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The mechanism of myocardial protection from ischemic arrest by intracoronary tetrodotoxin administration
The mechanism of myocardial protection from ischemic arrest by intracoronary tetrodotoxin administration Intracoronary injection of 14 meg. of tetrodotoxin into the ischemic isolated rat heart resulted in immediate cessation of mechanical activity. Upon reperfusion with oxygenated, modified Krebs-Henseleit bicarbonate buffer in a modified Langendorff apparatus, all hearts recovered normal rate, rhythm, and contractile vigor after up to 60 minutes of ischemia. In contrast, all hearts not administered tetrodotoxin showed bradycardia, irregular rhythm, and weak contraction upon reperfusion after 30 and 45 minutes of ischemia; after 60 minutes, no mechanical activity was evident. The improved cardiac function following ischemia in the tetrodotoxin-treated hearts was associated with persistence of normal adenosine triphosphate (ATP) levels after up to 30 minutes of ischemia and normal or elevated creatiiie phosphate (CP) levels after up to 60 minutes of ischemia. On the other hand, ATP and CP levels progressively declined to reach 50 per cent of normal values after 30 minutes in the ischemic hearts without tetrodotoxin. These findings indicate that postarrest A TP and CP levels play an important role in myocardial recovery after ischemic arrest.
G. Frank O. Tyers, M.D., F.R.C.S.(C), F.A.C.S., George J. Todd, M.D., James R. Neely, Ph.D., and John A. Waldhausen, M.D., F.A.C.S., Hershey, Pa.
J—/ow myocardial tolerance to ischemia may interfere with the effectiveness of treatment of heart disease. Since metabolic inhibition concurrent with the ischemic insult might limit the extent of myocardial injury, we studied the reversibility of tetrodotoxin arrest of the ischemic heart. The dose-dependent nerve blocking effects observed upon ingestion of the eggs of the From the Departments of Surgery and Physiology, College of Medicine, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033. This investigation was supported in part by the South Central Pennsylvania Chapter of the American Heart Association. Received for publication Jan. 4, 1974. Address for reprints: G. Frank O. Tyers, M.D., Associate Professor of Surgery, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033.
190
tetrodon, or puffer fish, have long been recognized,1 and a purified principle has been extracted from these eggs and termed tetrodotoxin.- Subsequent studies of neurologic tissue have detected that a selective blockade of the sodium ion inflow known to initiate membrane excitation is the mechanism of action of tetrodotoxin1 '; the external surface of the axon membrane is the site of action.'' Although the hypotensive effect of tetrodotoxin has been known since the late nineteenth century," its exact mechanism of action on the cardiovascular system was not elucidated until recently. Prior to the demonstration in 1968 by Feinstein and Paimre" of a combination of peripheral vasodilation and decreased cardiac output, theories on the mechanism of tetrodotoxin-
Volume 69
Number 2
Protection of myocardium with tetrodotoxin
19 1
February, 1975
induced hypotension included depression of central vasomotor areas,7 ganglionic or preganglionic blockade," and a direct relaxant action on vascular smooth muscle," wth little support provided for a direct action on the heart. Evidence that tetrodotoxin is indeed a direct cardiac depressant was provided by Cheng1" in 1970. His experiments with isolated perfused rat hearts showed that evanescent and reversible ventricular arrest occurred following intracoronary injection of tetrodotoxin, and he concluded that the mechanism of this cardiac depression was the same as that observed in studies on neuronal membranes, i.e., inhibition of membrane conduction by a selective blockade of the sodium channels. In this paper we report the effects of intracoronary tetrodotoxin administration on the ischemic isolated rat heart. We found an increased tolerance to ischemia and a marked preservation of myocardial energy reserves when tetrodotoxin was injected at the beginning of the ischemic period. Methods Rats of the Sprague-Dawley strain weighing 150 to 200 grams were fasted overnight before experimental use. Heparin sodium (500 units per rat) and sodium pentobarbital (30 mg. per rat) were injected intraperitoneally. When adequate anesthesia had been achieved, the abdominal cavity was entered via a transverse incision. This allowed easy access to the thoracic cavity by means of a diaphragmatic incision and bilateral incisions along the anterolateral aspect of the rib cage. The anterior chest wall was then folded cephalad, and the beating heart was lifted anteriorly and removed from the chest by means of a scissors cut along the posterosuperior aspect of the heart. Upon removal from the chest, the heart was immediately dropped into a beaker of chilled (10° C.) physiological saline. The heart was removed from the cold saline bath with fine-tipped forceps, and the aorta was slipped onto the grooved perfusion cannula of the modified Langendorff apparatus11 (Fig. 1). The aorta was secured
on the cannula with a silk ligature, and retrograde cardiac perfusion was begun at a pressure of 60 mm. Hg with oxygenated, modified Krebs-Henseleit bicarbonate buffer solution warmed to 37° C. The concentration, in millimoles, of the buffer solution constituents were as follows: sodium chloride, 118; potassium chloride, 4.7; calcium chloride, 2.5; magnesium sulfate, 1.2; potassium dihydrophosphate, 1.2; calcium ethylenediaminetetraacetic acid, 0.5; sodium bicarbonate, 25; and glucose, 5. The perfusate was oxygenated with a 95:5 oxygen: carbon dioxide mixture which was equilibrated with water at 37° C. Bubbling the gas mixture through the buffer solution in the perfusion reservoir produced arterial oxygen tensions in excess of 500 mm. Hg. The perfusate temperature was maintained at 37° C. by means of a water jacket which surrounded both the perfusate reservoir and the tubes leading from the reservoir to the aortic cannula. After cannulation of the aorta, retrograde perfusion was continued for 10 minutes to allow complete washout of all blood as well as recovery from the brief anoxic interval associated with removal of the heart from the animal. During this period all coronary venous effluent was discarded. Following the initial period of recovery and equilibration, a water-jacketed chamber was placed around the heart, and retrograde perfusion at 60 mm. Hg pressure was continued for another 15 minutes with recirculation of the oxygenated buffer. At the end of this interval, all hearts were rendered ischemic for 10, 20, 30, 45, or 60 minutes by cross-clamping the tube which carried buffer from the reservoir to the aortic cannula. Control hearts received no additional treatment, whereas experimental hearts received immediate retrograde coronary injection of 2 ml. of a 7 meg. per milliliter solution of tetrodotoxin by way of a self-sealing, multipleinjection adaptor located between the crossclamp site and the aortic root. The solution was prepared at room temperature by dissolving crystalline tetrodotoxin* in modified ♦Sankyo Co., Japan.
The Journal of
192
Tyers et al.
Thoracic and Cardiovascular Surgery
iQl _ _ RESERVOIR
— PERFUSATE
o 2 co2
AORTIC
CROSS
CLAMP
SITE
SELF SEALING INJECTION
AORTIC
HEART
SITE
CANNULA
IN CHAMBER
RECIRCULATI PUMP
CORONARY
VENOUS
EFFLUENT
Fig. 1. Modified Langendorff apparatus used for perfusion of the isolated rat heart. Krebs-Henseleit buffer or in a buffer solution differing only in that it contained 75 mM of sodium bicarbonate rather than 25 mM. After the designated ischemic interval, all hearts were reperfused with oxygenated buffer for 15 minutes without recirculation and were then removed from the aortic cannula with liquid nitrogen-cooled steel tongs. The hearts were flattened by the cold tongs, immersed in liquid nitrogen, and then analyzed for adenosine triphosphate (ATP) and creatine phosphate (CP) content.1-
Results Injection of tetrodotoxin into the aortic root effected a complete halt of all mechanical activity within 5 to 10 seconds. In contrast, the control hearts continued to contract for 5 to 8 minutes following occlusion of retrograde aortic flow. After 30 minutes of ischemia, the myocardial ATP levels of the control group had declined to less than 50 per cent of normal (Table I ) . The ATP levels of the tetrodotoxin-treated group were maintained at preischemic levels after the same arrest period
Volume 69
19 3
Protection of myocardium with tetrodotoxin
Number 2 February, 1975
Table I. Effect of ischemia and ischemia modified by intracoronary TTX on tissue A TP and CP Duration ischemic ai rest (min.) Ot 10 20 30 45 60 601
APT
(jiM/Gm. J
Ischemia 19± 1.5 18 ± 2.5 16 ± 0 . 4 9 + 0.3 7 ± 1.3 4 ±0.3 4 ±0.3
Legend: TTX, Tetrodotoxin. "Tetrodotoxin in modified tAfter a 10 minute washout ITetrodotoxin in modified
dry
weight)
(2) (4) (4) (4) (4) (4)
20 ± 0 . 3 18 ± 1.4 19 ± 4 . 0 9 ±0.9 6 ±0.5 10±0.8
dry
CP (liM/Gm.
Ischemia- TTX* (2) (4) (4) (4) (5) (3)
Ischemia 24 ±0.8 3612.4 39 ±0.3 15 ± 3 . 8 22 ± 3.4 16±3.8 16 ± 3 . 8
\
weigilt)
Ischemia-TTX*
— (2) (4) (4) (4) (4) (4)
31 35 38 20 20 38
±6.8 + 6.1 + 3.7 ±2.3 ±3.6 + 3.1
(2) (4) (4) (4) (5) (3)
ADP, Adenosine diphosphate. CP, Creatine phosphate. Krebs-Henselite buffer, 25 mM sodium bicarbonate. and 10 minutes as a working preparation. 13 Krebs-Henseleit buffer, 75 mM sodium bicarbonate.
(p < 0.05). The effect of tetrodotoxin on myocardial CP levels followed a similar trend. After 30 minutes of ischemic arrest, the tetrodotoxin group had CP levels which were somewhat greater than preischemic values and more than double those of the untreated group (p < 0.01). The preservative effect of tetrodotoxin on high-energy phosphate levels was less pronounced when the ischemic period was extended beyond 30 minutes, but even after 60 minutes of arrest the tetrodotoxintrcated group had 2 ^M more ATP (p < 0.02) and 4 rM more CP than the timematched control hearts. When the bicarbonate concentration of the tetrodotoxin solution was tripled, hearts arrested for 60 minutes had ATP levels 6 /xM higher (p < 0.001) and CP levels 22 rM higher (p < 0.01) than control hearts arrested for the same period (Table I ) . The cardioplegic effect of tetrodotoxin was readily reversible. Heart rate, rhythm, and contractile vigor were returned to the preischemic state within 5 minutes after release of the aortic cross-clamp in tetrodotoxin-treated hearts subjected to 10, 20, 30, 45, or 60 minutes of ischemic arrest. In the control group, on the other hand, ischemic periods of 30 and 45 minutes were characteristically associated with arrhythmias, weak contraction, and bradycardia, and no control heart demonstrated mechanical ac-
tivity upon reperfusion after 60 minutes of unmodified ischemic arrest. Discussion The ability to preserve myocardial integrity during extended periods of cardiac ischemia achieves practical significance in terms of cardiac surgery. The technical advantages of operating on a dry, flaccid heart have served to popularize the technique of cross-clamping the aorta just distal to the coronary arteries during cardiotomy procedures." However, this method of arresting the heart permits it to beat until it exhausts its energy reserves. Functional, histologic, and histochemical abnormalities follow.1"' On the other hand, cardioplegia and metabolic arrest are rapidly attained after the intracoronary injection of tetrodotoxin and are associated with improved maintenance of high-energy phosphate levels and an increased cardiac tolerance to ischemic insult. Increasing the buffering capacity of the tetrodotoxin solution further improves results when ischemic arrest is continued for over 30 minutes. The improved high-energy phosphate levels demonstrated in this study are probably primarily due to the instantaneous cessation of mechanical activity following tetrodotoxin administration, as similar maintenance of ATP and CP levels has
The Journal of
194
Tyers et al.
been observed after immediate potassiuminduced arrest of the ischemic heart."1 If critically low levels of high-energy phosphates arc allowed to develop, myocardial function may be irreversibly damaged,'7 and failure of contractile protein uncoupling with ischemic contracture (stone heart) may result.'"- '■' Although at first glance the ATP and CP levels seem high for the durations of ischemic arrest studied, this condition is due to the repcrfusion of each heart for 15 minutes at the end of the ischemic period to assess its functional state. Hearts were taken for analysis of high-energy phosphate at the end of the repcrfusion period. CP is preferentially preserved in the ischemic reperfused heart, as previously reported after potassium chloride-induced arrest.'" Only when the ATP level falls below 10 /M does the CP fall below base-line values. The results of this study indicate that postischemic ATP and CP levels are of central importance in the maintenance of myocardial viability and the recovery of myocardial function. They further suggest that intracoronary administration of tetrodotoxin concurrent with the onset of myocardial ischemia preserves high-energy phosphate levels, probably by a combination of mechanical and metabolic arrest. Similar functional protection from ischemia has been obtained with other metabolic inhibitors-" and with potassium-induced arrest.-1 Further studies will be required to determine the relative advantages of the various available methods of inducing cardiac arrest. This work was made possible by the advice and assistance of Dr. Howard E. Morgan of the Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033. The competent technical assistance of Miss Ida M. Niebauer is also acknowledged. REFERENCES 1 Kao, C. Y.: Tetrodotoxin, Saxitoxin and Their significance in the Study of Excitation Phenomena, Pharmacol. Rev. 18: 997, 1966. 2 Tahara, Y.: Uber das Tetrodongift, Biochem. Z. 10: 255, 1910.
Thoracic and Cardiovascular Surgery
3 Furakawa, T., Sasaoka, T., and Hasaya, Y.: Effects of Tetrodotoxin on the Neuromuscular Junction, Jap. J. Physiol. 9: 143, 1959. 4 Narahashi, T., Moore, J. W., and Scott, W. R.: Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons, J. Gen. Physiol. 47: 965, 1964. 5 Narahashi, T., Anderson, N. C , and Moore, J. W.: Tetrodotoxin Does Not Block Excitation From Inside the Nerve Membrane, Science 153: 765, 1966. 6 Feinstein, M. B., and Paimre, M.: Mechanism of the Cardiovascular Action of Tetrodotoxin in the Cat: Block of Conduction in Peripheral Sympathetic Fibers, Circ. Res. 23: 553, 1968. 7 Koizumi, K., Levine, D. G., and Brooks, C. M.: Effect of Tetrodotoxin (Puffer Fish Poison) on the Central Nervous System, Neurology 17: 395, 1967. 8 Kao, C. Y., and Fuhrman, F. A.: Pharmacological Studies on Tarichatoxin, a Potent Neurotoxin, J. Pharmacol. Exp. Ther. 140: 31, 1963. 9 Lipsius, M. R., Siegman, M. J., and Kao, C. Y.: Direct Relaxant Action of Procaine and Tetrodotoxin on Vascular Smooth Muscle, J. Pharmacol. Exp. Ther. 164: 60, 1968. 10 Cheng, C. P., Cheng. K. K., and Wang, J. C : The Action of Tetrodotoxin on the Heart, J. Pathol. 100: 121, 1970. 11 Neely, J. R., Liebermeister, H., Battersby, E. J., and Morgan, H. E.: Effect of Pressure Development on Oxygen Consumption by Isolated Rat Heart, Am. J. Physiol. 212: 804, 1967. 12 Bergmeyer, H. U.: Methods of Enzymatic Analyses, New York, 1963, Academic Press, Inc. 13 Rovetto, M. J., Whitmer, J. T , and Neely, J. R.: Comparison of the Effects of Anoxia and Whole Heart Ischemia on Carbohydrate Utilization in Isolated Working Rat Hearts, Circ. Res. 32: 699, 1973. 14 Messmer, B. J., Hallman, G. L., Liotta, D., Martin, C , and Cooley, D. A.: Aortic Valve Replacement: New Techniques, Hydrodynamics, and Clinical Results, Surgery 68: 1026, 1970. 15 Brachfeld, N.: Maintenance of Cell Viability, Circulation 40: 202, 1969 (Suppl. IV). 16 Levitsky, S., Merchant, F. J., and Feinberg, H.: Effects of KC1-Induced Cardiac Arrest on Energy Metabolism and Contractility of Ischemic Dog Heart, Fed. Proc. 33: 398, 1974. 17 Nowicki, J.: Induced Cardiac Arrest During Open Heart Surgery, J. Cardiovasc. Surg. 12: 157, 1971. 18 Coffman, J. D., Lewis, F. B., and Gregg, D. E.: Effect of Prolonged Periods of Anoxia on
Volume 69
Number
2
Protection of myocardium with tetrodotoxin
19 5
February, 1975
Atrioventricular Conduction and Cardiac Muscle, Circ. Res. 8: 649, 1960. 19 Lee. Y. C. P.. and Visscher. M. B.: Perfusate Cations and Contracture and Ca, Cr, PCr, and ATP in Rabbit Myocardium, Am. J. Physiol. 219: 1637, 1970. 20 Webb, W. R., Dodds, R. P., Unal, M. O., Karow, A. M., Cook, W. A., and Daniel,
C. R.: Suspended Animation of the Heart With Metabolic Inhibitors, Ann. Surg. 164: 343, 1966. 21 Gay, W. A., and Ebert, P. A.: Functional, Metabolic, and Morphologic Effects of Potassium-Induced Cardioplegia, Surgery 74: 284, 1973.
G. Frank O. Tyers, M.D., F.R.C.S.(C), F.A.C.S., George J. Todd, M.D., James R. Neely, Ph.D., and John A. Waldhausen, M.D., F.A.C.S., Hershey, Pa.
J—/ow myocardial tolerance to ischemia may interfere with the effectiveness of treatment of heart disease. Since metabolic inhibition concurrent with the ischemic insult might limit the extent of myocardial injury, we studied the reversibility of tetrodotoxin arrest of the ischemic heart. The dose-dependent nerve blocking effects observed upon ingestion of the eggs of the From the Departments of Surgery and Physiology, College of Medicine, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033. This investigation was supported in part by the South Central Pennsylvania Chapter of the American Heart Association. Received for publication Jan. 4, 1974. Address for reprints: G. Frank O. Tyers, M.D., Associate Professor of Surgery, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033.
190
tetrodon, or puffer fish, have long been recognized,1 and a purified principle has been extracted from these eggs and termed tetrodotoxin.- Subsequent studies of neurologic tissue have detected that a selective blockade of the sodium ion inflow known to initiate membrane excitation is the mechanism of action of tetrodotoxin1 '; the external surface of the axon membrane is the site of action.'' Although the hypotensive effect of tetrodotoxin has been known since the late nineteenth century," its exact mechanism of action on the cardiovascular system was not elucidated until recently. Prior to the demonstration in 1968 by Feinstein and Paimre" of a combination of peripheral vasodilation and decreased cardiac output, theories on the mechanism of tetrodotoxin-
Volume 69
Number 2
Protection of myocardium with tetrodotoxin
19 1
February, 1975
induced hypotension included depression of central vasomotor areas,7 ganglionic or preganglionic blockade," and a direct relaxant action on vascular smooth muscle," wth little support provided for a direct action on the heart. Evidence that tetrodotoxin is indeed a direct cardiac depressant was provided by Cheng1" in 1970. His experiments with isolated perfused rat hearts showed that evanescent and reversible ventricular arrest occurred following intracoronary injection of tetrodotoxin, and he concluded that the mechanism of this cardiac depression was the same as that observed in studies on neuronal membranes, i.e., inhibition of membrane conduction by a selective blockade of the sodium channels. In this paper we report the effects of intracoronary tetrodotoxin administration on the ischemic isolated rat heart. We found an increased tolerance to ischemia and a marked preservation of myocardial energy reserves when tetrodotoxin was injected at the beginning of the ischemic period. Methods Rats of the Sprague-Dawley strain weighing 150 to 200 grams were fasted overnight before experimental use. Heparin sodium (500 units per rat) and sodium pentobarbital (30 mg. per rat) were injected intraperitoneally. When adequate anesthesia had been achieved, the abdominal cavity was entered via a transverse incision. This allowed easy access to the thoracic cavity by means of a diaphragmatic incision and bilateral incisions along the anterolateral aspect of the rib cage. The anterior chest wall was then folded cephalad, and the beating heart was lifted anteriorly and removed from the chest by means of a scissors cut along the posterosuperior aspect of the heart. Upon removal from the chest, the heart was immediately dropped into a beaker of chilled (10° C.) physiological saline. The heart was removed from the cold saline bath with fine-tipped forceps, and the aorta was slipped onto the grooved perfusion cannula of the modified Langendorff apparatus11 (Fig. 1). The aorta was secured
on the cannula with a silk ligature, and retrograde cardiac perfusion was begun at a pressure of 60 mm. Hg with oxygenated, modified Krebs-Henseleit bicarbonate buffer solution warmed to 37° C. The concentration, in millimoles, of the buffer solution constituents were as follows: sodium chloride, 118; potassium chloride, 4.7; calcium chloride, 2.5; magnesium sulfate, 1.2; potassium dihydrophosphate, 1.2; calcium ethylenediaminetetraacetic acid, 0.5; sodium bicarbonate, 25; and glucose, 5. The perfusate was oxygenated with a 95:5 oxygen: carbon dioxide mixture which was equilibrated with water at 37° C. Bubbling the gas mixture through the buffer solution in the perfusion reservoir produced arterial oxygen tensions in excess of 500 mm. Hg. The perfusate temperature was maintained at 37° C. by means of a water jacket which surrounded both the perfusate reservoir and the tubes leading from the reservoir to the aortic cannula. After cannulation of the aorta, retrograde perfusion was continued for 10 minutes to allow complete washout of all blood as well as recovery from the brief anoxic interval associated with removal of the heart from the animal. During this period all coronary venous effluent was discarded. Following the initial period of recovery and equilibration, a water-jacketed chamber was placed around the heart, and retrograde perfusion at 60 mm. Hg pressure was continued for another 15 minutes with recirculation of the oxygenated buffer. At the end of this interval, all hearts were rendered ischemic for 10, 20, 30, 45, or 60 minutes by cross-clamping the tube which carried buffer from the reservoir to the aortic cannula. Control hearts received no additional treatment, whereas experimental hearts received immediate retrograde coronary injection of 2 ml. of a 7 meg. per milliliter solution of tetrodotoxin by way of a self-sealing, multipleinjection adaptor located between the crossclamp site and the aortic root. The solution was prepared at room temperature by dissolving crystalline tetrodotoxin* in modified ♦Sankyo Co., Japan.
The Journal of
192
Tyers et al.
Thoracic and Cardiovascular Surgery
iQl _ _ RESERVOIR
— PERFUSATE
o 2 co2
AORTIC
CROSS
CLAMP
SITE
SELF SEALING INJECTION
AORTIC
HEART
SITE
CANNULA
IN CHAMBER
RECIRCULATI PUMP
CORONARY
VENOUS
EFFLUENT
Fig. 1. Modified Langendorff apparatus used for perfusion of the isolated rat heart. Krebs-Henseleit buffer or in a buffer solution differing only in that it contained 75 mM of sodium bicarbonate rather than 25 mM. After the designated ischemic interval, all hearts were reperfused with oxygenated buffer for 15 minutes without recirculation and were then removed from the aortic cannula with liquid nitrogen-cooled steel tongs. The hearts were flattened by the cold tongs, immersed in liquid nitrogen, and then analyzed for adenosine triphosphate (ATP) and creatine phosphate (CP) content.1-
Results Injection of tetrodotoxin into the aortic root effected a complete halt of all mechanical activity within 5 to 10 seconds. In contrast, the control hearts continued to contract for 5 to 8 minutes following occlusion of retrograde aortic flow. After 30 minutes of ischemia, the myocardial ATP levels of the control group had declined to less than 50 per cent of normal (Table I ) . The ATP levels of the tetrodotoxin-treated group were maintained at preischemic levels after the same arrest period
Volume 69
19 3
Protection of myocardium with tetrodotoxin
Number 2 February, 1975
Table I. Effect of ischemia and ischemia modified by intracoronary TTX on tissue A TP and CP Duration ischemic ai rest (min.) Ot 10 20 30 45 60 601
APT
(jiM/Gm. J
Ischemia 19± 1.5 18 ± 2.5 16 ± 0 . 4 9 + 0.3 7 ± 1.3 4 ±0.3 4 ±0.3
Legend: TTX, Tetrodotoxin. "Tetrodotoxin in modified tAfter a 10 minute washout ITetrodotoxin in modified
dry
weight)
(2) (4) (4) (4) (4) (4)
20 ± 0 . 3 18 ± 1.4 19 ± 4 . 0 9 ±0.9 6 ±0.5 10±0.8
dry
CP (liM/Gm.
Ischemia- TTX* (2) (4) (4) (4) (5) (3)
Ischemia 24 ±0.8 3612.4 39 ±0.3 15 ± 3 . 8 22 ± 3.4 16±3.8 16 ± 3 . 8
\
weigilt)
Ischemia-TTX*
— (2) (4) (4) (4) (4) (4)
31 35 38 20 20 38
±6.8 + 6.1 + 3.7 ±2.3 ±3.6 + 3.1
(2) (4) (4) (4) (5) (3)
ADP, Adenosine diphosphate. CP, Creatine phosphate. Krebs-Henselite buffer, 25 mM sodium bicarbonate. and 10 minutes as a working preparation. 13 Krebs-Henseleit buffer, 75 mM sodium bicarbonate.
(p < 0.05). The effect of tetrodotoxin on myocardial CP levels followed a similar trend. After 30 minutes of ischemic arrest, the tetrodotoxin group had CP levels which were somewhat greater than preischemic values and more than double those of the untreated group (p < 0.01). The preservative effect of tetrodotoxin on high-energy phosphate levels was less pronounced when the ischemic period was extended beyond 30 minutes, but even after 60 minutes of arrest the tetrodotoxintrcated group had 2 ^M more ATP (p < 0.02) and 4 rM more CP than the timematched control hearts. When the bicarbonate concentration of the tetrodotoxin solution was tripled, hearts arrested for 60 minutes had ATP levels 6 /xM higher (p < 0.001) and CP levels 22 rM higher (p < 0.01) than control hearts arrested for the same period (Table I ) . The cardioplegic effect of tetrodotoxin was readily reversible. Heart rate, rhythm, and contractile vigor were returned to the preischemic state within 5 minutes after release of the aortic cross-clamp in tetrodotoxin-treated hearts subjected to 10, 20, 30, 45, or 60 minutes of ischemic arrest. In the control group, on the other hand, ischemic periods of 30 and 45 minutes were characteristically associated with arrhythmias, weak contraction, and bradycardia, and no control heart demonstrated mechanical ac-
tivity upon reperfusion after 60 minutes of unmodified ischemic arrest. Discussion The ability to preserve myocardial integrity during extended periods of cardiac ischemia achieves practical significance in terms of cardiac surgery. The technical advantages of operating on a dry, flaccid heart have served to popularize the technique of cross-clamping the aorta just distal to the coronary arteries during cardiotomy procedures." However, this method of arresting the heart permits it to beat until it exhausts its energy reserves. Functional, histologic, and histochemical abnormalities follow.1"' On the other hand, cardioplegia and metabolic arrest are rapidly attained after the intracoronary injection of tetrodotoxin and are associated with improved maintenance of high-energy phosphate levels and an increased cardiac tolerance to ischemic insult. Increasing the buffering capacity of the tetrodotoxin solution further improves results when ischemic arrest is continued for over 30 minutes. The improved high-energy phosphate levels demonstrated in this study are probably primarily due to the instantaneous cessation of mechanical activity following tetrodotoxin administration, as similar maintenance of ATP and CP levels has
The Journal of
194
Tyers et al.
been observed after immediate potassiuminduced arrest of the ischemic heart."1 If critically low levels of high-energy phosphates arc allowed to develop, myocardial function may be irreversibly damaged,'7 and failure of contractile protein uncoupling with ischemic contracture (stone heart) may result.'"- '■' Although at first glance the ATP and CP levels seem high for the durations of ischemic arrest studied, this condition is due to the repcrfusion of each heart for 15 minutes at the end of the ischemic period to assess its functional state. Hearts were taken for analysis of high-energy phosphate at the end of the repcrfusion period. CP is preferentially preserved in the ischemic reperfused heart, as previously reported after potassium chloride-induced arrest.'" Only when the ATP level falls below 10 /M does the CP fall below base-line values. The results of this study indicate that postischemic ATP and CP levels are of central importance in the maintenance of myocardial viability and the recovery of myocardial function. They further suggest that intracoronary administration of tetrodotoxin concurrent with the onset of myocardial ischemia preserves high-energy phosphate levels, probably by a combination of mechanical and metabolic arrest. Similar functional protection from ischemia has been obtained with other metabolic inhibitors-" and with potassium-induced arrest.-1 Further studies will be required to determine the relative advantages of the various available methods of inducing cardiac arrest. This work was made possible by the advice and assistance of Dr. Howard E. Morgan of the Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033. The competent technical assistance of Miss Ida M. Niebauer is also acknowledged. REFERENCES 1 Kao, C. Y.: Tetrodotoxin, Saxitoxin and Their significance in the Study of Excitation Phenomena, Pharmacol. Rev. 18: 997, 1966. 2 Tahara, Y.: Uber das Tetrodongift, Biochem. Z. 10: 255, 1910.
Thoracic and Cardiovascular Surgery
3 Furakawa, T., Sasaoka, T., and Hasaya, Y.: Effects of Tetrodotoxin on the Neuromuscular Junction, Jap. J. Physiol. 9: 143, 1959. 4 Narahashi, T., Moore, J. W., and Scott, W. R.: Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons, J. Gen. Physiol. 47: 965, 1964. 5 Narahashi, T., Anderson, N. C , and Moore, J. W.: Tetrodotoxin Does Not Block Excitation From Inside the Nerve Membrane, Science 153: 765, 1966. 6 Feinstein, M. B., and Paimre, M.: Mechanism of the Cardiovascular Action of Tetrodotoxin in the Cat: Block of Conduction in Peripheral Sympathetic Fibers, Circ. Res. 23: 553, 1968. 7 Koizumi, K., Levine, D. G., and Brooks, C. M.: Effect of Tetrodotoxin (Puffer Fish Poison) on the Central Nervous System, Neurology 17: 395, 1967. 8 Kao, C. Y., and Fuhrman, F. A.: Pharmacological Studies on Tarichatoxin, a Potent Neurotoxin, J. Pharmacol. Exp. Ther. 140: 31, 1963. 9 Lipsius, M. R., Siegman, M. J., and Kao, C. Y.: Direct Relaxant Action of Procaine and Tetrodotoxin on Vascular Smooth Muscle, J. Pharmacol. Exp. Ther. 164: 60, 1968. 10 Cheng, C. P., Cheng. K. K., and Wang, J. C : The Action of Tetrodotoxin on the Heart, J. Pathol. 100: 121, 1970. 11 Neely, J. R., Liebermeister, H., Battersby, E. J., and Morgan, H. E.: Effect of Pressure Development on Oxygen Consumption by Isolated Rat Heart, Am. J. Physiol. 212: 804, 1967. 12 Bergmeyer, H. U.: Methods of Enzymatic Analyses, New York, 1963, Academic Press, Inc. 13 Rovetto, M. J., Whitmer, J. T , and Neely, J. R.: Comparison of the Effects of Anoxia and Whole Heart Ischemia on Carbohydrate Utilization in Isolated Working Rat Hearts, Circ. Res. 32: 699, 1973. 14 Messmer, B. J., Hallman, G. L., Liotta, D., Martin, C , and Cooley, D. A.: Aortic Valve Replacement: New Techniques, Hydrodynamics, and Clinical Results, Surgery 68: 1026, 1970. 15 Brachfeld, N.: Maintenance of Cell Viability, Circulation 40: 202, 1969 (Suppl. IV). 16 Levitsky, S., Merchant, F. J., and Feinberg, H.: Effects of KC1-Induced Cardiac Arrest on Energy Metabolism and Contractility of Ischemic Dog Heart, Fed. Proc. 33: 398, 1974. 17 Nowicki, J.: Induced Cardiac Arrest During Open Heart Surgery, J. Cardiovasc. Surg. 12: 157, 1971. 18 Coffman, J. D., Lewis, F. B., and Gregg, D. E.: Effect of Prolonged Periods of Anoxia on
Volume 69
Number
2
Protection of myocardium with tetrodotoxin
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