J Mel Cell Cardiol

The

24, 106331077

Response to Ischemia Perfused Isolated

Henry L. Walters Julian R. Waggoner Division

(1992)

in Blood Perfused vs. Crystalloid Rat Heart Preparations

III, Stanley B. Digerness, III, Eugene H. Blackstone

David C. Naftel, and John W. Kirklin

of Cardiothoracic Surgery, Department of Surgecv, The lYniversit_y of Alabama at Birmingham, School of Medicine and Medical Center, UAB Station, Birmingham, Alabama, USA (Received 10 May 1991, accepted in revisedform

2 April

1992)

H. L. WALTERS 111, S. B. DIGERNESS, D. C. NAFTEL, J. R. WAGGONER III, E. H. BLACKSTONE AND J. W. KIRKLIN. The Response to Ischemia in Blood Perfused vs. Crystalloid Perfused Isolated Rat Heart Preparations.

3ournal of Molecular and Cellular Curdiolo~

(1992) 24, 1063-1077. With a research hypothesis that the behavior of blood perfused hearts was different from that of crystalloid perfused hearts, we tested the null hypothesis that the functional and metabolic status of blood-perfused (paracorporeal oxygenation) and Krebs-Henselcit (bubble oxygenation) perfused Langendorff isolated rat hearts is the same before, during and after global myocardial ischemia. Thirty isolated rat hearts were studied under identical conditions except that in equal numbers they were randomly assigned to either blood or crystalloid perfusion. In the blood perfused and crystalloid perfused hearts subjected to 22 min of normothermic ischemia and 30 min of reperfusion. mean respectively; coronary resistance increased systolic recovery was 72f3.9% (s.E.) and 20* 10% (P=O.OOl). for viscosity); mean water content after reperfusion was 21xlcl6% and 158f27% (P=O.O003) ( unadjusted 82.0+0.43%aand86.7f0.42% (P~0.000l),ATPcontentwas8.4fl.9and4.3i0.5~mol/gdrywt iP=O.O8). and energy charge was 0.74f0.114 and 0.59f0.048 (PzO.3). A major qualitative difference during reperfusion was spontaneous relaxation of contractwe and rapid resumption of sinus rhythm in blood perfused hearts, in contrast to continued contractwe and rise in intraventricular pressure in 9 of 10 crystalloid pertused hearts. One crystalloid perfused heart did not develop contractwe, and its phenomena during reperfusion were Gmilar to those of blood perfused hearts. The data support the research hypothesis, and suggest caution in extrapolating to blood perfused systems inferences from crystalloid perfused models. Better preservation of reactive hyperemia early in reperfusion may explain the better performance of blood perfused hearts. KEY WORDS:

Isolated

heart;

Blood perfusion;

Reperfusion;

Introduction The isolated rat heart model has been important in the development of knowledge of myocardial metabolism, the adverse effects of global myocardial &hernia and reperfusion, and the methods for protecting against these effects. Most of the knowledge has been obtained from asanguineously perfused preparations, although a few investigators have developed blood perfused models [I-q. The frequent extrapolation of the knowledge to human myocardial protection during cardiac surgery, where perfusion and reperfusion is largely by blood, make it important to know whether asanguineously perfused hearts behave differently from sanguineously perfused hearts. Please address all correspondence to: John W. Kirklin, Birmingham, U.4B Station, Birmingham, AL 35294, USA. rN2?~2828/92/101063

+ 15 $08.00/O

Myocardial

ischemia;

Ischemic

contracturr

Therefore, a study was undertaken to test the hypothesis that the functional and metabolic status of the isolated rat heart preparation is the same after ischemia and reperfusion, whether the perfusion during the non-ischemic periods is sanguineous or asanguineous. Materials

and Animal

Methods

models

Thirty Sprague-Dawley rats, weighing from 3 10 to 428 g were used in a randomized protocol study (Fig. 1). Five additional rats were used to obtain baseline biochemical values. They were anesDepartment

of Surgery,

The

Cnivrrsity

‘(-3 1992 Academic

of .\lahama

Prrss Limited

rlt

1064

H. L. Walters Isolated

Blood (n=l5)

III rat

et al.

hearts

Crystalloid (n=15)

perfused

/ Ischednly (n=5) I

i

Pre-ischemic function; chemical analysis, water content

(n=5’ J Chemical analysis ond water content (n=5)

FIGURE

1. Schematic

bhemio reperfusion (n= IO)

and

A only

I

I

Pre- and post-ischemic function, and (n= IO1

i Water content (n=2)

Ischemio (n=5)

perfused

i

Pre-ischemic function; chemical analysis, water content

Ischemia reperfusion (n q lO)

and

I

Preand function, (n= IO)

post-ischemic

\

presentation

Electron microscopy (n= 3)*

of protocol.

thetized, tracheotomized and ventilated with 100% 0,. A median sternotomy was made, the pericardium opened and the heart exposed. The heart was frozen within a few seconds by clasping it in precooled (by immersion in liquid nitrogen) aluminum tongs. The heart was quickly dissected away from its venoarterial connections and removed, while still in the tongs. The tissue was then processed and analyzed for high energy phosphates and their metabolites and percent water as described later, to obtain normal values. The hearts of four additional anesthetized rats were used in a blood perfused isolated heart preparation exactly as in the protocol study except that, after 20 min of the standard perfusion and without ischemia, the hearts were quick-frozen for determination of high energy phosphates and their metaboIites and water content. These additional experiments were interspersed in time among the protocol studies. All rats received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes

Chemical onolysis ond water content (n=5)

* indicates

specimens

Woter content (n=3)

lost during

Elktron microscopy (n=2f

processing.

of Health (NIH Publication No. 80-23, revised 1978). The first step in each study protocol was the administration intraperitoneally of 300 U of heparin and ( 10 min later) 18 mg of sodium pentobarbital. Crystalloid perfused model The heart and proximal aorta were quickly removed from the previously heparinized and anesthetized donor rat and immersed briefly in 4°C Krebs-Henseleit buffer while the aorta was surgically isolated. Within 60-90 s of being excised, the isolated heart was placed on the Langendorf apparatus, perfused with a modified Krebs-Henseleit (K-H) buffered solution (see Table 1) and instrumented with a left ventricular balloon (see section on Instrumentation). A constant aortic root pressure of 100 mmHg was achieved by maintaining the hydrostatic head of K-H buffer 135 cm above the level of the isolated heart at all times. The perfusate was bubbled with 95% O,-5% CO, (the PO, of the buffer perfusate, sampled from the distal portion of the arterial cannula, was 593 f 15.9 mmHg) and the temperature was maintained at 37°C with a temperature-controlled circulating water bath (Haake Inc., Saddle Brook, NJ, USA). Myo-

Blood

TABLE

Perfused

1. Electrolyte perfusates

Isolated

Rat

Heart

and glucose concentrations

in the crystalloid

Concentrations Substance Na+ K’ HCO, Glucose MgSO, PO, Ca” (ionized

1065

Model

(mMi

Blood perfusate (mean*s.n.) (n= 3) 146.5 ( zt 0.53) 3.9

only)

and blood

Crystalloid perfusatet 143

(f0.12)

5.9

* * * * 1.3 (ztO.15)

25

11 i170mg/dl) 1.2 1.2 1.12

*Not

measured. t Values known from the amounts added the Ca” which was titrated to the desired

cardial temperature was maintained at 37°C and was monitored with a thermistor (Webster Laboratories, Altadena, CA, USA) inserted retrogradely into the right ventricular outflow tract through an incision in the pulmonary artery. Blood perfused model The blood perfused isolated rat heart preparation was a modification of the paracorporeal rat heart preparation used by Hultman and his colleagues [Z-5]. A large SpragueDawley male support rat (500-600 g) was anesthetized with 30 mg of intraperitoneal sodium pentobarbital. The rat was placed supine on a copper heating plate which was maintained at 38°C by a temperature-controlled circulating bath (Haake Inc.). Through a midline neck incision, a tracheostomy was performed and the animal was ventilated and oxygenated with a rodent ventilator (Harvard Apparatus, South Natick, MA, USA). The arterial PO,, Pco, and pH of the support animal were > 100 mmHg, 3040 mmHg and 7.35-7.48, respectively, during the experiments. Sodium bicarbonate was not administered. The right and left carotid arteries and the left jugular vein were isolated and 600 units of intraperitoneal heparin were given. The left carotid artery was cannulated with a 0.034” I.D.jO.050” O.D. polyethylene catheter which was connected to the inflow side of a Cole-Parmer (Chicago) occlusion

to make level.

the

modified

K-H

buffer,

except

for

roller pump driven by a stepping motor whose RPM was precisely controlled at known rates. The outflow side of the pump was connected to the aortic cannula of the perfusion apparatus, and purged of air. The coronary flow was continually adjusted to maintain an aortic root pressure of 100 mmHg. The pump was calibrated with a graduated cylinder at the end of each experimental day in order to determine accurately the coronary blood flow associated with each setting of the stepping motor. The left jugular vein was cannulated with a 0.055” I.D./0.075” O.D. polyethylene catheter, for venous return to the support animal activated by a venous return pump (Critiko, Inc., Tampa, FL, USA) operating from the funnel reservoir. A PE 50 0.023” I.D./0.038” O.D. polyethylene catheter was introduced into the right carotid artery and attached to a pressure transducer for continuous pressure measurements. During the experiments the support animals’ mean arterial blood pressure was maintained between 50 and 80 mmHg (systolic/ diastolic pressure between 90/40 and 120/60) by varying the speed of the venous return pump. No drugs were administered. The heart donor rat was heparinized and anesthetized and the heart rapidly excised. The ascending aorta of the donor heart was cannulated and the heart suspended within a temperature-controlled plexiglass chamber. The arterial pump was activated to deliver oxygenated blood from the left carotid artery

1066

H. L. Walters

III et al.

of the support animal to the isolated heart’s ascending aorta at a rate which kept the aortic root and coronary perfusion pressure at 100 mmHg. The coronary effluent of the isolated heart was collected in the funnel and returned to the support rat by means of the venous return pump (Fig. 2). The temperature of the isolated rat heart was monitored with a thermistor (Webster Laboratories, Altadena, CA, USA) inserted into the right ventricular cavity through an incision in the pulmonary artery. This temperature was maintained at 37°C by altering the temperature of a water bath located within the plexiglass chamber. Shortly after excision of the donor’s heart, the donor rat’s blood was aspirated from the pericardium through a polypropylene filter by a syringe, and added to the perfusion circuit.

balloon was connected by a short polyvinyl tube to a pressure transducer (Gould-Statham Instruments, Inc., Hato Rey, PR, USA) and to a microliter syringe (Hamilton Co., Reno, NV, USA) [6J. All diastolic and systolic pressures were recorded from this tube. Prior to the experiment, the entire balloon system was filled with boiled water and de-aired. After the heart was placed on the perfusion apparatus, the latex balloon was placed in the left ventricular cavity through the left atrium and anchored with two 6-O prolene sutures, one placed in the mitral anulus and the other in the interatrial septum. This reliably held the balloon in place and prevented herniation through the mitral anulus. The microliter syringe allowed precise, incremental additions of boiled de-aired water to the intraventricular balloon in order to obtain pressure-volume function curves. The intraventricular balloon pressure was recorded continuously, using a Hewlett-Packard (Palo Alto, CA, USA) recorder at a paper speed of 1 cm/min. A bipolar pacing electrode was placed on the right atria1 appendage. Hearts in which the intrinsic (unpaced) rate just before the final function study was different from that during the pre-ischemic study were paced artrially at the pre-ischemic rate. The heart

Instrumentation Instrumentation was the same for all isolated rat hearts in the protocol study. Latex balloons were made by dipping aluminum molds in liquid latex (Killian Latex, Inc., Akron, OH, USA) and curing in an oven at 100°C. The molds were approximately twice the volume of the left ventricular cavity. Each

Intraventricular

balloon

Pressure

transducers

To intraventricular Isolated -

rot

Temperature

To left

FIGURE

2. Schematic

representation

syringes

balloon heart controlled

jugular

of the circuit

plexiglos

chamber

vein

in the blood

perfused

isolated

rat heart

preparation.

Blood

Perfused

Isolated

rate, unpaced, just before the pre-ischemic function study in the crystalloid and bIood perfused groups was 325 min f 26.1 (s.D.) f7.5 (s.E.) and 312minf27.2 (s.D.) Ifr 7.0 (LE.), respectively. Just before the post-ischemic function study, unpaced, it was 241 f 78.7 (S.D.) f26.2 (LE.) and 293f48.2 (s.D.) f 18.2 (s.E.). The crystalloid perfused hearts were in a poor functional state at that time isee Tables 2 and 3).

Rat

Heart

1067

Model

Immediately after the intraventricular balloon placement and the measurement of heart rate, and pacing if necessary, the intraventricular balloon was filled until the developed intraventricular pressure during systole (systolic minus end-diastolic pressure) was

increment”, a method employed by other investigators [7-H]; alternatively, the intraventricular balloon could have been emptied to obtain a true zero [II]. The pressures at peak systole and at end-diastole were measured at 0 increment volume and after incrementing the balloon volume by 10, 20 and 30 /AI successively. Zero increment balloon volume was then re-established volumetrically and maintained throughout the period of &hernia and reperfusion. For some reason, perhaps a mild, acute and transient change in ventricular compliance and/or contractility induced by the previous stepwise balloon inflation, the developed pressures were often not exactly 80 mmHg immediately after r-c-estahlishment of zero increment balloon volumt,. Thus, the mean pressure at this time in the blood perfused hearts was 87f 17.6 (S.D.J

80 mmHg,

f 4.5

Functional

‘I’ABLE

and

this

2. Developed

studies

volume

was

pressure

considered

with

“0

increasing

(S.E.)

intraventricular

mmHg,

and

balloon

in the

volumes

in

crystalloid

the

per-

prr-ischrmic

period Developed Intraventricular balloon volume inrremrnts (pl)

Blood

0

Intercept Rate of change

TABLE

(mmHg)

perfused (n= 10)

(mean

for different

3. Diastolic

1.79*0.202

(n=

with

a5 f 5.0 (P