Basic Res. Cardiol. 72, 636-650 (1977) 9 1977 Dr. Dietrich Steinkopff Verlag, Darmstadt ISSN 0300-8428
Max-Planck-Institut fi~r Physiologische und Klinische Forschung, W. G. Kerckhoff-Institut, Bad Nauheim
Dilatory capacity of the coronary circulation and its correlation to the arterial vasculature in the canine left ventricle Die koronare ihre Beziehung
Dilatationsreserve zur
B. W f t s t e n ,
arteriellen
D. D. B u s s ,
des linken
Ventrikels
Gef/il]versorgung
H. D e i s t ,
andW.
und
in Hundeherzen
Schaper
w i t h t h e t e c h n i c a l a s s i s t a n c e of M. C a r l With 4 figures and 2 tables (Received J u l y 22, 1977)
Summary The functional capacity of flow limiting myocardial conductance vessels was evaluated in canine hearts. In an isolated h e a r t preparation t r a n s m u r a l coronary flow distribution during m a x i m a l vasodilation was m e a s u r e d in the unloaded diastolic arrested left ventricle w i t h tracer microspheres. The ratio of subendocardial versus subepicardial (ENDO/EPI) flow in the left v e n t r i c u l a r free wall was 1.6. Measurements in 8 different wall layers showed a successive increase in m a x i m a l coronary flow from the subepicardium towards the deeper layers. A decreased subendocardial vascular resistance due to a better vascularization is f o r w a r d e d as a mechanism to compensate for the e x t r a v a s c u l a r compression during cardiac contraction. This statement contradicts the commonly accepted hypothesis that a diminished vascular tone with a reduction of the dilatory reserve in the subendocardium accounts for a homogeneous flow distribution in the normal beating heart. An augmentation of subendocardial supplying vessel capacity could be established from the angiographic determination of the coronary arterial volume of i n t r a m u r a l small arteries and arterioles. F r o m a strict parallelity in maximal coronary flow and coronary arterial volume within the wall, it becomes probable that these vascular structures are the flow-limiting factors which determine regional coronary flow reserve in the absence of e x t r a v a s c u l a r compressive forces.
T h e c o r o n a r y b l o o d flow in a n e s t h e t i z e d a n d c o n s c i o u s dogs at a n o r m a l p e r f u s i o n p r e s s u r e i s u n i f o r m l y d i s t r i b u t e d across t h e d e p t h of t h e left v e n t r i c u l a r w a l l (1-4) or is s l i g h t l y h i g h e r s u b e n d o c a r d i a l l y t h a n sube p i c a r d i a l l y (5-11). T h i s f i n d i n g s e e m s to c o n t r a d i c t t h e w e l l k n o w n f a c t t h a t a n e n h a n c e d i n t r a m y o c a r d i a l t i s s u e p r e s s u r e in t h e s u b e n d o c a r d i u m i m p e d e s c o r o n a r y flow e s p e c i a l l y i n t h e d e e p e r l a y e r s of t h e v e n t r i c u l a r w a l l (10-25). Moir a n d De Bra (1) h a v e p o s t u l a t e d t h a t c o r o n a r y a u t o r e g u l a t i o n in t h e s u b e n d o c a r d i u m a d j u s t v a s c u l a r t o n e to a l o w e r l e v e l 719
WiAsten et al., Dilatory capacity of the coronary circulation
637
f o r an a d e q u a t e s u b e n d o c a r d i a l p e r f u s i o n . D u e to such a m e c h a n i s m w h i c h i m p l i e s a l m o s t m a x i m a l u t i l i z a t i o n of a u t o r e g u l a t o r y c o m p e n s a t i o n b y t h e s u b e n d o c a r d i u m (24), it has b e e n s u g g e s t e d t h a t v a s o d i l a t i o n w o u l d r e s u l t in a r e l a t i v e u n d e r p e r f u s i o n of t h e d e e p e r l a y e r s (2). In t h e n o r m a l l y p e r f u s e d m y o c a r d i u m , h o w e v e r , w h e n c o r o n a r y p e r fusion pressure was normal, m a x i m a l vasodilation did not induce an i n h o m o g e n e o u s flow d i s t r i b u t i o n (26). F r o m such f i n d i n g s it h as b e e n c o n c l u d e d t h a t a u t o r e g u l a t i o n is n o t t h e o n l y m e c h a n i s m a v a i l a b l e f o r i n s u r i n g a d e q u a t e s u b e n d o c a r d i a l b l o o d s u p p l y , a n d a g r a d i e n t of vascularity favoring the subendocardium was postulated. However, there a r e no d a t a a v a i l a b l e w h i c h i n d i c a t e t h a t s u b e n d o c a r d i a l t i s s u e c o n t a i n s more arterioles. T h e p r e s e n t e d i n v e s t i g a t i o n w a s p e r f o r m e d in o r d e r to s t u d y t h e t r a n s m u r a l d i s t r i b u t i o n of t h e v a s c u l a r c o m p o n e n t of t h e c o r o n a r y c i r c u l a t i o n . F o r this p u r p o s e t h e f u n c t i o n a l c a p a c i t y of r e g i o n a l c o r o n a r y v a s c u l a r i t y in t h e l e f t v e n t r i c u l a r f r e e w a l l w a s m e a s u r e d a t m a x i m a l v a s o d i l a t i o n in the unloaded left ventricle and the anatomically determined arteriolar v o l u m e w a s e v a l u a t e d a n g i o g r a p h i c a l l y in t h e p o s t m o r t e m h e a r t .
Methods E x p e r i m e n ts were p e r f o r m e d in 16 adult mongrel dogs of either sex with a body weight of 25-35 kg.
Isolated heart experiments In 9 dogs anesthesia was p er f o r m ed with subcutaneous p i r i t r am i d e (5 mg/kg BW) and intravenous sodium pentobarbital (10 mg/kg BW). The chest was opened in the 5th left intercostal space during i n t e r m i t t e n t positive pressure respiration. A f t e r heparinization the h e a r t was fibrillated by increasing the voltage of a 50 cycle AC-stimulus until fibrillation occurred. The h ear t was rapidly excised, weighed, and connected to a similarly anesthetized "support" dog. Heparinized arterial blood from the support dog was delivered to the aorta of the isolation heart for cororary perfusion by a pressure controlled roller pump. Coronary sinus blood was collected and returned into the j u g u l a r vein of the support dog by a roller pump synchronized with the arterial pump. Blood t e m perature in the extracorporeal circuit was maintained at 37 ~ by heat exchangers. The isolated heart was defibrillated and during spontaneous beating the left atrium was opened, the chordae tendineae were dissected and closure of the aortic valve was proven by inspection. Total coronary inflow into the isolated h ear t was measured by an electromagnetic flowmeter which was calibrated in each experiment. The left ventricle was vented by a metal cannula inserted through the apex. A t a constant p e r f u sion pressure of about 80 m m Hg adenosine in a dose of 3-5 m g / m i n was infused into the arterial line to achieve m a x i m a l vasodilation. Maximal vasodilation was attempted, when an increase of the adenosine dose did not cause a f u r t h e r increase in coronary inflow. At a constant perfusion pressure and stabilized high coronary flow, the heart was arrested by an infusion of a 10% procain amide solution. When diastolic arrest had occurred, total coronary inflow into the isolated heart was measured f r o m the m e a n electromagnetic flow signal and regional coronary ftow was m e a s u r e d by the injection of radioactive labelled microspheres (TM). In these e x p e r i m en t s TM labelled with 1-125, Nb-95 (15# di-
Basic Research ~n Cardiology, Vol. 72, No. 6 (1977)
638
ENDO
EPI
Rv
~ RV
Fig. i. I l l u s t r a t e s t h e t e c h n i q u e for s e c t i o n i n g t h e h e a r t into t r a n s v e r s a l slices. S u b d i v i s i o n of l e f t v e n t r i c u l a r (LV) f r e e w a l l a f t e r d i s s e c t i n g the r i g h t v e n t r i c l e (RV) a n d t h e i n t e r v e n t r i c u l a r s e p t u m (SPT) into d i f f e r e n t t r a n s m u r a l l a y e r s is s h o w n in this d r a w i n g . ameter), and Ce 141, Cr-51, Sr 85, Sc-46 (8/~ diameter) were used randomized. The problem of TM aggregation was solved by suspending the spheres in i0 %~ dextran and by thorough dispersion with magnetic stirring and ultrasonic vibration before every injection. The bolus of 200 000 beads was injected directly into the arterial perfusion line at a distance of 20 cm from the coronary ostium to ensure good mixing. After fixation of the hearts in 4 ~ buffered formaldehyde the weights of the total heart, the left ventricle, the right ventricle, and the atria were taken and corrected for the slight changes produced by fixation. The total heart was dissected thereafter into approximately 200 - 250 samples (s. figure I). Sector samples from the left ventricle were subdivided into subepicardial, intermediate, and subendocardial pieces. Gamma-ray-spectrometry was carried out on each of the samples with a Nuclear data 4410-system software-controlled multichannel pulse height analysator. Details of the computer program were previously described (27). Samples of the left ventricular free wall from 2 medium slices (2nd and 3rd) were further subdivided across the depth of the wall. Each sector (approximately 6-8 sectors per slice) was thereby cut into 8 different layers from the epicardium to the endocardium with radioactivity of these tissue samples again subsequently measured. The total radioactivity of the entire heart was calculated from all sample activities. Flow of each tissue sample was calculated from the activity and the weight of the sample, since total activity and total coronary inflow at TMinjection are known. Mean flow of the entire left ventricle and mean flow to the left ventricular subendocardium and the subepicardium were calculated for each heart. From the left ventricular free wall sectors which were subdivided into 8 layers across the wall mean values of regional coronary flow of each wall layer were calculated.
W ~ s t e n et al., D i l a t o r y c a p a c i t y of the c o r o n a r y c i r c u l a t i o n
6:{9
A n g i o g r a p h i c s t u d i e s in p o s t m o r t e m h e a r t s I n 7 a n e s t h e t i z e d d o g s (5 m g / k g B W p i r i t r a m i d e s.c. a n d 10 m g / k g s o d i u m p e n t o b a r b i t a l i. v.) h e a r t s w e r e e x c i s e d a f t e r h e p a r i n i z a t i o n . A f t e r t r a n s e c t i o n of t h e a o r t a 3-4 c m a b o v e t h e a o r t i c v a l v e t h e m a i n o s t i u m of t h e left c o r o n a r y a r t e r y w a s e x p o s e d a n d a c a n n u l a of o w n c o n s t r u c t i o n w a s i n s e r t e d i n t o the o s t i u m and s e c u r e d by suture. T h e right c o r o n a r y a r t e r y w a s ligated, the h e a r t w a s i m m e r s e d in a w a t e r b a t h at a t e m p e r a t u r e of 40 C a n d t h e left c o r o n a r y c a n n u l a w a s c o n n e c t e d to a p r e s s u r e r e s e r v o i r c o n t a i n i n g a b a r i u m g e l a t i n e - m i x t u r e at t h e s a m e t e m p e r a t u r e . T h e c o n t r a s t m a t e r i a l w a s f r e s h l y p r e p a r e d before each e x p e r i m e n t .
Fig. 2. S h o w s a t y p i c a l p o s t m o r t e m c o r o n a r y a n g i o g r a m slice of t h e c a n i n e h e a r t .
of a l e f t v e n t r i c u l a r
640
Basic Research in Cardiology, -Col. 72, No. 6 (1977)
Twelve grams gelatine and 60 grams barium sulfate w e r e suspended in 100 ml saline. Before homogenisation by stirring with an u l t r a t u r r a x for 2 minutes, Sr-85 labelled TIVI were added to the b a r i u m - g e l a t i n e - m i x t u r e . The radioactivity of the contrast material was measured in several random samples to ensure good m i x i n g and for the evaluation of the specific activity, which ranged from 300 to 500 counts/rain, mg. P o s t - m o r t e m angiograms of the left coron a r y arterial system were carried out by pressure injection of the barium s u l f a t e - g e l a t i n e - m i x t u r e over a period of 6 minutes. During the first 4 minutes filling pressure was increased by steps from 80 to 130 m m Hg. The pressure was kept at 130mm Hg for f u r t h e r 2 minutes. Thereafter the connecting tube to the cannula was clamped and after disconnection the h ear t was kept in icecold saline for about 30 to 40 minutes. A f t e r fixation in 4 % buffered f o r m al dehyde, s t e r e o - X - r a y angiograms were done from the entire heart. The right ventricle and the atria were then dissected, and the left ventricle was sliced transversally into 7 to 8 slices of about 1 cm thickness. Angiograms of the LVslices were performed at 18 kV, 20 mA with exposure time of 1 minute (figure 2). F r o m two left v e n t r i c u l a r slices (2nd and 3rd) the large epicardial coronary arteries were r e m o v e d and the left v e n t r i c u l a r free wall was subdivided into 10 to 12 sector samples. Angiograms were again p er f o r m ed from the dissected LV-slices and tissue samples w i t h large i n t r a m u r a l arteries were excluded for further data processing. The rem ai n i n g wall sectors were dissected into 6 different layers from the epicardium to the endocardium. G a m m a - r a y - s p e c t r o m e t r y was carried out on each tissue sample. F r o m the weight corrected radioactivity of each sample, the specific weight of the m y o card• the specific activity, and the specific weight of the contrast material, the content of barium sulfate in each tissue sample was calculated and ex pressed in volume percent of myocardium.
Results Coronary flow m e a s u r e m e n t s in the diastolic arrested ventricle during m a x i m a l vasodilation
unloaded
left
I n t h e s e e x p e r i m e n t s w h e n t h e a c t i v e v a s c u l a r t o n e of c o r o n a r i e s in t h e i s o l a t e d h e a r t s w a s a b o l i s h e d d u e to a d e n o s i n e i n f u s i o n a n d w h e n extravascular compressive forces were avoided mean left v e n t r i c u l a r c o r o n a r y flow at a p e r f u s i o n p r e s s u r e of 76 + 5 m m H g ( m e a n • SD) w a s 486 m l / m i n 9 100 g. T h e a v e r a g e l e f t v e n t r i c u l a r c o r o n a r y flow a t t h e g i v e n p e r f u s i o n p r e s s u r e c o r r e s p o n d s to a m e a n r e s i s t a n c e of t h e l e f t v e n t r i c u l a r c i r c u l a t o r y s y s t e m of 0.157 m m H g - r a i n - 1 0 0 g . m1-1, w h e n r e s i s t a n c e is c a l c u l a t e d as t h e q u o t i e n t of p e r f u s i o n p r e s s u r e a n d m e a n flow. T h e r a t i o of s u b e n d o c a r d i a l v e r s u s s u b e p i c a r d i a l flow ( E N D O / E P I ) g r e a t e r t h a n 1 w a s f o u n d in each e v a l u a t e d h e a r t . T h e v a l u e s r a n g e d f r o m 1.11 to 1.85 w i t h a m e a n of 1.62 • 0.28 ( m e a n • SD). T h e r e a l t r a n s m u r a l d i s t r i b u t i o n of c o r o n a r y flow Can b e b e t t e r d e s c r i b e d f r o m t h e flow v a l u e s m e a s u r e d in t h e 8 d i f f e r e n t l a y e r s of t h e l e f t v e n t r i c u l a r w ai l . T h e c o r o n a r y flow in t h e m o s t s u p e r f i c i a l l a y e r 1 w a s 301 m l / m i n . 100 g. W i t h i n c r e a s i n g w a l l d e p t h m a x i m a l flow i n c r e a s e d p r o g r e s s i v e l y u p to 644 m l / m i n 9 100 g in l a y e r 6 a n d s h o w e d a s l i g h t d e c r e a s e in t h e d e e p e s t l a y e r s 7 a n d 8 (see also f i g u r e 3 a n d t a b l e 1). F r o m t h e s t a t i s t i c a l a n a l y s i s of t h e flow v a l u e s in t h e d i f f e r e n t w a l l l a y e r s b y t h e F r i e d m a n - t e s t it is d e m o n s t r a b l e t h a t t h e d i f f e r e n c e s w i t h i n
Wiisten et al., Dilatory capacity of the coronary circulation
641
T a b l e 1. M a x i m a l c o r o n a r y flow in t h e diastolic a r r e s t e d u n l o a d e d left ventricle. No. o f l a y e r
l
C o r o n a r y flow m l / m i n x 100 g SEM n
2
301 =[= 22 9
3
376 =t= 20 9
431 4- 24 9
4
5
6
7
8
474 • 36 9
537 ~ 44 9
644 -4- 58 9
613 ~ 47 9
532 ~ 39 9
6
7
Statistical evaluation "Wilcoxon-Wilcox-test" No. o f l a y e r ---> ~ 2
1
2
3
4
n.s.
-
.
3
n.s.
n.s.
-
4
n.s.
n.S.
i~.
5 6 7 8
p p p p
n.s. p < 0.001 p < 0.001 n.s.
n.s. p < 0.05 p < 0.05 n.s.
0.05), T h e r e g i o n a l m a x i m a l c o r o n a r y flow in t h e u n l o a d e d d i a s t o l i c a r r e s t e d l e f t v e n t r i c l e c a n be d e s c r i b e d as a l i n e a r f u n c t i o n of w a l l d e p t h . T h e r e g r e s s i o n e q u a t i o n is y = 309 § 41.7 x, w h e r e y ---- flow a n d x = n u m b e r of l a y e r 1-8. T h e c o r r e l a t i o n c o e f f i c i e n t r = 0.88. T h e c o r o n a r y arterial v o l u m e in the left v e n t r i c u l a r w a l t and its t r a n s m u r a l distribution T h e a v e r a g e of t h e a n g i o g r a p h i c a l l y d e t e r m i n e d c o r o n a r y a r t e r i a l v o l u m e of t h e l e f t v e n t r i c u l a r f r e e w a l l m e a s u r e d in 7 p o s t m o r t e m c a n i n e h e a r t s w a s 3.59 + 0.85 ( m e a n ___ SD) v o l u m e p e r c e n t . T h i s v a l u e does not i n c l u d e t h e e p i c a r d i a l a n d t h e l a r g e r i n t r a m u r a l a r t e r i a l vessels, since these w e r e excluded from the calculation and data processing. F r o m t h e a m o u n t of c o n t e n t of r a d i o a c t i v e l a b e l l e d c o n t r a s t m a t e r i a l in t h e m y o c a r d i u m in s i x d i f f e r e n t l a y e r s of t h e w a l l it can be s h o w n t h a t t h e v a s c u l a r c a p a c i t y i n c r e a s e s across t h e w a ! l d e p t h . C o r o n a r y a r t e r i a l v o l u m e w a s l o w e s t in t h e m o s t s u p e r f i c i a l l y l o c a t e d l a y e r w h e r e it was 2.21 _+ 0.43 v o l u m e p e r c e n t a n d it did i n c r e a s e up to 4.80 ___ 1.49 v o l u m e p e r c e n t in l a y e r 5. A r t e r i a l v o l u m e w a s s l i g h t l y l o w e r e d , i.e. 4.49 ___ 1.59 v o l u m e p e r c e n t in t h e d e e p e s t s u b e n d o c a r d i a l tissue, w h e n c o m p a r e d to th e m a x i m a l v a l u e in l a y e r 5 (see also f i g u r e 4 a n d t a b l e 2). D i f f e r e n c e s in c o r o n a r y a r t e r i a l v o l u m e w i t h i n t h e d i f f e r e n t w a l l l a y e r s w e r e s t a t i s t i c a l l y d i f f e r e n t (p % 0.001) w h e n d a t a w e r e a n a l y s e d b y the F r i e d m a n - t e s t . T h e a m o u n t of b a r i u m - g e l a t i n e in t h e l a y e r s 2, 3, 4, 5, 6 w a s s i g n i f i c a n t l y h i g h e r t h a n in t h e e p i c a r d i a l l a y e r 1. T h e s a m e r e s u l t s w e r e o b t a i n e d , w h e n t h e l a y e r s 3 to 6 w e r e c o m p a r e d to l a y e r 2, a n d also a s i g n i f i c a n t d i f f e r e n c e c o u l d b e e s t a b l i s h e d b e t w e e n l a y e r 5 a n d 3. Th e r e l a t i v e d e c r e a s e in l a y e r 6 w h e n c o m p a r e d to t h e m a x i m u m in l a y e r 5 Table 2. Coronary arterial volume in the left ventrieular myoeardium. No. of layer
1
2
3
4
5
Coronary arterial volume vol. % SEM n
2.21 0.16 7
2.74 0.12 7
3.40 0.22 7
3.91 0.27 7
4.80 0.56 7
Statistical evaluat,ion "Wileoxon-Wilcox-test" No. of layer -~:2 3 4 5 6
1
2
p
Max-Planck-Institut fi~r Physiologische und Klinische Forschung, W. G. Kerckhoff-Institut, Bad Nauheim
Dilatory capacity of the coronary circulation and its correlation to the arterial vasculature in the canine left ventricle Die koronare ihre Beziehung
Dilatationsreserve zur
B. W f t s t e n ,
arteriellen
D. D. B u s s ,
des linken
Ventrikels
Gef/il]versorgung
H. D e i s t ,
andW.
und
in Hundeherzen
Schaper
w i t h t h e t e c h n i c a l a s s i s t a n c e of M. C a r l With 4 figures and 2 tables (Received J u l y 22, 1977)
Summary The functional capacity of flow limiting myocardial conductance vessels was evaluated in canine hearts. In an isolated h e a r t preparation t r a n s m u r a l coronary flow distribution during m a x i m a l vasodilation was m e a s u r e d in the unloaded diastolic arrested left ventricle w i t h tracer microspheres. The ratio of subendocardial versus subepicardial (ENDO/EPI) flow in the left v e n t r i c u l a r free wall was 1.6. Measurements in 8 different wall layers showed a successive increase in m a x i m a l coronary flow from the subepicardium towards the deeper layers. A decreased subendocardial vascular resistance due to a better vascularization is f o r w a r d e d as a mechanism to compensate for the e x t r a v a s c u l a r compression during cardiac contraction. This statement contradicts the commonly accepted hypothesis that a diminished vascular tone with a reduction of the dilatory reserve in the subendocardium accounts for a homogeneous flow distribution in the normal beating heart. An augmentation of subendocardial supplying vessel capacity could be established from the angiographic determination of the coronary arterial volume of i n t r a m u r a l small arteries and arterioles. F r o m a strict parallelity in maximal coronary flow and coronary arterial volume within the wall, it becomes probable that these vascular structures are the flow-limiting factors which determine regional coronary flow reserve in the absence of e x t r a v a s c u l a r compressive forces.
T h e c o r o n a r y b l o o d flow in a n e s t h e t i z e d a n d c o n s c i o u s dogs at a n o r m a l p e r f u s i o n p r e s s u r e i s u n i f o r m l y d i s t r i b u t e d across t h e d e p t h of t h e left v e n t r i c u l a r w a l l (1-4) or is s l i g h t l y h i g h e r s u b e n d o c a r d i a l l y t h a n sube p i c a r d i a l l y (5-11). T h i s f i n d i n g s e e m s to c o n t r a d i c t t h e w e l l k n o w n f a c t t h a t a n e n h a n c e d i n t r a m y o c a r d i a l t i s s u e p r e s s u r e in t h e s u b e n d o c a r d i u m i m p e d e s c o r o n a r y flow e s p e c i a l l y i n t h e d e e p e r l a y e r s of t h e v e n t r i c u l a r w a l l (10-25). Moir a n d De Bra (1) h a v e p o s t u l a t e d t h a t c o r o n a r y a u t o r e g u l a t i o n in t h e s u b e n d o c a r d i u m a d j u s t v a s c u l a r t o n e to a l o w e r l e v e l 719
WiAsten et al., Dilatory capacity of the coronary circulation
637
f o r an a d e q u a t e s u b e n d o c a r d i a l p e r f u s i o n . D u e to such a m e c h a n i s m w h i c h i m p l i e s a l m o s t m a x i m a l u t i l i z a t i o n of a u t o r e g u l a t o r y c o m p e n s a t i o n b y t h e s u b e n d o c a r d i u m (24), it has b e e n s u g g e s t e d t h a t v a s o d i l a t i o n w o u l d r e s u l t in a r e l a t i v e u n d e r p e r f u s i o n of t h e d e e p e r l a y e r s (2). In t h e n o r m a l l y p e r f u s e d m y o c a r d i u m , h o w e v e r , w h e n c o r o n a r y p e r fusion pressure was normal, m a x i m a l vasodilation did not induce an i n h o m o g e n e o u s flow d i s t r i b u t i o n (26). F r o m such f i n d i n g s it h as b e e n c o n c l u d e d t h a t a u t o r e g u l a t i o n is n o t t h e o n l y m e c h a n i s m a v a i l a b l e f o r i n s u r i n g a d e q u a t e s u b e n d o c a r d i a l b l o o d s u p p l y , a n d a g r a d i e n t of vascularity favoring the subendocardium was postulated. However, there a r e no d a t a a v a i l a b l e w h i c h i n d i c a t e t h a t s u b e n d o c a r d i a l t i s s u e c o n t a i n s more arterioles. T h e p r e s e n t e d i n v e s t i g a t i o n w a s p e r f o r m e d in o r d e r to s t u d y t h e t r a n s m u r a l d i s t r i b u t i o n of t h e v a s c u l a r c o m p o n e n t of t h e c o r o n a r y c i r c u l a t i o n . F o r this p u r p o s e t h e f u n c t i o n a l c a p a c i t y of r e g i o n a l c o r o n a r y v a s c u l a r i t y in t h e l e f t v e n t r i c u l a r f r e e w a l l w a s m e a s u r e d a t m a x i m a l v a s o d i l a t i o n in the unloaded left ventricle and the anatomically determined arteriolar v o l u m e w a s e v a l u a t e d a n g i o g r a p h i c a l l y in t h e p o s t m o r t e m h e a r t .
Methods E x p e r i m e n ts were p e r f o r m e d in 16 adult mongrel dogs of either sex with a body weight of 25-35 kg.
Isolated heart experiments In 9 dogs anesthesia was p er f o r m ed with subcutaneous p i r i t r am i d e (5 mg/kg BW) and intravenous sodium pentobarbital (10 mg/kg BW). The chest was opened in the 5th left intercostal space during i n t e r m i t t e n t positive pressure respiration. A f t e r heparinization the h e a r t was fibrillated by increasing the voltage of a 50 cycle AC-stimulus until fibrillation occurred. The h ear t was rapidly excised, weighed, and connected to a similarly anesthetized "support" dog. Heparinized arterial blood from the support dog was delivered to the aorta of the isolation heart for cororary perfusion by a pressure controlled roller pump. Coronary sinus blood was collected and returned into the j u g u l a r vein of the support dog by a roller pump synchronized with the arterial pump. Blood t e m perature in the extracorporeal circuit was maintained at 37 ~ by heat exchangers. The isolated heart was defibrillated and during spontaneous beating the left atrium was opened, the chordae tendineae were dissected and closure of the aortic valve was proven by inspection. Total coronary inflow into the isolated h ear t was measured by an electromagnetic flowmeter which was calibrated in each experiment. The left ventricle was vented by a metal cannula inserted through the apex. A t a constant p e r f u sion pressure of about 80 m m Hg adenosine in a dose of 3-5 m g / m i n was infused into the arterial line to achieve m a x i m a l vasodilation. Maximal vasodilation was attempted, when an increase of the adenosine dose did not cause a f u r t h e r increase in coronary inflow. At a constant perfusion pressure and stabilized high coronary flow, the heart was arrested by an infusion of a 10% procain amide solution. When diastolic arrest had occurred, total coronary inflow into the isolated heart was measured f r o m the m e a n electromagnetic flow signal and regional coronary ftow was m e a s u r e d by the injection of radioactive labelled microspheres (TM). In these e x p e r i m en t s TM labelled with 1-125, Nb-95 (15# di-
Basic Research ~n Cardiology, Vol. 72, No. 6 (1977)
638
ENDO
EPI
Rv
~ RV
Fig. i. I l l u s t r a t e s t h e t e c h n i q u e for s e c t i o n i n g t h e h e a r t into t r a n s v e r s a l slices. S u b d i v i s i o n of l e f t v e n t r i c u l a r (LV) f r e e w a l l a f t e r d i s s e c t i n g the r i g h t v e n t r i c l e (RV) a n d t h e i n t e r v e n t r i c u l a r s e p t u m (SPT) into d i f f e r e n t t r a n s m u r a l l a y e r s is s h o w n in this d r a w i n g . ameter), and Ce 141, Cr-51, Sr 85, Sc-46 (8/~ diameter) were used randomized. The problem of TM aggregation was solved by suspending the spheres in i0 %~ dextran and by thorough dispersion with magnetic stirring and ultrasonic vibration before every injection. The bolus of 200 000 beads was injected directly into the arterial perfusion line at a distance of 20 cm from the coronary ostium to ensure good mixing. After fixation of the hearts in 4 ~ buffered formaldehyde the weights of the total heart, the left ventricle, the right ventricle, and the atria were taken and corrected for the slight changes produced by fixation. The total heart was dissected thereafter into approximately 200 - 250 samples (s. figure I). Sector samples from the left ventricle were subdivided into subepicardial, intermediate, and subendocardial pieces. Gamma-ray-spectrometry was carried out on each of the samples with a Nuclear data 4410-system software-controlled multichannel pulse height analysator. Details of the computer program were previously described (27). Samples of the left ventricular free wall from 2 medium slices (2nd and 3rd) were further subdivided across the depth of the wall. Each sector (approximately 6-8 sectors per slice) was thereby cut into 8 different layers from the epicardium to the endocardium with radioactivity of these tissue samples again subsequently measured. The total radioactivity of the entire heart was calculated from all sample activities. Flow of each tissue sample was calculated from the activity and the weight of the sample, since total activity and total coronary inflow at TMinjection are known. Mean flow of the entire left ventricle and mean flow to the left ventricular subendocardium and the subepicardium were calculated for each heart. From the left ventricular free wall sectors which were subdivided into 8 layers across the wall mean values of regional coronary flow of each wall layer were calculated.
W ~ s t e n et al., D i l a t o r y c a p a c i t y of the c o r o n a r y c i r c u l a t i o n
6:{9
A n g i o g r a p h i c s t u d i e s in p o s t m o r t e m h e a r t s I n 7 a n e s t h e t i z e d d o g s (5 m g / k g B W p i r i t r a m i d e s.c. a n d 10 m g / k g s o d i u m p e n t o b a r b i t a l i. v.) h e a r t s w e r e e x c i s e d a f t e r h e p a r i n i z a t i o n . A f t e r t r a n s e c t i o n of t h e a o r t a 3-4 c m a b o v e t h e a o r t i c v a l v e t h e m a i n o s t i u m of t h e left c o r o n a r y a r t e r y w a s e x p o s e d a n d a c a n n u l a of o w n c o n s t r u c t i o n w a s i n s e r t e d i n t o the o s t i u m and s e c u r e d by suture. T h e right c o r o n a r y a r t e r y w a s ligated, the h e a r t w a s i m m e r s e d in a w a t e r b a t h at a t e m p e r a t u r e of 40 C a n d t h e left c o r o n a r y c a n n u l a w a s c o n n e c t e d to a p r e s s u r e r e s e r v o i r c o n t a i n i n g a b a r i u m g e l a t i n e - m i x t u r e at t h e s a m e t e m p e r a t u r e . T h e c o n t r a s t m a t e r i a l w a s f r e s h l y p r e p a r e d before each e x p e r i m e n t .
Fig. 2. S h o w s a t y p i c a l p o s t m o r t e m c o r o n a r y a n g i o g r a m slice of t h e c a n i n e h e a r t .
of a l e f t v e n t r i c u l a r
640
Basic Research in Cardiology, -Col. 72, No. 6 (1977)
Twelve grams gelatine and 60 grams barium sulfate w e r e suspended in 100 ml saline. Before homogenisation by stirring with an u l t r a t u r r a x for 2 minutes, Sr-85 labelled TIVI were added to the b a r i u m - g e l a t i n e - m i x t u r e . The radioactivity of the contrast material was measured in several random samples to ensure good m i x i n g and for the evaluation of the specific activity, which ranged from 300 to 500 counts/rain, mg. P o s t - m o r t e m angiograms of the left coron a r y arterial system were carried out by pressure injection of the barium s u l f a t e - g e l a t i n e - m i x t u r e over a period of 6 minutes. During the first 4 minutes filling pressure was increased by steps from 80 to 130 m m Hg. The pressure was kept at 130mm Hg for f u r t h e r 2 minutes. Thereafter the connecting tube to the cannula was clamped and after disconnection the h ear t was kept in icecold saline for about 30 to 40 minutes. A f t e r fixation in 4 % buffered f o r m al dehyde, s t e r e o - X - r a y angiograms were done from the entire heart. The right ventricle and the atria were then dissected, and the left ventricle was sliced transversally into 7 to 8 slices of about 1 cm thickness. Angiograms of the LVslices were performed at 18 kV, 20 mA with exposure time of 1 minute (figure 2). F r o m two left v e n t r i c u l a r slices (2nd and 3rd) the large epicardial coronary arteries were r e m o v e d and the left v e n t r i c u l a r free wall was subdivided into 10 to 12 sector samples. Angiograms were again p er f o r m ed from the dissected LV-slices and tissue samples w i t h large i n t r a m u r a l arteries were excluded for further data processing. The rem ai n i n g wall sectors were dissected into 6 different layers from the epicardium to the endocardium. G a m m a - r a y - s p e c t r o m e t r y was carried out on each tissue sample. F r o m the weight corrected radioactivity of each sample, the specific weight of the m y o card• the specific activity, and the specific weight of the contrast material, the content of barium sulfate in each tissue sample was calculated and ex pressed in volume percent of myocardium.
Results Coronary flow m e a s u r e m e n t s in the diastolic arrested ventricle during m a x i m a l vasodilation
unloaded
left
I n t h e s e e x p e r i m e n t s w h e n t h e a c t i v e v a s c u l a r t o n e of c o r o n a r i e s in t h e i s o l a t e d h e a r t s w a s a b o l i s h e d d u e to a d e n o s i n e i n f u s i o n a n d w h e n extravascular compressive forces were avoided mean left v e n t r i c u l a r c o r o n a r y flow at a p e r f u s i o n p r e s s u r e of 76 + 5 m m H g ( m e a n • SD) w a s 486 m l / m i n 9 100 g. T h e a v e r a g e l e f t v e n t r i c u l a r c o r o n a r y flow a t t h e g i v e n p e r f u s i o n p r e s s u r e c o r r e s p o n d s to a m e a n r e s i s t a n c e of t h e l e f t v e n t r i c u l a r c i r c u l a t o r y s y s t e m of 0.157 m m H g - r a i n - 1 0 0 g . m1-1, w h e n r e s i s t a n c e is c a l c u l a t e d as t h e q u o t i e n t of p e r f u s i o n p r e s s u r e a n d m e a n flow. T h e r a t i o of s u b e n d o c a r d i a l v e r s u s s u b e p i c a r d i a l flow ( E N D O / E P I ) g r e a t e r t h a n 1 w a s f o u n d in each e v a l u a t e d h e a r t . T h e v a l u e s r a n g e d f r o m 1.11 to 1.85 w i t h a m e a n of 1.62 • 0.28 ( m e a n • SD). T h e r e a l t r a n s m u r a l d i s t r i b u t i o n of c o r o n a r y flow Can b e b e t t e r d e s c r i b e d f r o m t h e flow v a l u e s m e a s u r e d in t h e 8 d i f f e r e n t l a y e r s of t h e l e f t v e n t r i c u l a r w ai l . T h e c o r o n a r y flow in t h e m o s t s u p e r f i c i a l l a y e r 1 w a s 301 m l / m i n . 100 g. W i t h i n c r e a s i n g w a l l d e p t h m a x i m a l flow i n c r e a s e d p r o g r e s s i v e l y u p to 644 m l / m i n 9 100 g in l a y e r 6 a n d s h o w e d a s l i g h t d e c r e a s e in t h e d e e p e s t l a y e r s 7 a n d 8 (see also f i g u r e 3 a n d t a b l e 1). F r o m t h e s t a t i s t i c a l a n a l y s i s of t h e flow v a l u e s in t h e d i f f e r e n t w a l l l a y e r s b y t h e F r i e d m a n - t e s t it is d e m o n s t r a b l e t h a t t h e d i f f e r e n c e s w i t h i n
Wiisten et al., Dilatory capacity of the coronary circulation
641
T a b l e 1. M a x i m a l c o r o n a r y flow in t h e diastolic a r r e s t e d u n l o a d e d left ventricle. No. o f l a y e r
l
C o r o n a r y flow m l / m i n x 100 g SEM n
2
301 =[= 22 9
3
376 =t= 20 9
431 4- 24 9
4
5
6
7
8
474 • 36 9
537 ~ 44 9
644 -4- 58 9
613 ~ 47 9
532 ~ 39 9
6
7
Statistical evaluation "Wilcoxon-Wilcox-test" No. o f l a y e r ---> ~ 2
1
2
3
4
n.s.
-
.
3
n.s.
n.s.
-
4
n.s.
n.S.
i~.
5 6 7 8
p p p p
n.s. p < 0.001 p < 0.001 n.s.
n.s. p < 0.05 p < 0.05 n.s.
0.05), T h e r e g i o n a l m a x i m a l c o r o n a r y flow in t h e u n l o a d e d d i a s t o l i c a r r e s t e d l e f t v e n t r i c l e c a n be d e s c r i b e d as a l i n e a r f u n c t i o n of w a l l d e p t h . T h e r e g r e s s i o n e q u a t i o n is y = 309 § 41.7 x, w h e r e y ---- flow a n d x = n u m b e r of l a y e r 1-8. T h e c o r r e l a t i o n c o e f f i c i e n t r = 0.88. T h e c o r o n a r y arterial v o l u m e in the left v e n t r i c u l a r w a l t and its t r a n s m u r a l distribution T h e a v e r a g e of t h e a n g i o g r a p h i c a l l y d e t e r m i n e d c o r o n a r y a r t e r i a l v o l u m e of t h e l e f t v e n t r i c u l a r f r e e w a l l m e a s u r e d in 7 p o s t m o r t e m c a n i n e h e a r t s w a s 3.59 + 0.85 ( m e a n ___ SD) v o l u m e p e r c e n t . T h i s v a l u e does not i n c l u d e t h e e p i c a r d i a l a n d t h e l a r g e r i n t r a m u r a l a r t e r i a l vessels, since these w e r e excluded from the calculation and data processing. F r o m t h e a m o u n t of c o n t e n t of r a d i o a c t i v e l a b e l l e d c o n t r a s t m a t e r i a l in t h e m y o c a r d i u m in s i x d i f f e r e n t l a y e r s of t h e w a l l it can be s h o w n t h a t t h e v a s c u l a r c a p a c i t y i n c r e a s e s across t h e w a ! l d e p t h . C o r o n a r y a r t e r i a l v o l u m e w a s l o w e s t in t h e m o s t s u p e r f i c i a l l y l o c a t e d l a y e r w h e r e it was 2.21 _+ 0.43 v o l u m e p e r c e n t a n d it did i n c r e a s e up to 4.80 ___ 1.49 v o l u m e p e r c e n t in l a y e r 5. A r t e r i a l v o l u m e w a s s l i g h t l y l o w e r e d , i.e. 4.49 ___ 1.59 v o l u m e p e r c e n t in t h e d e e p e s t s u b e n d o c a r d i a l tissue, w h e n c o m p a r e d to th e m a x i m a l v a l u e in l a y e r 5 (see also f i g u r e 4 a n d t a b l e 2). D i f f e r e n c e s in c o r o n a r y a r t e r i a l v o l u m e w i t h i n t h e d i f f e r e n t w a l l l a y e r s w e r e s t a t i s t i c a l l y d i f f e r e n t (p % 0.001) w h e n d a t a w e r e a n a l y s e d b y the F r i e d m a n - t e s t . T h e a m o u n t of b a r i u m - g e l a t i n e in t h e l a y e r s 2, 3, 4, 5, 6 w a s s i g n i f i c a n t l y h i g h e r t h a n in t h e e p i c a r d i a l l a y e r 1. T h e s a m e r e s u l t s w e r e o b t a i n e d , w h e n t h e l a y e r s 3 to 6 w e r e c o m p a r e d to l a y e r 2, a n d also a s i g n i f i c a n t d i f f e r e n c e c o u l d b e e s t a b l i s h e d b e t w e e n l a y e r 5 a n d 3. Th e r e l a t i v e d e c r e a s e in l a y e r 6 w h e n c o m p a r e d to t h e m a x i m u m in l a y e r 5 Table 2. Coronary arterial volume in the left ventrieular myoeardium. No. of layer
1
2
3
4
5
Coronary arterial volume vol. % SEM n
2.21 0.16 7
2.74 0.12 7
3.40 0.22 7
3.91 0.27 7
4.80 0.56 7
Statistical evaluat,ion "Wileoxon-Wilcox-test" No. of layer -~:2 3 4 5 6
1
2
p
