Basic Research in

Cardiology

Basic Res Cardio185:9-20 (1990)

Acute hemodynamic effects of coronary artery ligation in conscious rats R. G. Schoemaker, J. U r q u h a r t , J. J. M. Debets, H. A. J. Struyker Boudier, and J. F. M. Smits*) D e p a r t m e n t of Pharmacology, University of Limburg, Maastricht, The Netherlands

Summary: Because of the growing interest in the use of coronary artery ligation (CAL) in rats as a model for studies on heart failure, we have investigatcd the acute hemodynamic changes following CAL in conscious rats. Animals were equipped for measurement of cardiac output (CO), arterial pressure (MAP), and central venous pressure (CVP). These parameters were measured before CAL, immediately after, and 24h after. Furthermore, peak CO, obtained by rapid infusion of 12 ml Ringer's solution (in 1 min) was measured 2 days before and 1 day after CAL. CAL resulted in immediate reduction of CO, because of reduced stroke volume (SV). CO as well as SV were inversely correlated with infarct size as determined 24 h after CAL. Heart rate (HR) and MAP did not change. Twenty-four hours later, CO was still reduced. MAP was now reduced, possibly as a result from resetting of nervous reflex mechanisms. Before CAL, peak CO and SV were similar in CAL and sham animals. At 24h after CAL, these parameters were greatly reduced in CAL rats. Peak values were strongly correlated to infarct size. Results indicate that CAL in rats leads to hemodynamic changes similar to the ones observed following myocardial infarction in man. Cardiac function is related to infarct size and is altered both at rest and during maximal stimulation. Key words: rats; m~vocardial infarction; cardiac function; infarct size

Introduction Ligation of the left anterior descending coronary artery ( C A L ) in rats is a widely used technique for induction of cardiac dysrhythmias (cf. 4, 6, 8, 14, 17, 23). T h e r e is now a growing interest for the use of C A L in rats in drug studies on congestive heart failure, among others. H o w e v e r , little is known about the early h e m o d y n a m i c consequences of C A L in rats. Most authors noted a reduction of m e a n arterial pressure (2, 4, 5, 14; review, cf. 8). Effects on heart rate range from bradycardia (4) to no effect (2, 9) or tachycardia (5). In one study (9) in which cardiac output was also m o n i t o r e d , no effect on resting hemodynamics was noted nor on exercise-stimulated h e m o d y n a m i c s 1 day following C A L . The aim of the present study was to investigate the acute h e m o d y n a m i c changes and consequent loss of contractile myocardium without interference of effects of acute surgery or anesthesia. T h e r e f o r e , C A L was p e r f o r m e d in conscious rats chronically instrumented for h e m o d y n a m i c m e a s u r e m e n t s and prepared with a loose ligature around the coronary artery. H c m o d y n a m i c s were studied at rest and peak cardiac function was obtained by rapid volume loading (22). Results from previous studies may have been obscured by the use of *) J. F. M. Smits is an Established Investigator for the Netherlands Heart Foundation 581

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Basic Research in Cardiology, VoL 85, No. I (1990)

anesthetics, acute surgery, and the variation in time after ligation at which h e m o d y n a m i c s w e r e studied. Results indicate that the h e m o d y n a m i c c h a n g e s are similar to those in m a n , suggesting that rats are well suited for studies into the p a t h o p h y s i o l o g y and p h a r m a c o l o g y of acute myocardial infarction, and associated cardiac failure.

Materials and Methods Male Wistar rats (Broekman, Borchem, FRG), weighing 250-300 grams, were used throughout this study. Animals were housed separately in perspex boxes on sawdust in climatized rooms with a 12-h light-dark cycle. Before and during all experiments, animals had free access to standard lab food and tap water (Hope Farms, Woerden, The Netherlands). All experiments were performed in adherence with local ethics codes.

Surgery Surgery was performed in two stages, which greatly improved survival. In the first stage of surgery, animals were submitted to both left and fight thoracotomy, in order to prepare the animals for coronary artery ligation and measurement of aortic blood flow, respectively. All thorax surgery was performed under strict aseptic conditions, Animals were anesthetized with pentobarbital (60 mg/kg i.p.) and the trachea was intubated (PE-240). First, the skin overlying the fourth left intercostal space was opened, the underlying muscles separated and cut, and the thorax was opened after starting respiration with oxygen-enriched room air (60 strokes/min, tidal volume 2.5 ml). The heart was prepared for later coronary occlusion as described by Lepr~in et al. (17). Briefly, the heart was carefully pushed to the left by applying gentle pressure to the right side of the thorax. A silk suture (6.0 with atraumatic needle; Ethicon K801H) was looped under the descending branch of the left coronary artery near the origin of the pulmonary cone. In order to obtain a range of in[arct sizes, no attempts were made to exactly reproduce the exact site of the suture between animals. In sham animals the suture was looped through the myocardium next to the left coronary artery. Both ends of the suture were passed through the lumen of a preshaped polyethylene (PE-100) cylinder (16). The polyethylene cylinder was placed between the heart and the ribs. Both ends of the ligature were left outside the thorax and the ribs were pulled together with 3-0 silk. Then, the ends of the suture were tied together with a piece of 3-0 silk without pulling. The 3-0 silk was inserted into a 9-cm piece of PE-100 and fixed with a metal obturator. The tube was guided s.c. to the neck, where it was exteriorized. The suture extended from the tube, so that, by pulling it at a later occasion, the left coronary artery could be occluded. Muscles and skin were sutured separately and negative pressure was restored by inserting a Silastic 602-175 drain through a stab wound between the sixth and seventh rib and application of a negative pressure of 10 cm H20. Next, an electromagnetic flow probe (Skalar, Delft, The Netherlands) was implanted on the ascending aorta, through a right thoracotomy, as described in detail elsewhere (24, 25). The connector of this probe was exteriorized in the neck and fixed to the skin. Negative pressure was again restored through a drain. Animals were allowed to recover 4-6 days from this preliminary surgery. Surgery was done aseptically and no antibiotics were given. Survival of this surgery was over 95 %. Following the recovery period, animals were re-anesthetized with ether and a catheter (PE-10 heatsealed to PE-50) was inserted into the abdominal aorta through the fight femoral artery for measurement of blood pressure. Two catheters were introduced into the vena cava through the left femoral vein. The first one of these (PE-10 heat-sealed to PE-50) was inserted for approximately 4 cm, locating its tip in the abdominal vena cava. The second venous catheter (Silastic 602-175) was advanced into the femoral vein for 9 cm, until its tip was in the thoracic vena cava. The earlier catheter was used for infusions, whereas the latter catheter was used to measure central venous pressure. All catheters were filled with normal saline, closed with metal plugs, and exteriorized in the neck. Furthermore, two subcutaneous electrodcs (0.25 mm silver coated with teflon) were implanted. One was over the sacral region, whereas the other was over the right scapula. These electrodes were soldered to a miniature connector that was exteriorized in the neck and were used to record ECG (13). Following surgery, animals were allowed another 2 days for recovery.

Schoemaker et al., Myocardial infarction in rats

11

Measuring protocol The total protocol for measurements encompassed 4 days. On the first day, animals were connected to the equipment to measure ascending aorta flow (Skalar MDL 600, Skalar, Delft, The Netherlands). This flow consists of cardiac output minus coronary flow and will be referred to as cardiac output (CO). Mean arterial pressure (MAP) and central venous pressure (CVP) were recorded with miniature low volume displacement pressure transducers (CPO1, Century Technology Company, lnglewood, California, USA). Measurements were always made with the animals in a normal position. The zero reference level was taken 2 cm above the floor of the cage. Heart rate (HR) was obtained from the flow signal. All signals were monitored on a Grass Model 7D polygraph. After equilibration for 45-60 min, resting values for all variables were obtained during 15 min. Then, an infusion of a warm (37~ Ringer's-type solution (154mM NaCI, 2.7 mM KCI, 1.8mM CaCI2; pH = 7.40) was given in a volume of 12 ml during 1 min. During this time, all hemodynamics were monitored continuously. Following the infusion, monitoring was continued during another 30 min. Two days later, animals were reconnected to the equipment and again allowed to stabilize for 45-60 min. After the stabilization period they received an i.v. injection of 2 mg/kg lidocaine to prevent complications by arrhythmias that might occur during ischemia. After 2-3 min, the coronary artery ligature was pulled cautiously until typical changes indicative of ischemia (ST elevation) occurred in the ECG. In sham animals, the ligature was pulled comparably. At this point, the metal obturator was again introduced into the PE-100 catheter containing the coronary ligature. This fixed the ligature so that it could not slip loose. Hemodynamics were monitored continuously during the first 5-10 min following coronary occlusion. Twenty-four hours later, animals were again connected to the equipment, baseline readings were obtained, and the volume infusion protocol described above was repeated.

Determination of infarct size After the last measurements, animals were killed with an overdose of iv pentobarbital. The hearts were excised, washed in saline, and vessels and atria were removed. Thcn the hearts were quickly frozen ( - 8 0 ~ 15 min) and cut in slices of 1 mm from apex to base. These slices were stained with nitro blue tetrazolium (NBT) as described elsewhere (17). Briefly, the slices were incubated for 15 min in 1 mg/ml NBT in 0.1 M SGrensen phosphate buffer (pH = 7.4) at 37 ~ and then put into distilled water to stop the reaction. This procedure stains all tissue that is vital at the time of death. Thus it gives no information about the area at risk. After staining the slices, the free wall of the right ventricle was removed, so that the left ventricle including the septum remained. Then the pale part of the slices, indicating the ischemic part of the ventricle, was cut out and ischemic and non-ischemic left ventricle was weighed separately. Infarct size is expressed as percentage of left ventricular weight. No other tissues were weighed.

Data analysis Hemodynamies were monitored continuously during 45-60 rain before and the first 5-10 min following CAL and again, 24h later. Since hemodynamics stabilized within 2-3 rain following CAL, hemodynamic readings were taken before, and 5 min and 24 h following CAL. This gives information on the very acute (5 min) and short-term (24h) changes of hemodynamics. Stroke volume (SV) was calculated from CO and HR. Total peripheral resistance (TPR) was calculated as (MAP - CVP)/CO. Stroke work (SW) was calculated as SV x (MAP - CVP). Results comprise data from 11 experimental animals and 9 sham animals. Data from five animals that died (cf. Table 1) within 24h after CAL were excluded from analysis. Comparisons between resting values on the three different occasions were made by one-way ANOVA and Bonferroni's test (27), Data obtained 5 min and 24 h after coronary occlusion were compared to resting values before occlusion by one-way analysis of variance and Dunnett's test (27). From the Ringer's infusion experiments, resting and peak values for CO and CVP were obtained. Since CO plateaued after infusion of 8-10 ml Ringer's-type solution, peak value for CO may be regarded as the maximally stimulated CO. Baseline and maximally stimulated CO were compared by one-way ANOVA and Bonferroni's test. Data arc presented as means + SEM, unless indicated otherwise. Differences were regarded to be statistically significant if p < 0.05.

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Basic Research in Cardiology, VoL 85, No. 1 (1990)

Results F r o m 25 rats submitted to the surgical procedure, five animals died b e t w e e n 5 rain and 24 h after tightening the ligature. H e m o d y n a m i c s before and after CA1 of these rats are presented in Table 1. Profound reductions of blood pressure and cardiac output rather than ventricular fibrillation was the cause of death. These five animals were excluded from further analysis. Baseline values for hemodynamics of the remaining rats, divided into a sham and C A L group, are summarized in Table 2. The sham and C A L groups were not significantly different in any respect.

Infarct size Left ventricular wet weight was significantly (p < 0.05) higher in C A L (0.84 + 0.02 g) than in sham animals (0.76 _+ 0.02 g). O t h e r tissues were not weighed. The size of the infarcted area ranged from 4 to 58 % of the left ventricular mass, with a m e a n value of 28 + 5 % . In all but one of the animals (infarct size 4 % ) infarct was transmural. The septum was n e v e r involved.

Effects o f ligation on resting hemodynamics Lidocaine was administered 2-3 min before pulling the sutures. In some animals, the injection caused a transient small reduction of M A P , which, however, n e v e r lasted m o r e

Table 1. Hemodynamic characteristics of the five animals that died within 24 h after CAL.

MAP(mmHg) CVP (cm H20) HR (min -1) CO (ml. rain -t) SV (~tl) TPR (mmHg. min- m1-1) SW(mmHg-ml)

Before CAL

5 min after CAL

110+_ 4 3.6_+ 1.3 375 +_ 10 89 + 3 237 4- 7 1.20 4- 0.08 25 4- 1

60+ 9 9.2_ 3.3 324 + 38 40 + 10 132 +__27 1.54 4- 0.64 7___ 3

Table 2. Hemodynamic variables (means ___SEM) before coronary artery ligation for the animals surviving the whole protocol.

N

Sham 9

CAL 11

Body weight (g) MAP (mm Hg) CVP (cm HaO) HR (min -1) C O ( m l - m i n -a) SV (~tl) T P R ( m m H g - m i n . m l -l) SW (mmHg.ml)

308 + 9 103 + 4 2.1 _ 0.8 376 + 11 93___ 3 249 4- 8 1.124- 0,06 24.8+ 1,5

299 + 5 109 + 4 1.9 +_ 0.7 377 + 7 89+ 4 239 4- 10 1.17+ 0.07 28.14- 0.9

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Schocmaker et al., Myocardial infarction in rats

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Fig. 1. Effects of tightening of thc ligature at 5 min and 24 h in sham (C)) and CAL ( 0 ) animals. MAP: mean arterial pressure; HR: heart rate; CO: cardiac output; SV: stroke volume; TPR: total peripheral resistance; CVP: central venous pressure. Significance of the difference between sham and CAL: *p