Hibernating myocardium is defined as a condition of persistent myocardial dysfunction at rest due to reduced coronary blood flow that can be partially or completely restored to normal by improving blood flow (1-3). Myocardial hibernation is thought to be an endogenous cardioprotective phenomena characterized by adaptation to myocardial ischemia; however, the underlying mechanisms remain unclear (4-6). The endogenous adenosine or activation of adenosine triphosphate (ATP)-sensitive K-channels are reported not to be involved (7). However, the effect of cardiac sympathetic nerves in the hibernating heart has not been well studied. It is supposed that cardiac sympathetic function may be decreased under reduced coronary flow and may contribute to the suppression of myocardial oxygen consumption.
In the present study, we aimed to clarify the change in cardiac sympathetic function in the hibernating heart. To impair coronary blood flow in dogs, we placed a tube via the carotid artery in the proximal portion of the left circumflex artery (LCx). The plasma catecholamine concentrations in the coronary sinus and the aorta were then measured before and 1 week after the tube placement to evaluate the change in catecholamine release from the heart.
Animals and experimental procedure
Beagle dogs weighing 10-14 kg were anesthetized with sodium pentobarbital (30 mg/kg intravenously), subjected to endotracheal intubation and ventilated using a respirator. Echocardiography was performed to evaluate left ventricular (LV) wall motion under baseline conditions. After an intravenous administration of antibiotics, the neck was incised and a catheter was advanced via the left jugular vein into the coronary sinus for coronary venous sampling. The left carotid artery was also dissected free and was cut down to insert a 7 Fr guiding catheter into the aorta for arterial blood sampling. To measure plasma catecholamine concentrations, blood samples were simultaneously obtained from the coronary sinus and the aorta.
We then placed a 4 Fr nylon tube via the carotid artery in the proximal portion of the LCx artery to make a coronary stenosis as follows: first, the tip of the guiding catheter was placed at the ostium of the left coronary artery; second, a 0.025 inch guidewire was inserted through the guiding catheter into the LCx artery; third, the guiding catheter was extracted; and, finally, a nylon tube was advanced along the guidewire and was placed in the proximal portion of the LCx artery. After tube placement, the guidewire and the venous catheter were extracted and the cervical incision was sutured. Echocardiography was repeated approximately 30 min after the tube placement, then the dogs were allowed to recover. Animals in the laboratory received every consideration for their comfort, including appropriate anesthetics during operation and veterinary care after operation.
Measurement of ventricular wall motion and regional myocardial blood flow 1 week after the tube placement
After an interval of 1 week, the dogs were returned to the laboratory, anesthetized with sodium pentobarbital, and ventilated with a respirator. The LV wall motion was evaluated by echocardiography. The cervical sutures were released, and the catheter for blood sampling was advanced via the left jugular vein into the coronary sinus. The right femoral artery was also cut down and a 7 Fr catheter was inserted into the aorta to record aortic pressure and withdraw arterial blood samples. Blood samples were simultaneously obtained from the coronary sinus and the aorta for measuring plasma catecholamine concentrations.
Thereafter, the dogs underwent left thoracotomy, and regional myocardial blood flow (RBF) was measured. A catheter was placed in the left atrium to inject the colored polystyrene microspheres. The colored microspheres, approximately 5 × 106 microspheres (red microspheres, 15 μm in diameter; E-Z Trac Inc., Los Angeles, CA, U.S.A.), were injected into the left atrium as a total volume of 3-4 ml. A reference blood sample was drawn from the catheter placed in the aorta, using a withdrawal pump at a flow rate 10 ml/min, 10 s before the microspheres injection and continuously for 90 s. Heart rate and aortic pressure were monitored continuously and recorded on a Macintosh personal computer with available hardware and software (MP100 and Acknowledge; Biopac Systems Inc., Santa Barbara, CA, U.S.A.).
At the end of the experiment, the hearts were excised and sectioned vertically to the long axis about 1.0 cm in width. Whether myocardial necrosis was present was assessed by trichlorotetrazolium chloride staining and also by histological observation. Tissue samples were obtained from the LCx and the left anterior descending (LAD) artery areas to measure RBF, respectively. When a myocardial necrosis was present, the dog was excluded from the analysis.
Measurement of ventricular wall motion after the tube extraction
The change in LV wall motion by extracting the tube placed in the LCx artery was studied in other dogs, in which the tube had been already placed for 1 week according to the procedure already described. The dogs were anesthetized with sodium pentobarbital and ventilated using a respirator. After echocardiograms were recorded, the cervical sutures were released and the LCx tube was extracted. The cervical incision was then resutured and the dogs were allowed to recover. One or 2 weeks after the tube extraction, the dogs were anesthetized, ventilated with a respirator and echocardiograms were repeated. After the experiment, the dogs were sacrificed by injecting excess doses of pentobarbiturate, and the hearts were excised and sectioned to examine for myocardial necrosis. When the dog had myocardial necrosis, the dog was excluded.
Echocardiographic measurement of ventricular wall motion. Two-dimensional echocardiography was performed, using a 3.5 MHz phased array transducer linked to an ultrasound system (SSA-250A; Toshiba Inc., Tokyo, Japan). Images were obtained from the epicardial surface of the right ventricle and the short-axis view of the LV was recorded. At the mid-papillary muscle level, the LV short-axis diameter was obtained as a line connecting the midseptum and the midpoint of the posterior wall between the two papillary muscles. The interventricular septal wall thickness and the posterior wall thickness were measured. The end-diastolic wall thickness was measured at the timing of the R wave on the electrocardiogram. End-systolic wall thickness was measured when the LV cavity was minimal. Regional wall thickening was calculated as end-systolic minus end-diastolic wall thickness divided by end-diastolic wall thickness, expressed as a percentage.
Regional myocardial blood flow. The detailed technique of sample resolution and the methods for microsphere counting and regional blood flow calculation have been reported previously (8,9). All reagents used for the processing of tissue and blood samples to quantify colored microspheres were obtained from E-Z Trac Company. In brief, the methods were as follows.
Tissue sample. The tissue samples were minced, placed in a tube together with Tissue Digest Reagent I, and boiled in a boiling water bath. The sample was diluted with Tissue Digest Reagent II and centrifuged. The supernatant was discarded, and the pellet was washed with Tissue Microsphere Counting Reagent. The samples were centrifuged, the supernatant was aspirated, and the final volume was measured.
Blood sample. The reference blood samples were centrifuged, and Blood Hemolysis Reagent was added to the sediment. The samples were mixed with distilled water and centrifuged. Blood Digest Reagent was added to the sediment, and the sample was boiled. The digest was washed with distilled water and Blood Microsphere Counting Reagent was added to the sediment. The samples were centrifuged, the supernatant was aspirated, and the final volume of the microspheres was measured.
Counting and calculation. Aliquots of the final solution were placed in a Fuchs-Rosenthal Hemocytometer. Sixteen chambers were counted for each sample, and the total number of microspheres in each reference blood and tissue sample was computed according to the formula: EQUATION 1 where "TM" is the total number of microspheres, "no. counted" is the number of colored microspheres counted, "no. chambers" is the number of chambers counted, "3.2 mm3" is the ruled volume of the chamber, and "ml suspension" is the final volume of the tissue or the blood samples. RBF was computed according to the formula: EQUATION 2 where CT is the total number of microspheres in the tissue sample, R is reference flow rate (ml/min), CR is the total number of microspheres in the reference blood sample, and WT is the weight of the tissue sample in grams. Results were expressed as milliliters per gram per minute.
Measurement of plasma catecholamine concentration. Blood samples obtained were placed in sodium edetic acid tubes in ice water. After the samples were centrifuged, the supernatant was aspirated and then frozen. Plasma concentrations of norepinephrine (NE) and epinephrine (E) were measured by means of liquid chromatography. The percent changes of NE or E release from the heart were calculated by dividing the difference in each concentration between the aorta and the coronary sinus by that of aorta.
Results are expressed as mean ± SE for plasma NE and E concentrations, and are expressed as mean ± SD for other values. Differences in wall thickening within the LCx or the LAD areas among experimental conditions were analyzed by analysis of variance using repeated measurements. A simultaneous multicomparison test (Bonferroni) was applied only if the F statistic indicated an overall statistically significant difference between the means. Differences in RBF between the LCx and the LAD areas 1 week after the tube placement were analyzed by the two-tailed paired t test. Differences in plasma catecholamine concentrations and percentage changes of NE or E release from the heart between the baseline condition and 1 week after the tube placement were also analyzed by the two-tailed paired t test. All statistical comparisons were performed with a commercially available statistical package for the Macintosh personal computer (StatView, version 5.0; SAS Institute Inc., Cary, NC, U.S.A.). The statistical significance was defined at p < 0.05.
Ventricular wall motion and regional blood flow 1 week after tube placement
The results of echocardiography and RBF were obtained in six dogs. As shown in Table 1, the wall thickening was decreased in the LCx area 30 min after the tube placement, but was not in the LAD area. One week later, the wall thickening was still significantly decreased in the LCx area.
The findings of RBF are shown in Fig. 1. The mean aortic pressure was 67 ± 11 mmHg at the microsphere injection. Compared with the LAD area, the RBF in the LCx area was decreased in the endocardium (0.82 ± 0.19 vs. 0.33 ± 0.20 ml/g/min; p < 0.05) but not in the epicardium (0.84 ± 0.22 vs. 0.72 ± 0.15 ml/g/min). Therefore, the tube was thought to obstruct the LCx blood flow and to impair the wall thickening in the LCx area.
Change in wall motion after tube extraction
To examine the reversibility of impaired wall motion in the LCx area, the LCx tube was extracted in the other five dogs after being placed for 1 week. Representative echocardiograms obtained from a dog are shown in Fig. 2. In all five dogs, the wall thickening of the posterior wall decreased 1 week after tube placement, which was not improved shortly after the tube extraction but was restored to normal levels 1 or 2 weeks after tube extraction (from 23 ± 14 to 51 ± 10%), whereas interventricular septal wall thickening was not significantly affected throughout the experiment (Fig. 3). The heart rate was similar among all experimental conditions (130 ± 23 at baseline, 131 ± 22 1 week after tube placement, and 134 ± 15 after tube extraction). Therefore, the impaired wall motion in the LCx area was reversed by extracting the tube.
Change in catecholamine release from the heart before and 1 week after tube placement
The findings of plasma NE and E concentrations in the coronary sinus and the aorta are shown in Table 2. The plasma NE and E concentrations in the coronary sinus were not significantly changed by the tube placement, but those concentrations in the aorta were mildly decreased after 1 week. Consequently, the percent change of NE release from the heart, calculated by dividing the difference in NE concentration between the aorta and the coronary sinus by that of aorta, was increased by the tube placement (59 ± 21 vs. 157 ± 25%; p < 0.05). The percent change of E uptake from the heart was not significantly different. Therefore, NE release from the hibernating heart was shown to be increased (Fig. 4).
We induced a persistent decrease in wall motion by placing a nylon tube in the LCx artery, which was associated with decreased subendocardial blood flow and could be restored to normal by extracting the tube. Therefore, dogs with the tube in place were considered to be models of myocardial hibernation. The role of catecholamine metabolism on hibernating myocardium has not been clarified; however, the present study suggested that NE release from the hibernating heart was increased and may contribute to the mechanism of myocardial hibernation.
To induce chronic but easily reversible coronary stenosis, we placed a tube in the LCx artery. A similar procedure was adopted by Berman et al. (10), although they placed a catheter in the LAD artery of swine models and carried out an acute experiment. They reported that their artificial stenosis reduced vessel diameter by about 80% in the proximal site of the LAD artery, with a pressure difference of approximately 20 mmHg between the mean arterial pressure and mean coronary pressure distal to the stenosis; whereas we preferred the LCx artery for placing the tube because the LAD artery is usually less developed in dogs, and expands and contracts more in response to ventricular wall motion. We also tried to induce not acute, but chronic coronary stenosis, so the pressure difference in the present study between mean femoral artery pressure and mean coronary pressure measured at the end of the tube was about 10-15 mmHg (data not shown) and the magnitude of the stenosis might be milder than that reported by Berman et al. (10).
Since we intended to prevent myocardial necrosis due to thrombus formation by tube placement, the interval of placing the tube in the LCx artery was limited to 1 week in the present experiment. Therefore, myocardial infarction was not found in the LCx area by pathological observation, and contamination with myocardial necrosis was excluded in the present study. The interval of 1 week was also considered to be favorable to prevent sufficient collateral development to the LCx artery area. It has been reported that coronary collateral growth is stimulated by coronary stenosis more than 80% of a luminal stenosis and collaterals work significantly more than 4 weeks after the production of coronary stenosis (11). However, in the present study, the magnitude of the LCx stenosis by the tube seemed to be at most 80%, as already mentioned, and the results were obtained 1 week after the tube placement. Consequently, it may be possible to reduce endocardial blood flow in the LCx area without associated myocardial infarction.
The present findings in RBF were compatible with those of Berman et al. (10) and Fallavollita et al. (12), which showed that stenotic-zone blood flow was decreased in the endocardium but not in the epicardium. However, the observed decrease in RBF was not so profoundly reduced to explain the regional myocardial dysfunction reported in acute coronary constriction (13). Why was there a discrepancy between myocardial dysfunction and regional myocardial blood flow in chronic myocardial hibernation? It seems that findings obtained in acute models cannot be easily applied to the chronic conditions of myocardial hibernation. Although many studies have used acute models of myocardial hibernation, it is difficult to produce chronic severe reduction in blood flow (4,6). Indeed, we preliminarily tried to insert the tube into a more distal portion of the LCx artery to induce more severe stenosis, but these dogs showed myocardial infarction. Therefore, it is suggested that the phenomenon of myocardial hibernation is time dependent and it would be inappropriate to extrapolate the short-term hypoperfusion-contraction relationship to the chronic condition of myocardial hibernation.
Hibernating myocardium was initially regarded as a consequence of low-flow ischemia, but this concept is now controversial (3-6). The majority of reversible dysfunctional myocardium are reported to be perfused at normal rest blood flows in clinical studies (14-16). Some animal studies have also demonstrated that chronically dysfunctional myocardium is characterized not by low resting flow, but by severely impaired coronary flow reserve (17-19). Those studies suggested that, when myocardial oxygen demand is increased in cases of limited coronary flow reserve, myocardial ischemia will develop regardless of the blood flow level under the baseline condition. Therefore, post-ischemic stunning due to intermittent episodes of ischemia may play a role in the development of chronic reversible myocardial dysfunction. However, the present study demonstrated that a mild decrease in subendocardial blood flow can exist and induce hibernating myocardium like the earlier studies (12,20). The findings of the present study suggest that the two conditions of myocardial hibernation and repetitive myocardial stunning can coexist in the presence of severe coronary artery disease and may induce the chronic condition of myocardial hibernation.
Myocardial hibernation is thought to be an endogenous cardioprotective phenomenon characterized by adaptation to myocardial ischemia, but the endogenous adenosine and activation of ATP-sensitive K-channels are reported not to be involved (7), whereas the changes in cardiac sympathetic function have not been studied in the hibernating heart. Some clinical studies used metaiodobenzylguanidine (MIBG) imaging, which can provide in vivo information on the NE re-uptake and storage system, and investigated the effect of myocardial ischemia on sympathetic nerve function. Tsutsui et al. reported that repetitive myocardial ischemia impairs regional MIBG uptake (21). Dae et al. suggested that myocardial ischemia acutely affects sympathetic nerves and may cause chronic sympathetic nerve denervation (22). The present study showed that norepinephrine release from the heart is increased in the hibernating heart and the present findings were similar to those studies. The detailed mechanism was unclear, but the increased NE release from the heart may have a significant role on the suppression of myocardial oxygen consumption.
In conclusion, the present study suggests that myocardial hibernation, defined by chronic matching between decreased myocardial blood flow and impaired regional function, can exist in endocardium perfused by a stenosed coronary artery, and also showed that norepinephrine release is increased in the hibernating heart and may contribute to the mechanism of myocardial hibernation.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.