ATP/ADP ratio was sustained at 50% and the value of EC was still maintained at 70% of the prepreservation level. However, in UW and GIK solutions, the ATP/ADP ratios declined to below 15% and the values of EC declined to 30% of the prepreservation levels after 8 hrs of storage. After 12 hrs of storage even in HTK solution, the ATP/ADP ratio also declined to 10% and the value of EC declined to below 30% of the prepreservative level and there were no significant differences in these values after 12 hrs of storage among the groups. DISCUSSION
The efficacy of HTK solution is attributed to the high buffering capacity provided by histidine, which suppresses ischemia-induced tissue acidosis.12 In addition, it has a low concentration of potassium and magnesium. Tryptophane and a-ketoglutarate have been added because of their membrane-protecting effect. In UW solution, Ca21 is also absent; however the potassium concentration is very high. During cold storage in the presence of a low intracellular pH, a high potassium concentration has been shown to
cause myocardial and endothelial damage.13,14 Therefore, UW solution, which has a high potassium concentration, may not be suitable for cardiac preservation compared to HTK solution. In addition, in the present study, the recovery of cardiac function of the hearts stored in GIK solution was impaired more than the recovery in UW solution. Intracellular solutions such as HTK and UW solutions have the potential advantage over extracellular solutions such as GIK solution of minimizing the reequilibration of normal ion gradients that are required during reperfusion.10 Cold storage causes energy exhaustion and leads to ischemic contracture of myocardium.15 In addition, a heart, which needs to perform mechanical work immediately, has a much greater energy demand upon reperfusion than other organs. In the present study, the myocardial ATP/ADP ratio stored in HTK solution was significantly higher than the ratios found in UW and GIK solutions and the recovery of cardiac function was well maintained at 70% to 80% of pre-preservative baseline function until 8 hrs of storage. The myocardial EC values of the hearts stored in HTK solution were also sufficiently maintained until 8 hrs of storage, whereas those values in UW and GIK solutions declined to below 30% at 8 hrs of storage. The suppression of ischemia-induced tissue acidosis and sustenance of cytosolic ATP pools by the high histidine buffering capacity might also lead to the better recovery of function of the
PROLONGED HYPOTHERMIC STORAGE
hearts stored in HTK solution.13 In the present study, even after 12 hrs of storage, the recovery of cardiac function stored in HTK solution was also significantly higher than the hearts stored in UW and GIK solutions; however, there was a remarkable energy deprivation between 8 and 12 hrs of storage and the recovery of cardiac function between 8 and 12 hrs of storage declined dramatically. Successful cold storage of the heart may thus be highly energy-dependent. In conclusion, HTK solution is much more effective than UW and GIK solutions for isolated rat heart preservation; however, successful cold storage of the heart is highly energy-dependent, and a dramatic breakdown of myocardial energy level which causes crucial decline of cardiac function occurs between 8 and 12 hrs of storage. REFERENCES 1. Plog RJ, Goossens D, McAnulty JF, et al: Transplantation 46:191, 1988 2. Jamieson NV, Sundberg R, Lindell S, et al: Transplantation 46:517, 1988 3. Wahlberg JA, Love R, Landegaard L, et al: Transplantation 43:5, 1987
3333 4. Fremes SE, Li RK, Weisel RD, et al: J Thorac Cardiovasc Surg 102:666, 1991 5. Gott JP, Pan-Chih, Dorsey LMA, et al: Ann Thorac Surg 50:348, 1990 6. Furukawa H, Todo S, Imventarsa O, et al: Transplant Proc 23:1550, 1991 7. Holscher M, Groenwoud AF: Transplant Proc 23:2334, 1991 8. Groenwoud AF, Buchholz B, Gubernatis F, et al: Transplant Proc 22:2212, 1990 9. Van Gulik TM, Reinders ME, Nio R, et al: Transplantation 57:167, 1994 10. Hendry PJ, Labow RS, Keon WJ: J Thorac Cardiovasc Surg 105:667, 1993 11. Gu K, Kin S, Saitoh Y, et al: Transplantation 61:1572, 1996 12. Kallerhof M, Blech M, Kehrer G: Urol Res 14:271, 1986 13. del Nido PJ, Wilson GJ, Mickle DAG, et al: J Thorac Cardiovasc Surg 89:689, 1985 14. Mankad PS, Chester AH, Yacoub MH: Ann Thorac Surg 51:89, 1991 15. Stringham JC, Southard JH, Hegge J, et al: Transplantation 53:287, 1992