The coronary microcirculation in the potassium chloride arrested heart

Jouwtal of Molecular and Cell&w Cardiology

The Coronary

Microcirculation Arrested

K. HELLBERG, Huntington

U&m&y

(1971) 2, 221-230

Mewwrial

A. RICKART,

in the Potassium Heart

H. WAYLAND

Hospital

AND R. J. BING

and The California Institute Pa.sadena,Cahfomia, U.S.A.

of Southern Califomk (Received 2 February,

Chloride

of

School of Medicine, Los Angela,

Technology

California

1971, and accepted 19 March, 1971)

K. HELLBERQ, A. RIOKART, H. WAYLAND AND R. S. BING. The Coronmy Microcirculation in the Potassium Chloride Arrested Heart. Journal of Molecular and Cellular Car&oZogy (1971) 2, 221-230. The experiments were concerned with the coronary capillary ciraulation of the left atrium in the potassium chloride arrested cat heart, using transillumination. Capillary red cell velocity was measured using cinematography and color 6lms (16 mm 24 frames/s) and frame to frame analysis of red cell progression. Optical mqmifkation on the f&n was from 20 to 30 times. Capillaries were visualized es lying on either side of the muscle fiber. Numerous intercapillary anastomoses which communicate through interconnecting loops of varying lengths were visualized. Capillary diameter varies from 4 to 8 q. Constancy of capillary red cell velocity was present at from 75 to 210 mmHg. With inoreesing perfusion pressure, the number of capillaries with discernible red cell movement increased as perfusion pressure rose (recruitment). In some capillaries, abrupt and extreme changes in red cell velocity were observed at the same perfusion pressure. About 10 to 30% of all capillaries showed counter-current flow in adjacent capillaries.

1. Introduction In a preceding paper, studies on the coronary microcirculation of the beating oat heart were described using transillumination of the left atrium and the determination of capillary red cell velocity by tine analysis [23]. Results obtained with nitroglycerin, methacholine, isoproterenol, norepinephrine and after vagal stimulation were reported. Variations in capillary red cell velooity were discovered after these drugs. There was evidence of autoregulation in the coronary microcirculation following nitroglycerin and norepinephrine, since capillary red cell velocity did not show a linear change with blood pressure [23]. Because of the cardiac contractions, it was not possible to keep the microcirculation in focus during all phases of the cardiac cycle. This prevented clear visualization of the finer structural arrangements of the capillary bed. Experiments were therefore performed to study the capillary circulation in the perfused cat heart arrested with potassium chloride. 221

222

E. HELLBERO

E!f’ AL.

2. Materials and Methods Cats weighing between 1.5 and 3.5 kg were anesthetized by the intraperitoneal injection of sodium pentobarbital(30 mg/kg wt). After tracheotomy and intubation, the animals were ventilated with oxygen by means of a Bird respirator. A left side thoracotomy was performed, the pericardium was incised and heparin (100 I.U./ kg wt) was given intravenously. A hollow glass tube (outside diameter 8 mm, Inside diameter 6 mm) was introduced into the left atrium through the left atria1 appendage and kept in position by a suture (Figure 1). The glass tube had a flat upper surface of 5 mm x 12 mm which could be directed gently against the atria1 muscle. Through this hollow light pipe a round quartz rod (diameter 3 mm) was introduced. This was 1 cm longer than the glass tube and had a reflecting surface of 45” angle which directed the light

Blood reservoir

coronary or1ery

FIGURE 1. Shows the perfusion systems, the polyethylene circumflex ooronwy artery and the transilluminating system.

tubing

introduced

into the left

perpendicular to the axis of the rod and through the atria1 muscle toward the objective of the microscope. By means of this light rod the left atria1 muscle was transilluminatecl. In experiments carried out to see whether the pressure of the tubing against the atria1 wall influenced capillary red cell movements, it was shown that red cell velocity diminished only at pressures of more than 500 N/ms. Care was taken to keep the pressure exerted on the atria1 muscle below this value. The light source consisted of an experimental xenon arc (Varian Associates) with an elliptical reflector forming a real image of the source exterior to a plane saphire window. The light rod was positioned so that the center of the image of the arc was formed just at the end of the quartz rod. The xenon arc was driven by a power supply (Chadwick-Helmuth) giving pulsed excitation of the arc of 30 to 50 ms duration, with a flasher synchronized with the shutter on a ciue camera by means of a reluctance pickup. In experiments where the temperature was measured at the atria1 end of the light pipe, the rise in temperature during 10 min observation was less than 1°C. After the accurate positioning of the light pipe in the left atrium, the heart was arrest.ed by intravenous infusion of potassium chloride (from 3 to 5 ems of a

CORONARY MICROC?IRCULATION

223

20% solution). A heparin-filled polyethylene catheter (PE-5O/C15, diameter of 1 mm) was then carefully introduced into the left circumflex artery so that its tip was about 2 to 3 mm within the vessel. This was kept in position by a ligature. Care was taken not to insert this tube too far so as to avoid interference with the blood supply to the left atrium. The time elapsed between cardiac arrest and perfusion of the left atrium was usually less than 10 min. The animal table and optical system were part of an intravital microscopic system developed by Wayland and associates. The basic animal table consisted of a heavy stainless steel platform capable of lateral movements by means of position screws which permitted the entire preparation consisting of animal, light pipe, pulsed light source and parts of the perfusion system to be moved as a unit with respect to the optical train. The objective system was optically coupled to the desired tine camera by means of a front surface mirror. Beam-splitter viewing was used to focus and to monitor the field for the motion pictures which were taken with a 16 mm tine camera at 24 frames/s. The perfusion system is shown in Figure 1. An occlusive calibrated Sigma pump delivered each preset flow constantly against pressures up to 1000 mmHg. The perfusion fluid was brought to body temperature with a heat exchanger; the electronically integrated mean perfusion pressure was obtained from a side tube placed near the polyethylene catheter previously inserted into the left circumflex artery. The perfusion pressure was measured by means of a pressure transducer (Statham P23Db) and monitored on a Sanborn recorder. The perfusion fluid consisted of oxygenated heparinized blood (100 I.U./lOO ems) drawn from anesthetized cats which had been bled immediately prior to the experiment. To prevent contraction of the heart muscle, 5 cm3 of 20% potassium chloride solution were added to each 100 cm3 of blood. The red cell velocity in the capillaries was measured by frame to frame analysis in a film analyser of motion microcinematographs taken on 16 mm color film at 24 frames/s. The optical magnification on the film was 20 to 30 times, verified in each instance with a reference scale. Because of the slight variations in red cell velocity between different capillaries, the following procedure was adopted in order to obtain mean red cell velocity: red cell velocity in every capillary was averaged over the period of observation (15 to 25 s) and the arithmetic mean was then calculated for all capillaries with discernible flow in each microscopic field.

3. Results The anatomical pattern of the coronary microcirculation of the left atrium of the potassium chloride arrested heart is illustrated in Plate 1. An artery branches into smaller arterioles dividing into individual capillaries. These capillaries lie on either side of the muscle fibers. The capillary diameter varied from 4 to 8 pm. Numerous interconnections between these capillaries at higher magnifications are seen. These

224

K. HELLBERG

TBLE Perfusion pressure

(mmHg)

Mean red cell velocity in open capillaries

(pm/s;

40 60 65 75 90

ET AL.

1

Open capillaries with flow to total capiky count

mean&S.E.)

115

155 180 210 260 305

96&15 83f15 113&13 18lf25 311f20

of

(%I

63f 12 2S&lO 68f16 97+14 117&5 102&20

Number

experiments

30 25 35 40 60 65 75 90 80 85 80

6 7 9 10 10

10 10

9 7 8 6

intercapillary anastomoses can form interconnecting loops of different lengths. The pattern on the venous side is similar to that observed on the arteriolar side. Table I and J?igure 2 illustrate the relationship of the perfusion pressure to mean red cell velocity in intramuscular capillaries. Mean red cell velocity was obtained

I

Venules

500 2 E *

400 -

P. 2 -0 t 300 = E x (r 200 -

P

100 -

Ip

Y

Capillaries

L

-

Perfusmn

1

I 200

I 100 pressure

( mmHg

300 )

FIGURE 2. Shows the relationship of perfusion pressure to red cell velocity. It may be seen that at perfusion pressures ranging from 75 to 210 mmI%g average red cell velocity in oapik’ies remains constant. In contrast, red cell velocity in venules increases as perfusion pressure rises.

PLATE intramuscular capillaries.

1. Shows a microphotograph of a coronary arkry branching into capillaries. It may be seen that each muscle fiber is surrounded x 200.

arterioles and by a pair of

[f&fig

page

2241

225

CORONARYMIOROCIRCULATION

as described above. No red cell movement was observed until the perfusion pressure had reached 40 mmHg. From 40 to 75 mmHg, red cell velocity incressed almost linearly with the perfusion pressure. From this level on up to 210 mmHg, however, mean red cell velocity in intramusculttr capillaries remained essentially constant (Table 1 and E’igure 2). However, when perfusion pressures exceeded 210 mmHg mean red cell velocity again increased linearly with the perfusion pressure. In contrast, mean red cell velocity in venules, as measured by a procedure identical to that used for the capillary mean red cell velocity, increased linearly with perfusion pressure (Figure 2). Figure 3 and Table 1 illustrate that the number

0

400

-

-80

Cl 0

T E 1

0

-3oo.5 0 5

f

0

B 0 200B IT - 20

100 -

IL 100 Perfusion

I

I

200

300

pressure

( mmHg

I

FIGURE 3. Shows the relrttionship of perfusion pressure to red cell velocity. As in Figure 2, the relationship of average red cell velocity in capillaries is illustrated. As perfusion pressure rose, the percentage of open capillaries increased (recruitment), 0, Percentage of open oapilIaries; 0, mean red cell velocity in capillwies.

of capillaries with discernible red cell movement increased as perfusion pressure rose. Apparently the discrepancy in red cell velocity between capillaries and venules which was observed with rising perfusion pressure, was the result of an increasing number of perfused capillaries feeding the venules (recruitment of capillaries) (Figure 3). The data illustrated in Figure 2 and Table 1 are average figures for red cell velocity in capillaries with discernible flow because minor fluctuations were observed in all vessels. However, in some capillaries, not included in these tables, abrupt and extreme changes in red cell velocity were observed at the same perfusion pressures. As illustrated in Table 2, in one specific capillary for instance, red cell velocity increased within 3 s from 0 to a,bout 700 pm/s.

226

H. RELLBERG

TABLE 2. Intermittent Time (s) o-3 4-6 7-9 IO-12 13-15

Capillary I ( wls) 458 0 53 0 900

ET AL.

flow (red cell velocity in capillaries).

Capillary II Capillary III (w/s) (f-43 0 0 0 0 0

0 0 0 0 688

Capillary IV (tLm/s) 63 248 156 125 655

Most of the red cells in intramuscular capillaries flowed in the same direction; however, in about 10 to 30% of all capillaries direction of red cell movements in adjoining capillaries occurred in opposite directions (counter current). This was seen particularly in capillaries joined together by large connecting loops. In some instances the rate of red cell velocity increased in one capillary branch while the velocity in a connecting branch decreased.

4. Discussion It was the purpose of these experiments to study the capillary circulation in the perfused left atrium of the cat heart arrested with potassium chloride; therefore the capillaries remained in focus and the coronary microcirculation could be observed uninfluenced by contractions of the heart muscle. It has previously been shown [la], that the potassium chloride arrested heart maintains its usage for at least 2.5 h. In addition, preservation of shortening of actomyosin bands persist for even longer periods of time. For these reasons, it is felt that interruption of perfusion for 10 min after arrest with potassium chloride is not detrimental to the viability of the preparation. The anatomical pattern of the coronary microcirculation of the left atrium in the potassium chloride arrested heart is illustrated in Plate 1. Larger arterioles branch into smaller vessels and divide into individual capillaries which lie on either side of the muscle fibers. There are numerous interconnections between the capillaries which form intercapillary anastomoses through interconnecting loops of different lengths. This pattern is similar to that described in skeletal muscle by Spalteholz [ZZ] in 1888, who mentioned freely branching arteries with numerous anastomoses which form a primary network; this in turn gives off anastomosiug small arteries which form a second network. The arterioles split up into a large number of capillaries which run parallel to the muscle fibers with numerous anastomoses. Clark [4] as well as Walls [24] found that the capillaries running longitudinally between muscle fibers are interconnected by transverse vessels which run over or under the intervening fibers and thus form a fine capillary network. Zweifach and Metz [ZS]

CORONARY MICROCECULATION

227

found in the mesentery many anastomoses between both arterioles and venules which form a series of arcades. In our experiments anastomoses between arterioles or between arterioles and venules could not be seen. The anatomical pattern in the myocardium appears to be similar to that in skeletal muscle. Wearn [25] in 1928 observed that almost every cardiac muscle fiber was in direct contact with one capillary and some fibers were touched by two or more. A similar pattern was observed in our experiments. However, since the fibers in our preparations were not stained, the accurate relationship between them and the surrounding capillaries could not be ascertained. Reynolds et al. [Zl] concluded from cross-sections of ventricular muscle that the capillary vessels running between heart muscle fibers, were elliptical rather than round. They found an average capillary diameter of from 3.5 to 7.2 pm, which is in general agreement with previous observations from this laboratory [23]. According to Johnson [lo], autoregulation is the ability of an organ to adjust its blood flow in accordance with its needs. The term however, also applies to the intrinsic tendency of an organ to maintain constant blood flow despite changes in arterial perfusion pressure [lo]. Autoregulation in the heart was observed by Berne [I], Olsson [18], Cross [5] and others [Y’, 273. Previously Tillich et al. [23] had noticed autoregulation of the coronary microcirculation under certain experimental conditions in the beating cat heart i?~ situ. In the experiments reported here, no autoregulation using this definition was observed. From 40 to 75 mmHg, red cell velocity increased linearly with the perfusion pressure. Up to 210 mmHg red cell velocity in intramusuclar capillaries remained essentially constant. Above 210 mmHg, mean red cell velocity again increased linearly with the perfusion pressure. Potassium chloride may paralyze the smooth muscles in the vascular wall. It may also produce vasodilation by depolarization of the vascular smooth muscles as shown by Overbeck et al. [19] and Dawes [6]. The finding that capillary red cell velocity remains constant, within a range of perfusion pressure of from 70 to 210 mmHg, could then be explained by a purely passive behavior of the vascular bed and by an increasing number of capillaries perfused with increasing pressure (recruitment of capillaries). The course of recruitment itself remains not understood. As illustrated in Figure 3 and Table 1 the number of capillaries with discernible red cell movements indeed increases as perfusion pressure rises (recruitment). Blesa et al. [2] also postulated the presence of recruitment from the dynamics of potassium exchange in the ventricular septum of rats. It is possible, as stated by Martini and Honig [IT] and by Kirk [15] that recruitment of capillaries also takes place in response to hypoxia. Apparently the number of perfused capillaries rises porportionally with the perfusion pressure, since red cell velocity in the venules increases linearly. Minor fluctuations in red cell velocity were observed in all capillaries, This tendency for spontaneous changes in red cell velocity with time appears to be a

228

K. HELLBERQ

Time

interval

ET

AL.

(?.I

FIGURE 4. Shows the relationship between the red cell velocity and time in seconds at a constant perfusion pressure of 170 mmHg. Marked variations in red ceil velocity at constant perfusion pressure are seen in some capillaries.

universal feature of the capillary circulation [ll, 12, 26,291. More extreme changes in red cell velocity were observed in some capillaries at the same perfusion pressure. This is particularly evident from Figure 4 in which red cell velocity increases from 0 to 688 p.m within 3 s. Webb and Nicoll [26] reported these spurts in the bat wing; Clark [3] in the rabbit’s ear and Zweifach [29] in the mesentery. Johnson and Wayland [ll] provided graphic evidence that flow velocity in the capillary bed wss remarkably non-uniform. In their experiments mean flow in adjacent capillaries differed as much as tenfold. The spurts seen in our experiments may be the results of an incomplete paralysis of the precapillary sphincter due to potassium chloride. It is also possible, that as Johnson [13] and Palmer [20] proposed an occasional plugging of the capillary lumen by leucocytes may alter red cell velocity in individual capillaries. In several instances counter-current flow in adjacent capillaries was observed. This occurred primarily in capillaries with interconnecting loops. Such counter currents were also observed by Grote et al. [8] and by Hubmann et al. [9] in the perfused rat heart. This counter-current flow may be of importance for the oxygenation of the heart muscle. Luebbers [lS] stresses that capillary structure as well as direction of capillary flow are important for the oxygenation of the tissues. In order to support his statement, he devised two different models: one on the basis of parallel capillaries with unidirectional flow, the second on the basis of parallel capillaries with some counter-current 0ow. He states that in the 6rst case, assuming constant oxygen supply many areas of the tissue hsve oxygen pressure below

CORONARYMICROCIRCXJLATION

229

1 mmHg. With counter-current flow and asymmetric capillary structure on the other hand, much more favorable oxygen distribution in tissues was found. Acknowledgments

This research was supported by grants from the Council for Tobacco Research, the American Medical Association Committee for Research on Tobacco and Health, the Los Angeles County Heart Association, and U.S. Public Health Services grant no. HE 08977. REFERENCES 1. BERNE, R. M. Cardiodynamics and the coronary circulation in hypothermia. Annals of the New York Academy of Sciences 80,365-383 (1959). 2. BLESA, E. S., LANCER, G. A., BRADY, A. J. & SERENA, S. D. Potassium exchange in rat ventricular myocardium : its relation to rate of stimulation. American Journal of Physiology 219, 741-754 (1970). 3. CLARK, E. R. & CLARK, E. L. Observations on living performed blood vessels as seen in a transparent chamber in the rabbit’s ear. American Journal of Anatomy 49,441478 (1932). 4. CLARK, W. E. LE GROS The Tissues of the Body, p. 144. New York: Oxford University Press (1952). 5. CROSS,c. E. Influence of coronary arterial pressure on coronary vasomotor tonus. Circulation Research 15, Sup$ement 1, 87-93 (1964). 6. DAWES, G. S. Vasodilator action of potassium. Jourrull of Phy&ology, London 99,224-238 (1941). 7. DRISCOL, T. E., MOE, T. W. & ECKSTEIN, R. W. Vascular effects of changes perfusion pressure in the nonischemic and ischemic heart. Circulation Research 15, Supplement 1,94 -102 (1964). 8. GROTE, J., HWMA~N, W. & NIESEL, W. Untersuchungen ueber die Bedingungen fuer die Sauerstoffversorgung des Myokards an perfimdierten Rattenherzen. II. Zur Fur&ion des Myoglobins. Pjliiger’s Archiv fiir die gesamte Phyaiologie des Mensohen und der Tiere 294, 256-264 (1967). 9. HUEIXANN, W., NIESEL, W. & GROTE, J. Untersuchungen ueber die Bedingungen fuer die Sauerstoffversorgung des Myokards an perfundierten Rattenherzen I. Zur Frage nach dem Vorliegen einer Gegenstromversorgung. Pfliiser’s Archiv fiir die gesam& Physiologic o!esMen&en und der Tiere 294, 250-255 (1967). 10. JOHNSON, P. C. Review of previous studies and current theories of auto-regulation. Circulation Research, 15, Supplement 1, 2-Q (1964). 11. JOFINSON, P. C. & WAYLAN~, H. Regulation of blood flow in single capillaries. American Journal of Physiology 212, 1405-1415 (1967). 12. JOHNSON, P. C. Autoregulation of blood flow in the intestine. Gastroenterology 52,435-443 (1967). 13. JOHNSON, P. C. Regulation of flow in the microcirculatory system. In Microcirculation, Perfusion, and Transplantation of Organs, pp. 44-58 (T. I. Malinin, Linn,B.S.,Callah~,A.B.&Warren,W.D.,Eds).AcademicPress:NewYork(1970). 14. KA~DESCH, M., HOGANCAMP, C. E. & BING, R. J. The survival of excitability energy production and energy utilization of the heart. Cimulation 18, 935-945 (1958). 15. Krn~, E. S. Capillary density: effect on K-42 from coronary blood. Physiologist, Washington 10,222 (1967).

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