Association Between Hemodynamic Parameters and the Degeneration of Sustained Ventricular Tachycardias into Ventricular Fibrillation in Rats

J Mol Cell Cardiol 29, 3091–3103 (1997)

Association Between Hemodynamic Parameters and the Degeneration of Sustained Ventricular Tachycardias into Ventricular Fibrillation in Rats Andreas Hagendorff, Christian Vahlhaus1, Werner Jung, Claus Martin1, ¨ Gerd Heusch1 and Berndt Luderitz Department of Cardiology, University of Bonn, 53105 Bonn, Germany; 1 Department of Pathophysiology, University of Essen, 45122 Essen, Germany (Received 16 April 1997, accepted in revised form 14 July 1997) ¨ A. H, C. V, W. J, C. M, G. H  B. L. Association Between Hemodynamic Parameters and the Degeneration of Sustained Ventricular Tachycardias into Ventricular Fibrillation in Rats. Journal of Molecular and Cellular Cardiology (1997) 29, 3091–3103. Sustained ventricular tachycardias (VT) often degenerate into ventricular fibrillation (VF). In the present study, the impact of VT on˙ mean arterial blood pressure (MAP), myocardial blood flow (MBF), and myocardial oxygen consumption (MVO2) was assessed. In addition, the degeneration of sustained VT into VF was analysed with respect to MAP. MBF was measured in 48 anesthetized rats with colored microspheres; arterial catecholamine levels were measured by HPLC in 16 additional rats during control conditions and VT. MBF (4.66±1.29 ml/g/min; mean±..) did not change with the onset of VT (5.37±1.92 ml/g/min, ..). Epinephrine (0.22±0.13 ng/ml) and norepinephrine (0.37±0.12 ng/ml) increased during VT (3.55±2.68 ng/ml, P<0.01; 0.88±0.44 ng/ml, P<0.05), respectively. VF was more frequent when MAP remained normal (MAP>80 mmHg: 26%) than with hypotension (MAP<80 mmHg: 2%, P<0.05). Mechanical failure was observed in 10% of rats with severe hypotension (MAP<60 mmHg), and 2% with moderate hypotension (MAP 60–80 mmHg). The endo-epicardial MBF ratio in the VF group was significantly lower than that in the non-VF group (0.94±0.17 v 1.11±0.24, P<0.05). Conclusions: severe hypotension predisposes to the occurrence of acute mechanical failure during VT; moderate hypotension during VT, however, serves as a protective mechanism against VF in structurally normal hearts. Subendocardial hypoperfusion in the presence of an increased energy demand during VT is suggested to be responsible for the initiation of VF.  1997 Academic Press Limited K W: Myocardial blood flow; Ventricular tachycardia; Ventricular fibrillation; Plasma catecholamines; Rat.

Introduction Predisposing and triggering factors of malignant ventricular arrhythmias are discussed in terms of a biological model, with elements derived from both structural pathology and functional pathophysiology (Verrier et al. 1974; Bernier et al. 1989; Myerburg et al. 1989). The structure/function concept implies that the initiation of fatal arrhythmias

requires a pre-existing structural abnormality which is modified by functional changes. Structural factors predisposing to ventricular tachycardia (VT) or ventricular fibrillation (VF) are myocardial infarction, fibrosis, aneurysm, hypertrophy, cardiac tumor, dilated cardiomyopathy, infiltrative and inflammatory heart disease as well as abnormalities of the specialized conduction tissue. Functional changes predisposing to VT/VF are myocardial

Please address all correspondence to: Andreas Hagendorff, Department of Cardiology, University of Bonn, Sigmund-Freud-Straße 25, D – 53105 Bonn, Federal Republic of Germany.

0022–2828/97/113091+13 $25.00/0

mc970537

 1997 Academic Press Limited

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ischemia, reperfusion, alterations in systemic hemodynamics, and sympathetic activation (Corr et al. 1986; Kopin, 1989; Priori and Schwartz 1989). An impairment of myocardial perfusion during tachycardia induced by sympathetic activation is a particularly dangerous scenario for the occurrence of VF in the experimental and clinical setting (Heusch and Deussen, 1983; Schwartz et al., 1984; Heusch et al., 1985; Heusch, 1990; Meredith et al., 1991; Wit and Janse, 1992). The aim of the present study was to analyse the degeneration of VT into VF using a previously described model of VT in rats simulated by rapid ventricular pacing (Hagendorff et al., 1994). In this model, myocardial blood flow (MBF) regulation is preserved during moderate hypotension within the first minutes after the onset of VT. During pacinginduced tachycardia, MBF is predominantly increased in the epimyocardial layers (Fedor et al., 1980). Sympathetic activation during sustained VT, however, may shift the lower limit of MBF autoregulation to higher values of mean arterial blood pressure (MAP) and may limit myocardial perfusion, particularly in the subendocardium secondary to a-adrenoceptor mediated vasoconstriction (Dole and Nuno, 1986). A dysbalance of energy demand and consumption during hemodynamically compromising VT is then caused by the reduction of perfusion pressure, the compressing forces induced by the raised intracavitary pressure, and by the increase of energy consumption. Therefore, we hypothesized that MAP during VT influences the outcome and the degeneration of VT into VF. Measurements of left ventricular pressure, aortic pressure, MBF, myocardial oxygen con˙ sumption (MVO2), and plasma catecholamines were performed during sustained VT, and the results correlated to the incidence of VT-degeneration into VF.

Materials and Methods The experimental protocol conforms to the guidelines of the American Physiological Society, and it was approved by the authorities of the district of Cologne.

General preparation In 48 anaesthetized (thiobarbital, 100 mg/kg i.p.) Sprague–Dawley rats, weighing 250–350 g, two

polyethylene catheters were inserted into the femoral arteries for continuous blood pressure recording (Gould Statham transducer, Gould, digital recording oscilloscope DRO 1604, USA), intermittent blood sampling for blood gas analysis (ABL 505, Radiometer Copenhagen, Denmark), determination of hemoglobin concentration and serum electrolytes (OSM3, Radiometer Copenhagen, Denmark), and for the reference withdrawal during the microsphere injection. A polyethylene catheter was introduced into the left ventricle via the right common carotid artery. During this preparation, the right common carotid artery was ligated in all animals, including the sham-operated controls. An epicardial lead was positioned at the right ventricular apex for rapid ventricular pacing. ECG (limb leads) was continuously recorded (Gould, digital recording oscilloscope DRO 1604, USA; Mingograf 82, Siemens, Germany). Pacing as well as pulse rate were 600/min. In previous experiments lower pacing rates than 600/ min rarely compromised systemic hemodynamics; normally, the higher the pacing rate, the higher the MAP-reduction. At a pacing rate of 600/min the frequency of normotensive, moderately hypotensive or severely hypotensive animals was approximately equal.

Myocardial blood flow Regional myocardial blood flow (MBF) was measured with colored polystyrene microspheres of 15 lm diameter (Triton Technologies, Inc., San Diego, USA) (Kowallik et al., 1991; Hagendorff et al., 1994; Hakkinen et al., 1995; Hiller et al., 1996). About 300 000 microspheres were dissolved in 0.3 ml physiological sodium chloride containing 0.02% Tween 80. White-, yellow-, red-, blue-, or violet-colored microspheres were randomly assigned to the measurements and were injected into the left ventricle (LV) through the LV-catheter within a time frame of 20 s. Systemic hemodynamics remained unchanged during the injection. The reference flow was withdrawn starting 10 s before, and then during, and for 30 s after the microsphere injection at a rate of 0.65 ml/min (withdrawal pump: Braun, Melsungen, Germany). At the end of the experiment, animals were killed and their hearts removed. The left ventricle was cut into two slices in a plane parallel to the basis; each slice was divided into a subendocardial and a subepicardial layer by cutting the left ventricular wall through the mid-myocardium. After weighing each tissue sample (BP 310S,

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Sartorius, Germany) the colored microspheres were quantified by their dye content. Microspheres were filtered through a polyester filter (Costar, Bodenheim, Germany: pore size, 8 lm; diameter 25 mm; Nucleopore) after digestion of the tissue samples with 4  KOH and the blood samples with 16  KOH solution. A special high-grade steel vacuum filtration chamber was constructed in order to avoid loss of microspheres during filtering. The dye was recovered from the microspheres by adding 100 ll dimethylformamide. The dye solution was then transferred into 0.3-ml glass tubes and separated from additional particles and microspheres ¨ by centrifugation (Rotina 48, Hettich, Dusseldorf, Germany, 10 min, G=1000). Then, spectrophotometry was performed using an uv/visible ¨ spectrophotometer (model DU64, Beckman, Dusseldorf, Germany, wave length range: 300–820 nm with 1 nm optical band width). Since microspheres with five different colors were used for multiple MBF measurements in each animal, the total spectrum between 300 and 820 nm was measured. The spectra were transferred to a personal computer (soft¨ ware: Beckman, Dusseldorf, Germany). The composite spectrum of each dye solution was resolved into spectra of single constituents by a matrix inversion technique using the Dye-Trak-MISS software package (Triton Technologies, Inc., San Diego, USA). MBF was calculated according to the following equation:

where ArI, autoregulation index; d MBF= MBFA−MBF; MBFA, MBF during control condition; MBF, MBF during VTgroup II or III; dMAP= MAPA−MAP; MAPA, MAP during control conditions; and MAP, MAP during VTgroup II or III. Calculation was performed using the mean MBF- and MAP-values of the groups A, II and III. A trans-sternal thoracotomy was performed in 12 additional, artificially ventilated (respirator pump: Braun, Melsungen, Germany) rats to expose the great cardiac vein for coronary venous blood sampling for the determination of myocardial oxygen ˙ consumption (MVO2). A needle tip (diameter 0.05″) was inserted into the great cardiac vein, and the minimal distance to the right atrium was 1 mm during coronary-venous blood sampling. Blood samples were taken with a small syringe during sinus rhythm ˙ as well as twice during pacing conditions. MVO2 was determined from the oxygen saturations of arterial and coronary-venous blood samples by the following equations:

MBF (ml/g/min)=As×Vref×Aref−1× Ws−1

where AvdO2, arterio-coronary-venous oxygen content difference; and MBF, myocardial blood flow.

where MBF, myocardial blood flow; As, absorption of tissue sample; Aref, absorption of reference flow; Vref, reference flow; and Ws, weight of tissue sample. The subendo-subepicardial MBF-ratio (R) was calculated by the equation: R=MBFendo/MBFepi where MBFendo, regional blood flow of the left ventricular subendomyocardial layers; and MBFepi, regional blood flow of the left ventricular subepimyocardial layers. Coronary resistance (CR) was calculated by the equation:

AvdO2 (ml/ml blood)=0.134×Hb ×(SaO2−ScvO2)

[1]

where AvdO2, arterio-coronary-venous oxygen content difference; Hb, hemoglobin concentration; SaO2, arterial oxygen saturation, and ScvO2, coronary-venous oxygen saturation. ˙ MVO2 (ml/g/min)=AvdO2×MBF [2]

Left ventricular function The left ventricular pressure was continuously recorded. The maximal dP/dt and peak left ventricular systolic pressure (LVSP) were determined to estimate systolic function. Peak negative dP/dt and left ventricular enddiastolic pressure (LVEDP) were measured to assess diastolic function. In order to estimate cardiac work, the pressure-rate-product (PRP) was calculated.

CR (mmHg×g×min/ml)=MAP/MBF where MAP, mean arterial blood pressure; and MBF, myocardial blood flow. Coronary autoregulation was characterized using the autoregulation index (ArI). ArI was calculated according to the following equation: ArI=1−dMBF×dMAP−1×MAP×MBF−1

Catecholamines For the measurement of plasma epinephrine and norepinephrine 1 ml arterial blood samples were obtained from the femoral artery. The blood samples were centrifuged immediately after withdrawal (10 min, G=1000), and the plasma was collected.

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The plasma samples were frozen in liquid nitrogen and stored at−70°C. After thawing, 1 ng/ml DHBA (3,4-dihydroxy-benzylamine: Sigma Chemie GmbH, Deisenhofen, Germany) was added as an internal standard. The samples were then deproteinized (trichloro-acetic acid, 0.3  final concentration), and the catecholamines were absorbed to purified alumina (Anton and Sayre, 1966) at pH 8.6 (2.5  Tris/HCl buffer containing 10 m mercaptoethanol). The alumina-bound catecholamines were washed three times with 0.8  acetic acid (HAc)/sodium acetate buffer pH 7.5 and once with water (deionized and double distilled in a quartz still). Then, the catecholamines were dissolved in 200 ll 1.0 N HAc. An aliquot of this solution was used for HPLC. Separation was achieved on a NovaPak-C18 3.9×150 mm column (Millipore GmbH, Eschborn, Germany), the mobile phase consisting of 1.0 N HAc, 2.2 m OSS (octane-sulfonic acid-Nasalt: Fluka Feinchemikalien, Neu-Ulm, Germany), 0.2 m EDTA-Na2, with a flow of 1.3 ml/min (pump: waters M510, Millipore GmbH, Eschborn, Germany; pulse dampener: LP-21, Scientific system Inc., State College, PA, USA). A glassy carbon electrode set to 650 mV against Ag/AgCl was used for electrochemical detection (model 400, EG&G Princeton Applied Res. Corp, Lawrenceville, USA). Peak areas were determined.

Experimental protocols In a first series of experiments MBF was measured in 48 spontaneously breathing rats. In each rat, up to three repetitive MBF measurements were performed. MBF was determined during control conditions at normal sinus rhythm (control group A: n=6; m=18, where n=number of animals and m=number of measurements) and during VT induced by rapid ventricular pacing at a rate of 600/min (VT group: n=42; m=63). Microspheres were injected during control conditions as well as at 5, 10, 15 min after the onset of VT. Arterial blood gas analyses were performed before and after the MBF measurements. Maximal dP/dt, LVSP, peak negative dP/dt and LVEDP were determined during control conditions and after the onset of VT to characterize myocardial function. ECG and arterial blood pressure were continuously monitored (Gould, digital recording oscilloscope DRO 1604, USA; Mingograf 82, Siemens, Germany). ˙ In a second series of experiments, MVO2 was determined in 12 additional, artificially ventilated

rats after thoracotomy. Arterial and coronary venous blood samples were collected during control conditions and 5 min after the onset of VT. In a third series of experiments, catecholamine measurements were performed in 16 additional rats. Blood samples were withdrawn twice during control conditions (n=8, m=16) as well as after 5 and 10 min during VT conditions (n=8, m=16). Blood samples were collected during moderately hypotensive VT (m=8) and during normotensive VT (m=4). Animals of the second and third series were not included in the analysis of cardiac events because withdrawal of blood for MBF measurements fol˙ lowed by blood sampling for MVO2 and catecholamine measurements caused uncomparable conditions for a reliable analysis of arrhythmias or mechanical failure. Table 1 illustrates the study design and the retrospective subgrouping according to the hemodynamic effect of VT as well as the occurence of cardiac events (see Results).

Statistics For statistical analysis, animals were grouped according to the following criteria: (1) According to the time duration of VT data determined in the VT group were grouped into the data obtained at 5 (B1: m=31), 10 (B2: m=18) and 15 min (B3: m= 14) after the onset of VT; (2) With respect to coronary autoregulation (Mosher et al., 1964; Becker, 1976; Rouleau et al. 1979; Dole et al., 1986; Dole, 1987), three MAP pressure ranges were defined: normotension with mean MAP values between 80 and 130 mmHg (I), moderate hypotension with a drop of MAP to values between 60 and 80 mmHg (II), and severe hypotension with MAP values below 60 mmHg (III). According to this definition data of the VT group were a posteriori grouped into the data obtained at normotensive VT (group I: m=26), moderately hypotensive VT (group II: m=21) and severely hypotensive VT (group III: m=16); (3) With respect to the occurrence of VF, data of the VT normotensive group were further grouped into the data obtained at normotensive VT immediately before VT-degeneration into VF (VF group: n=11; m=11) v normotensive VT without VT-degeneration into VF (non-VF group: n=8; m=15). A one-way ANOVA was used for multiple comparisons of the data determined in group A and group’s B1, B2 and B3; in group A and groups I, II and III, as well as in A and in the VF group

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Table 1 Diagram of the study design, the a posteriori grouping of the data according to the hemodynamic effect of VT and the correlation to the occurence of fatal cardiac events (ventricular fibrillation, mechanical failure) during pacing induced tachycardia in rats Protocol

Control group

VT group

1. seriesMBF

n=6

n=42

2. ˙series− MVO2

n=3

n=9

3. series− catecholamine

n=8

n=8

Subgroups during VT I: II: III: I: II: III: I: II: III:

n=19 n=13 n=10 n=3 n=3 n=3 n=3 n=5 —

and non-VF group. A paired Student’s t-test was performed for the comparison of plasma catecholamines measured at control conditions and at 3 and 10 min after the onset of pacing. The Fisher’s exact test was used to compare the incidence of VF with respect to MAP. The MAP value immediately before the onset of VF was used in the VF group. In the non-VF group, the MAP value was the mean value during the VT period of 20 min. Data are given as mean values±.. P values of <0.05 were considered statistically significant.

Results Hemoglobin, sodium and potassium concentrations, blood gas tensions and pH The hemoglobin concentration (Hb) was 14.1±0.8 g/dl during control conditions. Hb decreased with the number of MBF measurements in a linear fashion. At the end of the experiments, in which three MBF measurements were performed, Hb was reduced to 13.3±0.9 g/dl (P<0.05 v control; paired t-test). Serum electrolytes, arterial oxygen tensions, arterial pH, buffer bases and base excess remained unchanged in normal range during the experiments. No significant changes of arterial blood gases were observed during hemodynamically compromising, but stable tachycardias. Arterial oxygen tension was 129±10 mmHg, arterial carbon dioxide tension was 32±6 mmHg, and arterial pH was 7.35±0.03 in artificially ventilated animals. The decrease of coronary venous oxygen tensions (PcvO2) (25.1±2.8 mmHg in controls v 17.0±2.4 mmHg during severely hypotensive VT) was accompanied by an increase of AvdO2

No events

Fatal cardiac events

Ventricular fibrillation

Mechanical failure

n=8 n=11 n=6 n=3 n=3 n=3 n=3 n=5 —

n=11 n=2 n=4 — — — — — —

n=11 n=1 n=0 — — — — — —

n=0 n=1 n=4 — — — — — —

(67.0±12.2 ml/ml blood in controls v 79.8± 5.5 ml/ml blood during VT, P<0.05).

Myocardial function MAP was 105±13 mmHg during sinus rhythm in the control group. Spontaneous heart rate ranged from 300–490/min (386±28/min). In all experiments an initial, distinct MAP-drop directly after the onset of VT was observed. One type of MAPtime course was characterized by a subsequent stabilization within the next 3 min with normotension, moderate hypotension or severe hypotension; a second type of MAP-time course was characterized by a gradual decrease of MAP several min after the onset of sustained VT, resulting in acute mechanical failure, and a third type of MAPtime course was characterized by the occurence of sudden VF (Fig. 1). LVSP and maximal dP/dt decreased during VT (Table 2). The reductions of LVSP and maximal dP/ dt paralleled the reduction of MAP during sustained VT (Tables 2 and 3). PRP (43 042±10 704 mmHg/min in controls) was significantly elevated within the first interval after the onset of VT (60 161±14 278 mmHg/min) (P<0.05), and normalized within 10 min of VT (47 091±11589 mmHg/min). PRP was significantly higher during normotensive VT (60 966±9148 mmHg/min) and significantly lower during severely hypotensive VT (29 798±5380 mmHg/min) than in controls (Fig. 2). PRP did not significantly differ between the VF group and the non-VF group (Fig. 2). LVEDP increased significantly with sustained VT (B2, B3) (P<0.05) (Table 2). During normotensive

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MAP [mmHg]

ECG

200 100

-ECG-documentation of VF

-VT-degeneration into VF -Calibration

-End of pacing

(b) 1s

MAP [mmHg]

ECG

200 100

End of pacing

-ECG-documentation of VF

Figure 1 (a) Original recordings of surface electrograms (ECG) and femoral artery pressure (MAP) during pacinginduced normotensive VT. Calibration of arterial blood pressure and the moment of the VT-degeneration into VF are marked by arrows. (b) Detail of an original recordings of ECG and MAP during VF at higher paper speed.

and severely hypotensive VT, LVEDP was increased, whereas during moderately hypotensive VT LVEDP was within the normal range (Table 3). Peak negative dP/dt increased during sustained VT (P<0.05) (Tables 2 and 3).

Myocardial blood flow and oxygen consumption Tables 2 and 3 display the results of the MBF measurements. Three repetitive microsphere measurements did not significantly alter MBF by hemo-

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Degeneration of tachycardias Table 2 Systemic and coronary hemodynamics and myocardial oxygen consumption during sinus rhythm and normotension (A) and at 5, 10, and 15 min of ventricular pacing ˙ (B1–B3). MAP, mean arterial blood pressure; MBF, myocardial blood flow; CR, coronary resistance; MVO2, myocardial oxygen consumption; LVSP, left ventricular peak systolic pressure; dP/dtmax, maximum dP/dt; LVEDP, left ventricular end diastolic pressure; dP/dtmin, peak negative dP/dt. Group

A

MAP (mmHg) MBF (ml/g/min) MBF/beat (1/g) CR˙ (mmHg min g/ml) MVO2 (ml/g/min) LVSP (mmHg) dP/dtmax (mmHg/s) LVEDP (mmHg) dP/dtmin (mmHg/s)

105±13 4.88±1.18 12.7±3.5 21.9±9.3 0.66±0.14 123±13 7001±1631 1.3±1.7 −4226±1462

B1 103±22 5.91±1.81 10.2±3.0∗ 17.1±5.5 0.83±0.24 118±19 4950±944∗ 1.2±2.2 −2143±1484

B2

B3

82±21∗ 4.75±1.81 8.2±2.3∗ 18.2±5.2 0.65±0.23 94±17∗ 4949±1744∗ 4.6±3.3∗ −4049±1061

72±26∗ 4.19±2.08 7.4±3.5∗ 16.9±7.0 0.62±0.27 85±17∗†‡ 4800±1023∗†‡ 4.4±3.2∗ −3902±905∗

Pressure-rate-product (mmHg/min)

Data are mean values±.. ∗ P<0.05 v A; † P<0.05 v B1; ‡ P<0.05 v B2.

80 000 70 000 P < 0.05

60 000 P < 0.05

50 000 40 000 30 000 A

I

II

III

Non-VF VF group group

Figure 2 Bar diagram of pressure-rate-product (PRP) during control conditions (A), normotensive (I), moderately hypotensive (II) and severely hypotensive VT (III). PRP of the non-VF and VF group is displayed by the bars on the right side. Mean values are given±..

dilution in controls (4.78±1.77 ml/g/min v 5.06±1.35 ml/g/min (P=..). No significant ˙ time-dependent MBF and MVO2 differences were observed during sustained VT (Table 2) MBF was significantly lower in animals with severely hypotensive VT than in all other groups, especially than the normotensive VT-group. No significant differences were observed between MBFendo and MBFepi in controls and all other groups. MBFendo was 5.05±1.29 ml/g/min, MBFepi was 4.85±1.36 ml/g/min in controls. Significant MBFdifferences were found in MBFendo and MBFepi during normotensive VT (6.00±1.92 and 5.58±1.75 ml/ g/min) as compared to severely hypotensive VT (3.83±1.86 and 3.25±1.25 ml/g/min, respectively) (P<0.05). The subendo-subepicardial MBF-ratio (1.06±0.23) did not change at 5, 10

and 15 min after the onset of VT. Also, the subendosubepicardial MBF-ratio was not different when animals were grouped by MAP during VT (I: 1.06±0.17, II: 1.13±0.23, III: 1.18±0.34) (Fig. 3). No significant time- and MAP-dependent changes of CR were observed during VT (Table 2). ArI comparing group A with group II as well as group III were 0.5 and 0.4, respectively. Table 4 shows the differences in hemodynamic parameters in the non-VF group and VF group. Using ANOVA MBF of the VF group was not different from that of the non-VF group during normotensive VT. The data of the rat with the occurrence of sudden VF during moderately hypotensive VT was not included into the VF group, because normotensive VT conditions were ˙ only compared. MVO2 in the VF group, however, was significantly lower than in the non-VF group (P<0.05). The rats of the VF group ˙ represented a subpopulation of low MBF- and MVO2-conditions despite the observed values were within the normal range. Furthermore, the subendo-subepicardial MBF-ratio was significantly reduced in the VF group in comparison to the non-VF group (P<0.05) (Fig. 3).

Fatal cardiac events Restoration of spontaneous circulation after VT with sudden normalization of systemic blood pressure and normal blood gas tensions was observed in 25 rats (59% of the animals). Acute mechanical failure defined as the inability to

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A. Hagendorff et al. Table 3 Systemic and coronary hemodynamics and myocardial oxygen consumption during normotensive VT (I–MAP: 80–130 mmHg), moderately hypotensive VT (II–MAP: 60–80 mmHg) and during severely hypotensive VT (III–MAP: <60 mmHg). ˙ MAP, mean arterial blood pressure; MBF, myocardial blood flow; CR, coronary resistance; MVO2, myocardial oxygen consumption; LVSP, left ventricular peak systolic pressure; dP/dtmax, maximum dP/dt; LVEDP, left ventricular end diastolic pressure; dP/dtmin, peak negative dP/dt. Group

Rapid ventricular pacing

MAP-range MAP (mmHg) MBF (ml/g/min) MBF/beat (1/g) CR˙ (mmHg min g/ml) MVO2 (ml/g/min) LVSP (mmHg) dP/dtmax (mmHg/s) LVEDP (mmHg) dP/dtmin (mmHg/s)

80–130 mmHg I 105±17 5.64±1.68 9.7±2.8∗ 18.6±5.5 0.72±0.27 107±12∗ 4860±1135∗ 4.1±3.5∗ −3612±1229∗

60–80 mmHg II 73±6∗† 4.12±1.22 7.1±2.1∗ 19.0±5.6 0.62±0.23 80±4∗ 3241±1043∗ 2.7±2.9 −2236±826∗

<60 mmHg III 52±8∗†‡ 3.42±1.21† 6.0±2.4∗ 15.9±7.5 0.62±0.27 61±12∗† 2300±498∗ 4.7±3.3∗ −1475±532∗†

Subendo-/subepi-myocardial ratio

Data are mean values±.. ∗ P<0.05 v A; † P<0.05 v I; ‡ P<0.05 v II.

1.2

P < 0.05

1.1 1.0 0.9 0.8 0.7 A

I

II

III

Non-VF VF group group

Figure 3 Bar diagram of endomyocardial-/epimyocardial blood flow ratio during control conditions (A), normotensive (I), moderately hypotensive (II) and severely hypotensive VT (III). The subendo-subepicardialratio of the non-VF- and VF group is displayed by the bars on the right side. Mean values are given±..

restore normofrequent sinus rhythm with normotension after 20 min of rapid ventricular pacing occured in 5 of 42 rats (12%). MAP values during VT below 60 mmHg were related to a higher risk of mechanical failure (P=0.01). VF

occurred in 12 of 42 rats (29%) within 20 min of rapid ventricular pacing. The onset of VF was related to hemodynamic changes. The moment of VT-degeneration into VF was not predictable, occuring between the sixth and twentieth min of ventricular pacing (meantime±..: 10.5±4.8 min). VF was initiated in 11 rats during normotensive VT and in one rat during moderately hypotensive VT (Table 1). MAP values above 80 mmHg were related to a higher risk of VT degeneration into VF (P=0.02).

Plasma catecholamines Figure 4 displays the results of the plasma catecholamine determinations. Plasma epinephrine and norepinephrine concentrations (0.22±0.13 and 0.37±0.12 ng/ml during control conditions, respectively) increased 5 and 10 min after the onset of VT (0.71±0.42 and 0.71±0.24 ng/ml; P<0.05 and 3.55±2.68 and 0.88±0.44 ng/ml; P<0.01, respectively). There

Table 4 Mean arterial blood pressure (MAP), myocardial ˙ blood flow (MBF), subendocardial-subepicardial MBF -ratio and myocardial oxygen consumption (MVO2) determined in the VF group and in the nonVF group during normotensive VT. ˙ Group MAP mmHg MBF (ml/g/min) MBF/beat (l/g) MBF-ratio MVO2 (ml/g/min) Non-VF group VF group

106±14 104±16

Data are mean values±..

5.69±2.19 4.28±1.34

9.7±2.80 7.6±2.87

1.21±0.3 0.94±0.2

0.71±0.28 0.51±0.24

Plasma (ng/ml)

6.0

1.8

5.0

1.5

1.2

P < 0.01

4.0

P < 0.05

3.0

0.9

P < 0.05

Plasma (ng/ml)

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0.6

2.0

P < 0.05

1.0

A

0.3

5 min VT

10 min VT

A

5 min VT

10 min VT

Figure 4 Bar diagram of plasma epinephrine (left) and plasma norepinephrine (right) concentrations determined during control conditions (A) as well as 5 and 10 min after onset of ventricular tachycardia (VT). Mean values are given±..

were no differences between plasma catecholamine concentrations during normotensive and moderately hypotensive VT (epinephrine: 0.61±0.57 v 0.75±0.36 ng/ml; norepinephrine: 0.78±0.46 v 0.67±0.07 ng/ml).

moderately ˙ hypotensive VT, and by (II) the retained MVO2 balance at increased heart rate and reduced afterload.

Critique of methods

Discussion The major findings of this study are (I) that the degeneration of VT into VF is more frequent during normotensive VT than during hypotensive VT, and (II) that acute mechanical failure occurs more frequently during severely hypotensive VT than during moderately hypotensive and normotensive VT. Because plasma catecholamine concentrations are equal during normotensive and moderately hypotensive VT, it can be assumed that sympathetic activation is not the main cause, that facilitates the VT-degeneration into VF. Moderate hypotension during VT seems to protect against fatal cardiac events in normal hearts. Thus, it can be concluded that normotension and hypertension during VT are risk factors for the degeneration into VF, and severe hypotension, in turn, predisposes to acute mechanical failure. The underlying pathophysiological mechanisms are characterized by (I) the preservation of myocardial perfusion pressure during

The present experimental set-up permits us to obtain data in an in vivo model, both considering the inherent non-steady-state nature of ventricular arrhythmias and the steady-state requirements of MBF-measurement techniques. The incidence of fatal cardiac events in this experimental setting is only due to functional changes, caused by the coronary and systemic hemodynamic responses to VT, because the results were obtained in structurally normal hearts. In this communication, data from two experimental settings are combined. MBF measurements and catecholamine analysis were ˙ performed in spontaneously breathing rats, MVO2 was determined in artificially ventilated rats. Thoracotomy was necessary to obtain coronary venous blood samples from the great cardiac vein. However, systemic acid-base-balance as well as hemodynamics did not differ significantly in spontaneously breathing and artificially ventilated rats. Organ perfusion might be influenced by the repetitive MBF measurements due to hemodilution.

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However, five consecutive MBF measurements during control conditions revealed no significant MBF changes. The distinct MBF changes observed during sustained tachycardia were attributed to the VT effect per se. The tachycardia in the present model was induced and maintained by ventricular pacing. The clinical correlate is a focal VT rather than a re-entry VT. Both, hemodynamic consequences and electrophysiological characteristics of a focal and re-entrant VT may be different. ST segment changes and conduction alterations are difficult to assess in the rat heart at a rate of 600 beats/ min. Most importantly, with ventricular pacing the ST segment is not useful for the detection of ischemia. Therefore, electrophysiological studies, that characterize subendocardial ischemia, were not performed.

Why does moderate hypotension during VT present degeneration into VF? MBF under normal conditions is autoregulated and correlated to myocardial function (Mosher et al., 1964; Becker 1976; Rouleau et al., 1979; Dole et al., 1986; Dole, 1987; Canty, 1988; Mommura et al., 1991). MBF during VT depends on VT rate and duration (Saksena et al., 1984; Baron et al., 1990). In the present experiments, coronary resistance is not different between control and VT conditions. Thus, regulatory function of the coronary resistance vessels seems to be impaired during hemodynamically compromising VT. The autoregulation index, however, calculated for MAP-MBF-changes during moderately hypotensive VT indicates an almost preserved MBF autoregulation. Myocardial perfusion is matched to myocardial function and depends on coronary resistance, which is determined by extravascular and vascular compounds. Because myocardial function is markedly altered during VT, reactions of the resistance vessels can be assumed despite unchanged CR values. VT-degeneration into VF predominantly occured in a subpopulation of rats with borderline low MBF conditions per se. The degeneration of VT into VF appears to be associated with myocardial ischemia because the subendo-subepicardial MBF ratio in the VF group was significantly lower than that of the non-VF group. This decrease of the endo-epicardial MBF ratio reflects a transmural steal phenomenon, which is attributed to increased extravascular compression of subendocardial layers and a further vasodilation of subepicardial layers in the face of

exhausted dilator reserve in subendocardial layers (Becker 1976; Guyton et al., 1977; Gallagher et al., 1982; Canty, 1988). The association of the subendo-subepicardial MBF ratio to normotensive VT might be comparable to experimental VFconditions with normal perfusion pressure (Hottenrott et al., 1974; Buckberg and Hottenrott, 1975). In Hottenrott’s experiments, a low subendosubepicardial MBF ratio and subendocardial ischemia were caused by VF. In the present experiments, however, the changes of the subendosubepicardial MBF ratio were induced by the VT, and these changes were correlated to a higher incidence of VF. Therefore, we assume, that subendocardial ischemia was the cause for the degeneration of VT into VF.

Why does ischemia occur during normotensive VT but not during moderately hypotensive VT in structurally normal hearts? The increase of energy demand secondary to the elevation of heart rate is identical in all groups as is the reduced duration of diastole. The reduction of arterial pressure reduces energy demand during hypotensive VT, but possibly also reduces energy supply. During moderately hypotensive VT myocardial energy demand is reduced secondary to reduction of afterload, whereas the reduced perfusion pressure does not result in reduced blood flow (Hagendorff et al., 1994). During severely hypotensive VT, energy demand is further reduced by afterload reduction, however, energy supply is now reduced at exhausted coronary autoregulation, and myocardial ischemia and mechanical failure develop. The impairment of diastolic function – as documented by the significant increases of LVEDP and peak negative dP/dt – is most marked during severely hypotensive VT, and therefore obviously related to ischemia. Taken together, during moderately hypotensive VT, the reduction of coronary perfusion pressure does not result in an insufficiency of subendocardial blood flow in structurally normal hearts. Coronary autoregulation seems to be preserved in the presence of reduced energy demand and reduced extravascular compression. Progressive sympathetic activation in the present study is evident from the increase of plasma catecholamines with the duration of VT. However, it can be assumed that normotension during VT is not caused by progressive sympathetic activation, because catecholamine levels during moderately hypotensive and normotensive VT do not differ.

Degeneration of tachycardias

Other unknown factors inducing borderline low MBF conditions in the VF group may play an important role in the scenario of the VTdegeneration into VF. Apart from the hemodynamic effects discussed above, direct electrophysiological effects of catecholamines, such as delayed after-depolarization due to the increase in intracellular calcium, the shortening of the action potential duration due to the increase of potassium outward currents and/or inhomogeneous changes in the refractory period predispose to triggered activity and favor re-entry ¨ mechanisms (Schomig et al., 1987; Priori et al., 1988; Winfree, 1993). We did not determine whether increased plasma catecholamines were of neuronal or adrenal origin. Baroreflex activation does not appear to be a major mechanism in the present study, because plasma catecholamine concentrations were not higher during moderately hypotensive VT compared with during normotensive VT. Chemoreflexes from the ischemic myocardium and mechanical stimuli due to the asynchronous myocardial contraction pattern could possibly explain the sympathetic activation, through a spinal cardiocardiac sympathetic excitatory reflex (Malliani et al., 1969; Lombardi et al., 1984; Kopin, 1989; Heusch, 1990). In addition, asynchronous contraction pattern and increased heart rate during VT modulate the disposition of neuronally released norepinephrine in cardiac tissue due to mechanical stretching and compressing (Euler, 1980, Masuda and Levy, 1985). This “cardiac massaging” during VT might facilitate the transport of neuromediators from neurons into myocardial capillaries. Finally, electrical stimulation causes the release of autonomic mediators by excitation of intramyocardial nerve fibers (Euler, 1980). Alternatively, or in addition, a stress-induced discharge of epinephrine from the adrenal medulla into the systemic circulation causes elevated plasma catecholamine concentrations. Norepinephrine release from the heart due to chemical and mechanical stimuli and stress-induced epinephrine release from the adrenal gland both contribute to the sympathetic activation during VT. Sympathetic activation precipitates and aggravates myocardial ischemia, largely by a-receptor mediated vasoconstriction (Heusch, 1989, 1990). Although sympathetic activation itself is arrhythmogenic (Priori and Schwartz, 1989), we suggest that in structurally normal hearts, increased adrenergic activity triggers the degeneration of VT into VF in the presence of regional ischemia at high energy demand.

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Clinical implications and study limitations The impact of systemic hemodynamics on the incidence of VF in structurally normal hearts is obvious from the present study. It can be concluded that during tachycardia, optimal energy supply conditions are present during moderate hypotension. In conditions with regional myocardial hypoperfusion due to severe coronary stenoses or incomplete collateralization after coronary occlusion, however, hypotension and even moderate hypotension may exacerbate or precipitate regional myocardial ischemia. The optimal pressure range for cardioprotection found in the present study is not directly applicable on the clinical setting. Therefore, the present findings are helpful in a clinical set-up, when the severity of VT has to be assessed. The relation between systemic hemodynamics and fatal arrhythmias as well as the propensity of arrhythmias to degenerate during hemodynamic deterioration support our hypothesis that transient systemic hemodynamic alterations are involved in the genesis of sudden cardiac death. In addition, specific arrhythmogenic mechanisms are involved in the failing myocardium. The effect of vasodilator therapy on clinical outcome in patients with chronic heart failure suggests that normotension and hypertension during tachyarrhythmias in combination with LV-overload and sympathetic activation are crucial for the electrical destabilization of the heart (Lombardi et al, 1983; CONSENSUS Study Trial Group, 1987; SOLVD Investigators, 1991; Vanoli et al., 1994). Nevertheless, moderate hypotension can also be dangerous in heart failure and cardiomyopathy, because of the reduction of coronary perfusion pressure. Therefore, a general extrapolation of the present results to the diseased heart is not possible. There is a need for further studies both in structurally normal and in diseased hearts of larger mammals, where local heterogeneity of electrophysiology, blood flow and function may be more pronounced than in intact rat hearts.

Acknowledgement This study was supported by DFG grants (Ha 1922/ 1–1, 1–2 and 1–3) and by the Herbert-Reeck Foundation. We wish to acknowledge the technical contribution of Andrea Gey and Marcus Lassau.

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