] Mol Cell Cardiol

Changes

Heinz-Gerd

22, 1231-1243

in Heart

Zimmer,

(1990)

Function and Cardiac Cell Chronic Myocardial Infarction A. Martin

Gerdes’,

Sylviane

Size in Rats

Lortet2,

and Gerhard

with

Mall3

Department of Physiology, University of Munich, Munich, FRG, 1Department of Anatomy, University qf South Florida, Tampa, Florida, USA, 2Laboratoire de Physiologie Cellulaire Cardiaque, Universite Joseph Fourier, Grenoble, France, and 3Department of Pathology, University of Heidelberg, Heidelberg, FRG (Received 6 December 1989, accepted in revisedform

16 May 1990)

H.-G. ZIMMER, A. MARTIN GERDES, S. LORTET AND G. MALL. Changes in Heart Function and Cardiac Cell Six in Rats with Chronic Myocardial Infarction. Journal of Molecular and Cellular Cardiology ( 1990) 22, 1231-1243 Myocardial infarctions were induced in rats by ligation of the left anterior descending (LAD) coronary artery. After 4 weeks, parameters of left (LV) and right ventricular (RV) function and of peripheral circulation were measured in the intact, anesthetized animals. The morphology of the heart chambers was examined at the macroscopic and cell size level. In animals with slight reduction in LV function, LVEDP was elevated from 3.4 + 0.8 to 8.8 i 1.5 mmHg, LV stroke work was reduced by 14”/b, and dP/dt,., of both ventricles was depressed by lOa, compared with sham-operated controls. Myocytes isolated from the LV and RV were elongated to some extent and had a greater cell volume, but there was no change in heart weight. These rats had infarctions that were small or medium in size. In rats with severe depression in left heart function, cardiac output, LVSP, LV stroke work, mean arterial pressure, and the LV weight/body weight ratio were markedly lower than in sham-operated controls. LVEDP was elevated up to 32 + 2 mmHg. These rats had large infarctions. RVSP. RV weight/body weight ratio, and the volume of myocytes isolated from the RV were doubled. RV stroke work was increased by 58?&. Myocytes from the LV, RV and from the septum were elongated to about the same extent. The septum had developed a 23 9; hypertrophy. Histological examination of the lungs revealed marked thickening of the tunica medica of small pulmonary arteries with narrowing of the lumen. These changes are considered to represent the morphological basis for the increased pulmonary vascular resistance that was associated with RV pressure overload and hypertrophy. KEY WORDS: Cardiac output; hypertension; Right ventricular

Heart catheterization hypertrophy.

in rats; Myocardial

Introduction Complete ligation of the left anterior descending (LAD) coronary artery in rats leads to ischemia that is transmural. The resulting infarcts of the left ventricular (LV) free wall are variable in size from small to large. They develop rapidly and completely, since there is very low collateral flow (Schaper, 1984; Hearse et al., 1988). Due to the loss of viable myocardium, LV function is immediately diminished as evidenced by the depression in LVSP, elevation in LVEDP and decrease in All correspondence should be sent to: Heinz-Gerd Pettenkoferstr. 12, D-8000 Munich 2, FRG.

infarction;

Myocyte

size; Pulmonary

cardiac output (Zimmer et al., 1989). In this experimental situation, the non-ischemic myocardium has to compensate for the tissue loss both in metabolic and functional terms. It has been shown previously that the ATP content in the non-ischemic area becomes depleted due to augmented contraction of this zone (Noma et al., 1988; Zimmer et al., 1989). Moreover, long-chain fatty acyl-CoA-esters are increased and adenine nucleotide translocase is lower in the non-ischemic area of the dog heart (Shug et al., 1975). These changes in

Zimmer,

Department

of Physiology,

University

of Munich,

Supported by grant ROI HL30696 from the National Heart, Lung, and Blood Institute, a DAAD (Deutscher Akademischer Austauschdienst) grant from the Federal Republic ofGermany, travel grant CRG.0521/88 from NATO, grants Zi 199/4-5 and Zi 199/8-l from the Deutsche Forschungsgemeinschaft and grant No 89.006. I from the Wilhelm Sander-Stiftung. 0022-2828/90/

11123 I + 13 $03.00/O

0

1990 Academic

Press Limited

1232

H.-G. Zimmer

the non-ischemic heart become more pronounced with the duration of ischemia thus contributing to the decline of ATP. In addition, within 24 h subsequent to coronary artery ligation, there was an increase in adenine nucleotide and protein synthesis in the non-ischemic area indicating an immediate metabolic adaptation (Zimmer and Gerlach, 1982) that has been regarded as one of the first signs indicative of myocardial hypertrophy that develops after some time has elapsed (Norman and Coers, 1960; Hort et al., 1964; Turek et al., 1978; Pfeffer et al., 1979; Rubin et al., 1983; Anversa et al., 1986). It was one of the objectives of the present study to examine how frequent cardiac hypertrophy is in rats with chronic LAD coronary artery ligation and which particular heart chamber develops the most marked degree of cardiac hypertrophy. There are conflicting reports on this point in the literature. While there was a proportional increase of left and right ventricular weight in one study (Anversa et al., 1986), predominant hypertrophy of the right ventricle was found in other investigations (Hort et al., 1964; Turek et al., 1978). Another objective was to correlate the functional changes in the left and right ventricle, as well as, in the peripheral circulation with the morphological alterations both at the macroscopic and cell size level. For the characterization of left and right heart function we have used high-fidelity ultraminiature catheter pressure transducers for heart catheterization in intact, anesthetized rats. For the cell size measurements, cardiac myocytes were isolated from three parts of the infarcted hearts and sham-operated controls. Finally, the histology of the lung was examined to get information on the possible changes that may occur in the pulmonary vascular bed.

Materials

and Methods Animal

model

Female Sprague-Dawley rats, obtained from Savo-Ivanovas GmbH (Brunnthal, FRG), were used in this study. They were fed a pelleted diet (Altromin GmbH, Lage, FRG) and had free access to tap water. Myocardial infarction was induced under ether anesthesia.

et 41. The fourth intercostal space was opened, the heart was exteriorized, and the pericardium was cut. The apex of the heart was held with a small surgical forceps while the descending branch of the left anterior descending (LAD) coronary artery was ligated between the left auricle and the pulmonary outflow tract with a dermafil monofil thread (USP: S/O, HR 8, obtained from Dr Ruhland Nachf., Neustadt/Donau, FRG). Thereafter the rats were ventilated artificially through the nose with room air, and the chest was closed (Bechtelsheimer et al., 1971). The entire procedure from opening to closing the chest lasted not more than 60 s. Mortality was 42% within the first 24 h after surgery. Sham operation was done in the same way except that no ligation was performed. Hemodynamic measurements Heart and circulatory function was measured in closed-chest rats anesthetized with thiobutabarbital sodium (Inactin@ Byk Gulden, Konstanz, FRG, 80 mg/kg, i.p.) four weeks after coronary artery ligation. The depth of anesthesia was evaluated by eliciting reflexes in the legs with a forceps. If necessary, the animals received an additional i.p. injection of the anesthetic. After tracheotomy, a cannula was placed in the trachea through which the animals were breathing spontaneously. Through this cannula, the airways could be cleared, and artificial respiration could be instituted in an emergency situation. Left and right heart function was measured with ultraminiature catheter pressure transducers. Model PR-249 (Millar Instruments, Inc., Houston, TX, USA) was used for left heart catheterization and model PR-291 for right heart catheterization via the right jugular vein. The latter catheter is angled in such a way as to facilitate the advancement of the catheter tip from the right atrium into the right ventricle. The catheter model PR-249 was inserted into the right carotid artery and advanced upstream to the aorta and into the left ventricle. The catheterizations of the left (Zimmer, 1983) and right heart (Zimmer et al., 1988) were done successively. The ultraminiature catheter pressure transducers were attached to a Millar transducer control unit (model TC-100) which was connected to an

Myocardial

Infarction

HSE electromanometer (Hugo Sachs Elektronik, March-Hugstetten, FRG). Heart rate was monitored with the HSE Digi-Pulsratemeter MP (Hugo Sachs Elektronik), the maximal rate of rise in ventricular pressure and RV dP/dtmDx) with an WV Wdt,,,,, electronic differentiation system (PhysioDifferentiator, Hugo Sachs Elektronik). The recording system was a Gould Brush 2600 recorder. Calibration of pressure and of was done using a mercury manWdtm,, ometer (type 376, Hugo Sachs Elektronik) and the calibration device that is built into the Physio-Differentiator, respectively. Cardiac output was measured using the thermodilution technique. A thermodilution microprobe (1.5 French, Columbus Instruments, Inc., Columbus, OH, USA) was positioned in the ascending aorta via the right carotid artery. A bolus of 100 ,ul saline at 18°C was injected into the right atrium via a catheter placed in the right jugular vein. From the recorded temperature change (Gould Brush 280 recorder) cardiac output index was determined by a computer (Cardiomax IIR, Columbus Instruments). From the recorded parameters the pressurerate-product and the developed pressure were calculated, the latter being the difference between systolic and end-diastolic pressure. Also the pressure-volume-work of the left and right heart was obtained by multiplying the developed pressure by the stroke volume thus giving the stroke work. The experimental animals were divided into two groups: The first group (MI 1) consisted of rats which had only a slight reduction in left heart function. These rats had small or medium size infarctions. The second group of animals (MI 2) had an average reduction in LV developed pressure to 100 mmHg or below, an elevation of LVEDP to 25 mmHg or above, and a significant decline in cardiac output. These rats had large myocardial infarctions. Morphological

measurements

‘l‘he hearts of animals in which the hemodynamic parameters had been measured were divided into two groups. In one group, the hearts were removed and cut into three parts: left vrntricular free wall (including the entire necrotic area), septum and right ventricular

in Rats

1233

free wall. The weights of these regions were measured and related to the body weight of the respective animals. The size of isolated cardiac myocytes was determined from the other group of rats. To do this, the chest was opened, and the hearts were quickly removed. The aorta was immediately cannulated and attached to a modified Langendorff apparatus. Isolated myocytes were prepared by perfusion with collagenase in calcium-free Joklik medium as described by Bishop and Drummond (19791. The right and left ventricles were separated from the septum by sharp dissection and minced in calcium-free Joklik media containing 0.01 mM ethylene glycol bis @-aminoethyl ether)-N,N-tetracetic acid and poured through nylon mesh (250 pmj. Freshly isolated cells were centrifuged through 4% Ficoll (Sigma Chemical Company, St Louis, MI, USA) in 0.15 M phosphate buffer to remove unwanted debris. A Coulter Channelyzer (model C256) was used to determine volume of cells obtained from the hearts of sham-operated controls and from experimental animals (Gerdes et al.. 1986; Gerdes et al., 1987). The Coulter system determines cell volume by measuring the change in electrical resistance due to displacement of electrolyte as cells move through an aperture. The shape factor (1.05) employed in this study was based on a cell length/width ratio of 7 according to Hurley ( 1970). Cell length was measured directly using a phase microscope. Crosssectional area was calculated from cell volume divided by cell length. The histology of the lung was examined in 4 pm thin paraffin sections of formalin-fixed tissue obtained from sham-operated controls (n = 6) and from rats with severe depression in LV function (n = 6). A set of six sections through each lung at a distance of 0.5 mm was prepared. The elastica van Giesson stain was chosen for assessment of the lung vessels. A total of 180 small vessels/lung were analyzed in both experimental groups. Statistical analysis All data are presented as mean values + SAM. unless otherwise indicated. The unpaired ttest was used for calculating statistical dif-

1234

H.-G. Zimmer

et al.

ferences. A P-value of < 0.05 was considered was decreased by 16%. The septum to denote a significant difference. weight/body weight ratio was increased by 23%, and the RV free wall weight/body weight ratio by 90% (Table 2). Results In both the MI 1 and MI 2 group there Four weeks after LAD coronary artery lig- were no changes in heart rate and in total ation, the rats with slight reduction in LV peripheral resistance. In the MI 2 group, function (MI 1) had the samebody and heart mean arterial pressure and cardiac output weights as the sham-operated controls. How- were lower than in sham-operated controls ever, all animals with severe depressionin LV (Table 3). function (MI 2) had lost someweight (Table An original record from a rat with severe 1). In these animals, the weight of the LV was depressionin LV functional parameters shows about 25% lower than in sham-operated con- that LVSP and aortic pressurewere reduced. trols due to the loss of viable myocardium. LVEDP was elevated markedly (Fig. 1). The The septum had gained some weight, while mean values of all measurementsof LV functhe right ventricular free wall had a 100% tion are given in Table 4. In the MI 1 group, weight increase. When corrected for body LVSP and the pressure-rate-product were not weight, there were no significant changes in significantly altered, while LVEDP wasmodeheart weight in animals with slight reduction rately elevated, and LV dP/dt,,, was slightly, in LV function. In the MI 2 group, the total but significantly diminished. In the MI 2 heart weight/body weight ratio was elevated group, LVSP had declined by 25%. There by 25%. The LV weight/ body weight ratio was an elevation in LVEDP from 3.4 + 0.8 to

TABLE

1. Changes in body weight and in heart weights of rats 4 weeks after sham operation or LAD coronary artery ligation Body weight

Heart weight (mg)

n

(9)

LV

Septum

RV

Total

so

7

MI1

17

273 k 4 269 + 3 249 + 11*

376 f 23 347 * 12 281 f 24+

284 + 16 273 f 13 322 _+ 27

156 f 8 147 + 18 321 f 15**

808 + 25 767 + 18 924 + 45*

MI2

9

LV: Left ventricle; RV: Right All values are means f s.E.M.;

ventricle. n = number

of experiments.

*P < 0.05; +P < 0.01; **p < 0.0005 vs. so. SO: sham operation; MI depression in LV function.

TABLE

I: rats with

slight

reduction

MI

2: rats with

severe

2. Regional and total heart weight/body weight ratios for rats 4 weeks after sham operation and LAD coronary artery ligation Heart weight/body

SO MI1 MI2

in LV function;

weight (mg/g)

n

LV

Septum

RV

Total

7 17 9

1.37 f 0.07 1.30 f 0.04 1.15 * 0.07*

1.04 _+ 0.06 1.02 f 0.05 1.28 f O.lO*

0.67 f 0.03 0.55 f 0.02 1.27f0.05+

2.96 _+ 0.05 2.87 + 0.06 3.71*0.14+

All values are means f s.E.M.; n = number

of experiments.

*P < 0.05; +P < 0.0005 vs. so. SO: sham operation; MI depression in LV function.

I: rats with slight

reduction

in LV function;

MI

2: rats with severe

Myocardial

TABLE

3. Hemodynamic parameters coronary artery ligation

SO MI 1 MI 2 All

in Rats

1235

in rats 4 weeks after sham operation

and LAD

Heart rate (beats/min)

Mean arterial pressure (mm%)

Cardiac output index (ml/kg.min)

Total peripheral resistance index (mmHg.kg.min/ml!

328 f- 9 (20) 336 + 5 (30) 310 + 6 (15)

137 * 3 (20) 132 + 3 (29) 104 f 3* (15)

352 + 12 (20) 339 f 17 (14) 270 + 14* (13)

0.398 k 0.017 (20) 0.400 * 0.020 (14) 0.417 + 0.032 (12;

values

are means

*P < 0.0005 vs. so. SO: sham operation; depression

Infarction

in LV

TABLE

so MI 1 MI2

f

S.E.M. Number MI

1: rats

of animals with

slight

in parentheses. reduction

in

LV

function;

MI

2: rats

with

sevcrr

function.

4. Left ventricular functional parameters operation and coronary artery ligation

in rats 4 weeks after sham

II

LVSP CmmHg)

LVEDP (mmfk)

LV dP/dt,., (mm&/s)

LVSP x HR !mmHg/min)

20 29 14

154+ 3 150* 3 115 + 3*

3.4 k 0.8 8.8 * 1.5** 32 + 2*

8568 k 218 7687 + 250+ 4848 f 243*

49436 f 1800 51310 + 1671 35969 k 1203*

LVSP: Left ventricular systolic pressure; LVEDP: Left ventricular end-diastolic sure; LV dP/df ,,,a; maximal rate of rise of left ventricular pressure; LVSP x HR: Heart rate-product. iill values are means k s.E.M.; n = number of experiments.

presPressurr-

*P < 0.0005; **p < 0.005; +P < 0.01 vs. so. SO: sham operation; severe depression in LV

MI 1: rats function.

with

slight

reduction

Control

FIGURE el and

from

I. Original record ofleft heart a rat with severe depression

functional parameters obtained in LV function 4 weeks after

in LV

function;

MI

2: rats

with

Myocardlal lnfarctlon from coronary

a sham-operated artery ligation

control (right

rat (left hand hand side).

1236

H.-G. Zimmer

32 f 2 mmHg. LV dP/dt,., and the pressurerate-product were significantly lower than in sham-operated controls. An example of the changes in right ventricular function in a rat with severe depression in LV function is given in Figure 2. The most conspicuous alteration was the increase in RVSP. The mean values are compiled in Table 5. In the MI 1 group, RVSP and the RV pressure-rate-product were entirely normal, whereas RV dP/dt,,, was 10% lower than in sham-operated controls. In animals of the MI 2 group, RVSP was twice as high as in sham-operated controls. RVEDP was not altered (data not shown). Since heart

et al. rate did not change, the pressure-rate-product was doubled. RV dP/dt,,, was increased by about 40%. When LV and RV function was measured in the same animals, there was no change in RVSP over a wide range of LVEDP up to about 25 mmHg (Fig. 3). These rats belonged to the MI 1 group. However, when LVEDP was elevated above 25 mmHg, all animals had an increase in RVSP (MI 2 group). When the LV developed pressure was between 100 and 160 mmHg, RVSP was within the normal range (Fig. 4). When LV developed pressure was lower than 100 mmHg, RVSP was elevated (MI 2 group).

Control FIGURE 2. Original tracings of right n LV function of 4 weeks’ duration. TABLE

heart function

Myocordlal inforctlon in a sham-operated

rat and in an animal

5. Functional parameters of the right heart weeks after sham operation and LAD artery ligation

n

SO

19

MI1

13

MI2

15

RVSP (mm%)

RV dP/dt,,,

37 * 1 36+ 1 79 &y 2’

2290 + 89 2062 + 93+ 3264 f 155*

(mm&+)

in rats 4 coronary

RVSP x HR (mmHg/min)

11865 f 382 11770 f 506 23979 + 1007*

RVSP: Right ventricular systolic pressure; RV dP/dr ms; maximal rate of rise of right ventricular pressure; RVSP x HR: Pressure-Heart rate-product. All values are means + s.E.M.; n = number ol’experimrnts. *P < 0.0005: +P < 0.05 vs. so. SO: sham operation; MI 1: rats with slight reduction in LV function: MI 2: rats with severe depression in LV function.

with severe depression

Myocardial

Infarction

I”‘17

in Rats

mmHg

100

0

0 0 80

0

-

0

0

2 SD

0

0 &

T

lJ 0

t 2 SD 1

0

60-

2

. 40

-

.

2:D

.

. . . 20

”

:

2)sD I

-

I

I

,

I

IO

20

30

40

mmHg

LVEDP FIGURE 3. Right ventricular systolic pressure (RVSP) and left ventricular end-diastolic with slight reduction in LV function (closed circles: MI 1 group) and in animals with severe ‘open circles: MI 2 group). Individual data are compiled from those experiments in which measurrd in the same animal. Also indicated are the respective mean values + 2 S.D.

pressure reduction LV and

:LVEDP in rats in LX’ function RV fl1n(-tinn was

mmHg 100

0

80

0 0

-

0

T

0

0 0 00

2 SD

0

t 2 SD 1

OO

a

60-

2 (L

r

40

.

.

l .

. .

2TSD .

2$D

20

60

80

100

120

140

160

180

mmHq

LVDP FIGURE 4. Right ventricular systolic pressure (RVSP) and left ventricular developed pressure slight reduction in LV function (closed circles) and in animals with severe reduction in LV function values are from those experiments in which both LV and RV function was waluatrd in the samr next to the respectivr mean values.

iL\‘DP) in rats with (open c-irrlcsi. Thr rat. + 2 S.D. is qi\.rn

H.-G. Zimmtr

1238

The changes in RV and LV developed pressure in animals of the MI 2 group were correlated with the changes in the weights of the RV, septum and the LV which were related to body weight (Fig. 5). The pressure developed by the RV was of the samemagnitude as that generated by the LV. The increase in RV developed pressure was of a similar extent as the elevation in the RV free wall weight/body weight ratio. There was a parallel decrease in LV developed pressure and in the LV weight/body weight ratio. The changes in LV and RV function were also clearly evident when the stroke work was calculated (Table 6). In rats of the MI 1 group, only the stroke work of the LV was slightly diminished, and that of the RV was not significantly different from that of shamoperated controls. In animals of the MI 2 group, stroke work of the LV was depressedby 58%, however, that of the RV was increased by 58% so that it was as high as that of the LV. The resultsof the cell size measurementsare presented in Table 7. Cardiac myocytes iso-

1

Right

ventrde

et al.

lated from the RV and LV of rats of both groups were all significantly elongated and had a greater volume, though to a different degree. Myocytes isolated from the septum of rats of the MI 1 group were not altered, whereas those obtained from rats of the MI 2 group showed a 150/belongation. The most conspicuous result was the doubling of the volume of cells isolated from the RV free wall of rats in the MI 2 group. The cross-sectional area of these cells was increased by about 70%.

The histological examination of the lungs of rats with severe depression in LV function (n = 6) revealed that there was a marked thickening of the media of small pulmonary arteries with obstruction of the lumen (Fig. 6) when compared to sham-operated controls (n = 6). On average, 8 out of 30 small vessels analyzed showed this marked alteration. Infiltration of inflammatory cells had occurred to a similar extent in the lungs of both the sham-operated controls and the experimental rats.

Septum

Left

ventricle

(20)

l

P