LABORATORY INVESTIGATION MYOCARDIAL METABOLISM Evidence that ischemic cell death begins in the subendocardium independent of variations in collateral flow or wall tension

Irreversible ischemic injury occurs after coronary artery occlusion in vivo, first in the subendocardium and progressing toward the subepicardium over time. presumably due to transmural variations in collateral flow or wall tension. In this study, 10 left ventricular globally ischemic slabs were created that were free of wall tension and collateral flow. The onset and completion of ischemic contracture were identified by means of a new tissue compressibility gauge designed for these studies. Transmural samples were obtained at 15 min intervals for determination of high-energy nucleotide levels and for ultrastructural analysis. The results show that there is a statistically significant gradient of ATP depletion, with the subendocardium consistently showing accelerated energy utilization compared with the subepicardium (p < .05). Ultrastructural evidence of irreversible injury first appeared in the subendocardium at the onset of ischemic contracture and occurred when ATP levels declined to less than 1 ,umol/g wet weight. In summary, these data show that during total ischemia in vitro, cell death begins in the subendocardium at the onset of ischemic contracture and progresses toward the subepicardium over time. These changes occurred independent of variations in collateral flow or wall tension. The results suggest that the increased risk of the subendocardium to ischemic injury previously noted in vivo may occur not only because of variations in collateral flow and wall tension, but may also be secondary to an increased metabolic rate of the subendocardium resulting in faster ATP use during the period of ischemia. Circulation 68, No. 1, 190-202, 1983. GLOBAL myocardial ischemia, either in vivo or in vitro, eventually leads to the development of ischemic contracture or cardiac rigor mortis. There is significant evidence suggesting that the development of rigor or ischemic contracture is closely associated both with the severe depletion of high-energy phosphate compounds and the onset of cell death.1 Therefore the detection of ischemic contracture may represent an online experimental method to identify when myocardial cells pass from a state of reversible to irreversible ischemic injury. A variety of measuring devices have been used to detect the onset of ischemic contracture. A model system with an intracavitary left ventricular balloon has been most commonly used, which is a From the Department of Surgery, Duke University Medical Center, Durham, NC. Supported by American Heart Association Grant-in-Aid 82933 and NIH grant HL 093 15-17. Address for correspondence: James E. Lowe, M.D., Duke University Medical Center, Durham, NC 27710. Received Jan. 18, 1983; revision accepted March 24, 1983. Presented in part at the 55th Scientific Session of the American Heart Association, November 1982. 190 sensitive indicator of the onset and the completion of this process.' However, contracture onset, which causes an increase in tissue stiffness, has been detected by the use of weights gently lowered onto the surface of the heart, with the distance traveled recorded by a linear transducer; ultrasonic crystals that indicate an increase in wall thickness with the onset of contracture2 I have also been used. A variety of interventions have been shown to increase or decrease the time period of global ischemia required to produce severe depletion of high-energy phosphate compounds and the subsequent formation of rigor complexes. Hypothermia, potassium cardioplegia, and decreased electrical activity all appear to delay the onset of contracture, whereas pacing and increased temperature appear to hasten the process. I 4 Thus this model system appears to be a simple experimental means to investigate interventions that delay or hasten the process of ischemic injury. Although the onset of ischemic contracture appears to be associated with severe ATP depletion and with CIRCULATION by gest on July 6, 2017 http://ciajournals.org/ D ow nladed from LABORATORY INVESTIGATION-MYOCARDIAL METABOLISM the onset of cell death, the exact sequence of events explaining the onset and completion of ischemic contracture have not been well defined. Until now it was unclear whether cell death during global ischemia occurred simultaneously and uniformly or whether there was a transmural progression of cell death from the subendocardium to the subepicardium. It is well established that irreversible ischemic injury occurs after experimental coronary artery occlusion in vivo, first in the subendocardium and extending toward the subepicardium over time.5 Presumably, the progression of this injury is primarily related to the transmural distribution of coronary collateral flow in vivo and perhaps secondarily to variations in transmural wall tension.51-0 Studies by Dunn and Griggs 10, 11 and Allison et al.12 suggest that during in vivo ischemia, there may be a transmural gradient of metabolic rate, with the greatest energy use occurring in the subendocardium. However, these metabolic gradients observed in vivo were attributed to variations in wall tension or to collateral flow.s012 The present studies were designed to determine whether or not there is a transmural progression of metabolic and ultrastructural changes that occur during total ischemia in vitro. In such a model system, collateral flow is no longer a variable, since the entire heart is suddenly rendered completely ischemic. Of critical importance in the experimental methods required to further investigate this question was the development of a new means to accurately identify when ischemic contracture occurs. Previous detection devices, including the left ventricular intracavitary balloon and ultrasonic crystals, have required an intact heart. These experimental models do not allow free access to the myocardium for transmural tissue sampling at various time intervals because sampling interferes with the monitoring process. The myocardial probe and linear force transducer described by Gavin et al.2 requires that the heart be positioned on firm support to make serial determinations of tissue stiffness over time. Therefore, for the present studies a new tissue compressibility gauge was designed to accurately determine the onset of ischemic contracture in free wall slabs of left ventricular myocardium. Such a compressibility gauge can be used in vitro with a slab of left ventricular myocardium, while an adjacent slab of left ventricular myocardium from the same heart can be sampled at various intervals for determination of highenergy phosphate levels and for ultrastructural analysis. Comparative studies with the tissue compressibility gauge have shown that this device is capable of identifying an increase in stiffness of left ventricular myocardium, which correlates directly with the pressure rise noted in an intracavitary left ventricular balloon placed in intact hearts. The advantage of this model system is that it allows myocardium to be sampled transmurally at multiple intervals during total ischemia and that the tissue studied is under no wall tension and receives no collateral blood flow. Materials and methods Ten dogs anesthetized with pentobarbital and weighing between 18 and 22 kg were intubated and ventilated for 30 min before a left thorocotomy and rapid excision of the heart. The right ventricle, base, and apex were removed and the left ventricle was opened along the epicardial course of the left anterior descending coronary artery, creating a large, left ventricular slab. The slab was then divided into two equal halves (figure 1). Half of the slab was immediately positioned in the tissue compressibility gauge designed and built for these studies (figure 2). The remaining half was available for transmural sampling at control (3 to 5 min after removal of the heart), at 15, 30, 45, and 60 min, and at the onset of ischemic contracture, midischemic contracture, and completion of ischemic contracture. Tissue samples were obtained at each interval from the subendocardium, midmyocardium, and subepicardium and were submitted for determination of ATP, ADP, and AMP levels and for