Temperature Threshold and Preservation of Signaling for Mitochondrial Membrane Proteins during Ischemia in Rabbit Heart

Abstract

Temperature modulates both myocardial energy requirements and production. We have previously demonstrated that myocardial protection induced by hypothermic adaptation preserves expression of genes regulating heat shock protein and the nuclear-encoded mitochondrial proteins, the adenine nucleotide translocator isoform 1 (ANT 1 ), and the ␤ subunit of F 1 -ATPase (␤F 1 -ATPase). This preservation is associated with a reduction in ATP depletion similar to that noted in cardioplegic arrested hearts preserved at a critical temperature (30°C) or below. We tested the hypothesis that expression of these genes may also be subject to this temperature threshold phenomenon. Isolated perfused rabbit hearts were subjected to ischemic cardioplegic arrest at 4, 30, or 34°C for 120 min. Cardiac function indices and steady-state mRNA levels for ANT 1 , ␤F 1 -ATPase, and HSP70-1 were measured prior to ischemia (B) and after 45 min of reperfusion. Cardiac function was significantly depressed in the 34°C group. Ischemia at 34°C reduced steady-state mRNA levels for ANT 1 and ␤F 1 -ATPase from B, but these levels were similarly preserved at 4 and 30°C. HSP70-1 levels were mildly elevated (fourfold) above B to similar levels at all three temperatures. These results indicate that mRNA expression for ANT 1 and ␤F 1 -ATPase is specifically preserved in a pattern consistent with the temperature threshold phenomenon. HSP70-1 expression is not influenced by ischemic temperature. Preservation of gene expression for these mitochondrial proteins implies that signaling for mitochondrial biogenesis or resynthesis is maintained after ischemic insult. © 1998 Academic PressKey Words: adaptation; ANT 1 ; ␤F 1 -ATPase; cardioplegia; HSP70-1; hypothermia; myocardial ischemia; myocardial reperfusion; threshold.Temperature protects ischemic myocardium (2,3,6,7,15,34,36,38) and modulates both myocardial energy requirements and production (27). The relationship between energy supply and demand in the cardioplegic arrested heart remains fairly constant in the temperature range between 4 and 30°C (27), which is frequently used during cardiac surgical procedures. The myocardial supply/demand ratio demonstrates a threshold phenomenon by deteriorating rapidly at temperatures above 30°C (27). Accelerated ATP depletion and inability to reestablish highenergy phosphate stores during reperfusion accompany this deterioration. Thus, 30°C represents a critical temperature point (39), which triggers acceleration of metabolic energy utilization rate above the energy production rate. Provision of glucose as substrate in the cardioplegia solution can partially ameliorate this metabolic imbalance by promoting anaerobic ATP production (27).Recently, we have demonstrated that hypothermic exposure at 30°C prior to warm ischemia preserves myocardial function and ATP stores after reperfusion (28). This response represents an adaptation, whereby the heart becomes tolerant to a second exposure of the same or alternative type of stress. Hypothermic exposure induces a metabolic dow...

Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia

“…, where CF is coronary flow (ml ⅐ min Ϫ1 ⅐ g wet tissue Ϫ1 ), PaO 2 Ϫ PvO 2 is the difference in the partial pressure of oxygen (PO 2, mmHg) between perfusate and coronary effluent, and c is the Bunsen solubility coefficient of O 2 in perfusate (22). Oxygen extraction was calculated as O2 Ext ϭ MVO2/O2 content in the perfusate.…”

“…RNA samples were tested by ultraviolet absorption at an analytical wavelength of 260 nm to determine the concentration. The quality and concentration of the RNA samples were further confirmed by electrophoresis on denatured 1% agarose gels (22).…”

“…A reduction in ATP utilization rate initiated by hypothermia persists during rewarming and plays a central role in preserving myocardial function after ischemia and reperfusion (20). Hypothermic ATP preservation may promote specific molecular responses observed during reperfusion in the isolated rabbit heart model, including stabilization of steady-state mRNA levels for nuclear-encoded mitochondrial membrane proteins, enhanced induction of the stress protein heat shock protein-70 (HSP70) (19,21,22) and promotion of signaling for antiapoptotic pathways (18).…”

Mitochondrial protein and HSP70 signaling after ischemia in hypothermic-adapted hearts augmented with glucose

“…The relative amount of mRNAs was evaluated using a PhosphorImager (model 400S, Molecular Dynamics, Sunnyvale, CA). The same size area at each band was taken to measure the intensity, and the same size area at the closest upstream position of each band was taken as the background of the image, respectively (12). The blots were exposed on Kodak X-omat film (Eastman Kodak, Rochester, NY) at Ϫ70°C.…”

“…Similar coordination occurs in hibernating or cold-adapted tissues subjected to cold stress. The mechanisms responsible for this coordination have not been studied, although injury reduction and enhanced ATP preservation appear to be necessary for maintenance of steady-state mRNA levels for these mitochondrial proteins after ischemia (10,12). Thus we postulated that ATP availability might play a role in determining these signal levels.…”