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The effects of uremic compounds on cardiac function and metabolism
Journal of Mole&r
and Cell&r
The Effects
Cardiology (1973) 5, 287-300
of Uremic Compounds Function and Metabolism
JAMES SCHEUER Mpardial
AND
S. WILLIAM
on Cardiac
STEZOSKI
Metabolism Luboratol-y, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152 13, U.S.A. (Received 14 November 1972, and accepted 26 January
1973)
J. SCHEUER AND S. W. STEZOSIU.The Effects of Uremic Compounds on Cardiac Function and Metabolism. Journal of Molecular and Cellular Cardiology (1973) 5, 287-300. In order to study the effects of some compounds known to be elevated in the uremic syndrome, hearts of normal rats were perfused in an isolated working heart apparatus. High concentrations of urea, creatinine, methyl guanidine, and guanidinosuccinic acid were used alone or in combination. Urea at 20, 10, and 2.5 mM all decreased the cardiac output response to increasing atria1 pressure. A combination of urea 20 rnq creatinine 0.88 mM, and guanidinosuccinic acid 0.31 rnM caused a depression in coronary flow, cardiac output and maximum left ventricular dP/dt. These three agents with methyl guanidine 0.46 mM caused depressions in cardiac output, coronary flow, left ventricular pressure and dP/dt, and was associated with a rise in end-diastolic pressure. With these four agents, myocardial oxygen extraction, lactate production and lactate pyruvate ratios were increased. Hearts perfused with methyl guanidine, guanidinosuccinic acid, and creatinine but without urea had the same metabolic and dynamic performance as control hearts. Thus acute exposure to high concentrations of uremic compounds depresses cardiac pumping function in the rat heart. The depressant effect is related to the number of agents present, but is not seen when urea is absent. Depression appears to be partially due to a limitation in oxygen delivery and to the resultant hypoxia. KEY
WORDS:
Cardiac function;
Lactate;
Myocardial
mechanism;
Myocardial
metabo-
lism.
1. Introduction
Altered cardiac function has been reported in uremic patients. These include both decreased or increased hemodynamic function [Z, 4, 7,11,14]. Histological changes in the heart have also been described which are presumably characteristic of uremia [9]. However, it is not known whether these effects are primarily due to the accumulation of the retained products of protein breakdown such as urea, creatinine or guanidine, or are secondary to related factors such as anemia, hypertension, hypervolemia or electrolyte disorders. Recent studies from our laboratory demonstrated that hearts of rats made moderately uremic had increased ventricular performance when studied in the isolated heart apparatus [13]. Hearts from rats with profound, acute uremia had normal cardiac function when measured in the same manner. The previous investigation evaluated the responses of hearts from uremic rats
288
J. SCHEUER
AND
S. W. STEZOSKI
when they were perfused in the absence of the products of uremia. Plasma levels of a variety of compounds increase to abnormal levels in the blood of uremic subjects [5]. Methyl guanidine and guanidino-succinic acid are thought to be particularly toxic [I, 31. The purpose of the present investigation was to study the effects of some of these compounds on the function and metabolism of hearts from normal rats.
2. Methods The hearts of male Wistar rats weighing between 320 and 400 g were studied in an isolated working rat heart apparatus as described in its modified form by Scheuer and Stezoski [ 251. In this apparatus, the heart is perfused through the left atrium, and it ejects fluid from the left ventricle into the aorta. The overflow of the aorta drips into a calibrated aortic chamber that is used to measure aortic flow. The effluent fluid that drips out of the right ventricle is the coronary flow, and this is also measured in a calibrated chamber. In the present experiments, the perfusion medium contained 143 mM-sodium, 123 mu-chloride, 25 mM-bicarbonate, 6 mM-potassium, 1.2 mM-magnesium, 1.2 mhl-phosphate, 2.0 mM-calcium, 0.5 mM-disodium EDTA, and 5 mr+glucose. The gas bubbled through the solution was 95% 0~5% COz. This maintained the oxygen tension of the perfusate above 600 mmHg and the pH close to 7.4. The pH was monitored in each perfusion reservoir and kept the same on each side (control and uremic) by adjusting the rate of gassing. Special adjustments were not necessary with the addition of the small amount of compounds used in these experiments. To maintain cardiac output during exposure to “uremic toxins,” the height of the aortic column was 62.5 cm, which was lower than we originally used in this apparatus [13]. Also, there were two parallel systems either of which could be switched into the atria1 cannula for an abrupt change from normal to ?iremic” medium. In both systems the apparatus was in a waterjacket so that perfusion was at 37 “C. Left ventricular pressures were monitored through a flanged 17-cm polyethylene-90 catheter which pierced the left ventricular wall. The catheter was attached to a Statham P23Gb strain gauge. The system had a frequency response of 21 Hz. The rate of left ventricular pressure rise (dP/dt) was recorded with a resistancecapacitance differentiating channel on an Electronics for Medicine photographic recorder. The time constant for the differentiating circuit was 0.5 msec, and the response was linear within 5% from 1 to 57 Hz. Although the frequency response of the recording system may be borderline for the measurement of dP/dt at rapid heart rates, such a limitation would tend to mask any potential differences between control and uremic perfusions. The system was sensitive enough to demonstrate significant differences in this apparatus in our previous studies [13,15].
UREMIC
AGENTS
IN CARDIAC
FUNCTION
289
Oxygen tension in the influent and effluent perfusion media was monitored as described previously [ 151. To avoid a change in heart rate, hearts were paced through a platinum wire attached to a Grass SD 5 stimulator. The active lead was placed on the right atrium and the ground lead was placed on the aortic cannula. The constant pacing rate was 330 beatslmin, which is the minimal rate necessary to constantly take over the pacemaker role in perfused rat hearts. The square wave stimulus had an amplitude of5 V and a duration of 4 msec. Thii apparatus permits assessment of performance at a controlled, paced ventricular rate with a constant aortic diastolic pressure. Coronary flow, cardiac output, myocardial oxygen extraction and oxygen consumption, left ventricular pressure, and the rate of left ventricular pressure rise (dP/dt) can all be measured. Since there is an air trap in the aortic tubing system, the aortic column contains an inertial component that permits left ventricular systolic pressure transiently to exceed that hydrostatic pressure which would be imposed by the height of the aortic column. Since pressures upon which analyses are based are recorded from the left ventricle, these provide an accurate reflection of forces that the left ventricle perceives. In the current experiments, the purpose was to determine if high levels of uremic products would affect cardiac function. The experimental side of the perfusion apparatus contained medium without any uremic products (control hearts) or medium containing urea 20 mu (120 mg/lOOml), creatinine 0.88 mM (10 mg/ 100 ml), methyl guanidine (MG) 0.46 mu (5 mg/lOO ml) or guanidinosuccinic acid (GSA) 0.31 mu (5.5 mg/lOO ml).* These agents were used alone or in combination. All solutions were made up freshly each day. The protocol was to perfuse the hearts in a retrograde manner through the aorta for a lo-minute period while the atria1 cannula and ventricular catheters were placed. Antegrade perfusion through the left atrium was begun for an initial IOminute control period at 5 cm atria1 pressure.t Then the experimental side was abruptly switched into the perfusion cannula at 5 cm atria1 pressure. With the experimental perfusion medium still being used, the atria1 pressure was raised to 20 cm in order to evaluate the Starling response of the heart. Finally control perfusion conditions were returned (normal medium at 5 cm atria1 pressure) for a recovery period. Each step was maintained for 5 min and all measurements were made during the final minute at any one level. In order to determine performance during isovolumic contraction, the tubing just above the aortic cannula was clamped. At least five beats were recorded once the heart had reached a steady state of isovolumic performance. At the end of the experiment hearts were removed and a portion of the ventricles * All of these compounds were purchased from Sigma Chemical Company, St Louis, Missouri. 7 Pressure units are cm of perfusion medium.
290
J. SCHEUER
AND
S. W. STEZOSKI
was then taken for determining the dry weight. For lactate and pyruvate determinations, per&sate was delivered into tubes containing 1 ml of cold 6% perchloric acid, and the substances were determined enzymically [S, 171. In these experiments cardiac work was not calculated but an approximate correlate was determined by multiplying cardiac output and peak left ventricular systolic pressure (CO x PLVSP) . Control hearts and hearts exposed to uremic products were perfused on the same day in random order. Each set of “uremic” conditions was compared with its own set of control hearts. Results of dynamic performance are presented as percentage of performance prior to imposing the experimental conditions. Initial values are given in Table 1. Statistical significance was determined by analysis of variance, and when paired data were compared interaction was employed [l8]. All flow-related data are expressed per gram dry heart weight. 3. Results
Table 1 shows the body weights and heart weights in each group and the baseline values for dynamic performance for the hearts used in these experiments. Baseline values were obtained in the minute prior to switching to “uremic” perfusion. There was some variation between groups with regard to cardiac output, CO x PLVSP and maximum dP/dt. The greater initial performance in some studies, such as the control and matched urea, GSA, creatinine group, was probably related to the lesser heart weight in this series of experiments. It has been noted previously that performance per gram of heart measured in this apparatus tends to be inversely related to the heart weight [12]. I n any case in terms of dynamic performance each experimental group was well matched with its control group at the start of experimental perfusion. Studies with urea
Figure 1 shows the results of perfusion with 20 nm-urea. Performance was generally similar to hearts perfused under control conditions. However, coronary flow remained elevated in control hearts after return to the original perfusion conditions at the end of the experiment. Hearts perfused with urea returned close to the initial value for coronary flow. The cardiac output response to increased atria1 pressure (20 cm) was slightly but significantly depressed in hearts perfused with urea, and these hearts failed to demonstrate a rise in maximum dP/dt with an increasing atria1 pressure. A significant rise in dP/dt (P< 0.05) was present in control hearts. The responses to isovolumic clamping were similar in control hearts and those perfused with urea and there was no difference in the CO x PLVSP response in the two groups. Table 2 shows that oxygen consumption, lactate production and the effluent lactatelpyruvate ratios were similar in hearts perfused with urea and in controls.
361 f 15 376 & 8
6
7
382 f 8
11
222 * 3
228 f 8
228 & 4
225 f 5
199&5
200 f 7
220 f 6 216 & 5
51 f2
52 f 1
51 f2
52 &2
51 f2
50 f2
55 *4 52 f2
Dry heart Coronary weight flow (ml glmin-l) k>
Results are mean + S.E.Flow related data are per gram dry heart weight PLVSP = peak left ventricular pressure CO x PLVSP = cardiac output x PLVSP Max dP/dt = maximum rate of left ventricular pressure rise
Control GSA, MG Creatinine
385 f 8
10
338 f 5
8
Control Urea, GSA, MG Creatinine
344&8
7
Control Urea, GSA Creatinine
385 f 14 386 f 16
8 11
Body weight k)
Control Urea
Number of hearts
159 f6
157 f 8
154 f6
146f7
203 f 9
197 * 10
149 f4 138 f 13
Cardiac output (ml grmir-1)
80 f 3
82 rt 3
88 f 2
84 &2
80 f 2
84 f 4
80 & 2 75 f2
PLVSP (mmHg)
1. Weight relationships and initial control dynamic values for perfused hearts
Conditions
TABLE
12 650 f 671
12 870 f 1000
13 460 f 476
11900*630
16 530 f 910
16 680 f 1070
11830 &300 11300 f460
co x PLVSP (ml x mmHg grmin-1)
3700 f 132
3520 f 196
3224 f 70
3414 f 168
3858 f 130
3954 f 206
3690 f 108 3580 f 172
Max dP/dt (mmHg/sec)
292
J. SCHEUER 1
';i 1202 b IIOloo95-
AND
S. W. STEZOSKI
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I
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I 1 5
I I 5
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1 20
I_1 5
Atrial pressure (cm)
FIGURE 1. The effects of urea 20 rn~ ( 120 mg/ 100 ml) on cardiac dynamics. Perfusion during the initial and the final periods were in the absence of urea. Perfusion during the second and third periods was with urea for the experimental group. CF = coronary flow; CO = cardiac output; LVSP = left ventricular pressure; max dP/dt = maximum rate of left ventricular pressure rise. Panel A indicates LVSP and dP/dt during isovolumic beats, and panel B shows these variables during ejecting beats. Results are mean f S.E. as percent of the initial 5 cm value. The number of hearts are shown in the parenthesis. *indicates P-c 0.05. (0) Control, 8; (0) urea, 11. Atria1 pressures are in cm of perfusion medium, ventricular pressures are in mmHg.
In order to test whether the response to 20 m-urea represented a toxic effect, hearts were perfused with lower concentrations of this agent. In general the hearts performed similarly to the hearts perfused with 20 mM-urea. At 20 cm atria1 pressure, performance in the presence of urea always tended to be less than in controls. The changes in cardiac output and CO x PLVSP at 20 cm atria1 pressure are shown in Table 3.
Studies with urea, creatinine and guanidinosuccinic acid (GSA)
Figure 2 shows the effects of adding creatinine and GSA to urea. More marked differences in performance were noted with this mixture. Coronary flow fell with
UREMIC
TABLE
2.
AGENTS
IN CARDIAC
293
FUNCTION
Metabolic values for perfused hearts
Control Urea Control Urea, GSA Creatinine Control Urea, GSA, MG Creatinine Control MG, GSA Crealinine
Control Urea Control Urea, GSA Creatinine Control Urea, GSA, MG Creatinine control MG, GSA Creatinine
G5CM 0.84 & 0.03 0.78 f 0.03 0.81 & 0.03
Oxygen consumption ml g-lmin-l 5CM 20CM 0.86 f 0.05 1.12 & 0.10 0.79 * 0.03 1.02 f 0.08 0.84 & 0.02 1.10 & 0.08
0.81 f 0.04 0.80 * 0.03
0.80 & 0.03 0.81 f 0.03
0.91 f 0.06 0.92 5 0.04
0.80 & 0.03 0.84 f 0.02
0.80 f 0.04 0.85 f 0.02
0.79 f 0.05 0.82 5 0.03
0.81 f 0.03 0.93 f 0.02
0.75 f 0.04 0.81 f 0.02
0.82 f 0.02
0.81 & 0.03 1.04 f 0.05 Lactate production ymol g-l min-l ___.__ 5CM 20CM 3.1 f 0.4 6.7 f 0.5 3.2 + 0.7 7.7 -j= 0.6 2.4 f 0.3 7.6 * 0.8
0.80 & 0.03
C4CM 3.2 f 0.5 3.6 f 0.6 2.9 * 0.4
.__.-~~
~-
R-5CM 0.94 & 0.08 0.82 & 0.04 0.90 f 0.04
R-5CM 3.2 5 0.64.6 f 0.6 2.7 & 0.4
2.7 f 0.3 3.2 +- 0.7
2.7 f 0.4 2.0 + 0.5
9.0 + 1.0 6.6 & 0.4
3.3 f 0.6 2.5 & 0.6
3.6 f 0.6 3.2 & 0.7
4.0 & 0.8* 2.0 f 0.3
9.8 f 0.9* 6.5 f 0.8
5.8 & 1.3* 2.5 f 0.5
2.6 f 0.5
3.4 f 0.8 7.6 f 0.8 3.6 Lactate/Pyruvate 5CM 20CM ~-- -Rx6 7.2 f 1.2 13.4 f 0.7 6.9 7.0 & 1.1 15.7 f 1.0 9.4 5.7 f 0.7 15.5 & 2.0 6.4
& 0.8
C-SCM -__ Control f 7.1 f 0.9 Urea & 6.6 f 1.0 Control 6.1 f 0.7 f Urea, GSA Creatinine 4.8 f 0.7 6.0 i 0.6 15.9 f 2.0 6.9 & Control 7.0 f 1.2 5.2 f 0.3 15.4 * 1.3 5.7 f Urea, GSA, MG Creatinine 7.1 f 1.2 9.8 f 1.27 25.1 f 2.5t 12.1 & Control 5.8 f 0.9 5.1 & 0.6 13.8 -& 1.2 5.9 & MG, GSA Creatinine 4.9 f 0.9 8.5 f 1.5 16.4 f 1.4 8.2 f Results are mean f S.E.QOs and lactate are per gram dry weight. C = initial control period; R = recovery period; 5 CM and 20 CM indicate atria1 pressures. * P< 0.05. t P< 0.01.
1.1 1.0 0.7 0.9 1.2 2.5* 0.7 1.8
294
J. SCHFXJER
TABLE
3.
Performance
Concentration b-4
AND
S. W. STEZOSKI
at 20 cm atria1 pressure-urea Number of hearts
0
9
2.5 10
6 8
20
9
Cardiac .~ -~ .-@--266 f 217 f 200 f 213 f
perfusions output
co -
12 12* 15t 14t
x PLVSP
~-~~ -~-~~ (%I ~~~~ 295 + 25
211 f21* 232 121:: 237 & 23*
Results for urea at 20 cm atria1 pressure are mean -J= S.E. as y0 of initial values recorded at 5 cm atria1 pressure with urea absent. CO x PLVSP = the product of cardiac output and peak left ventricular pressure. * PcO.05 t P< 0.01 $ P< 0.2
the switch from initial conditions (5 cm, no uremic compounds) to perfusion with the mixture (P < 0.05). Coronary flow also failed to rise with increasing atria1 pressure when exposed to this mixture. At 20 cm atria1 pressure coronary flow
115 I IOE
Atrial pressure (cm)
FIGURE 2. The effects of urea 20 mu, creatmine 0.88 mM, and guanidinosuccinic acid 0.31 m~ on cardiac dynamics. The experimental design and format are the same as in Figure 1. **indicates PcO.01. (a) Control, 7; (0) uremicmixture, 8.
UREMIC
AGENTS
IN CARDIAC
FUNCTION
295
was significantly higher in control hearts than in those perfused with the mixture. Cardiac output was also depressed by the mixture at 5 cm atria1 pressure, and at 5 cm, 20 cm and during recovery cardiac outputs were lower in the experimental than in the control hearts. Maximum dP/dt failed to rise with increasing atria1 pressure in hearts perfused with the mixture, and isovolumic maximum dP/dt was lower during the recovery period in the experimental group. That this combination was more depressant than urea alone is shown by the CO xPLVSP value, at 20 cm atria1 pressure. CO xPLVSP rose to 265% of the initial value in controls and only 174% in experimental hearts (P
Studies with urea, creatinitu, guanidinosuccinic acid and methyl guanidine (MG)
Figure 3 shows the results of perfusions with an urea, MG, GSA, and creatinine mixture. The performance with this mixture generally showed the most marked
Atrial pressure km)
FIGURE 3. The effects of urea 20 mu, creatinine 0.88 mM, guanidinosuccinic acid 0.31 mly and methyl guanidine 0.46 mM on cardiac dynamics. The format is the same as in Figures 1 and 2. (a) Control, 10; (0) mixture, 11.
296
J. SCHEUER
AND
S. W. STEZOSKI
differences from control perfusions. Coronary flow was significantly lower with the mixture at 20 cm atria1 pressure and during the recovery period. Peak left ventricular pressure and maximum dP/dt during both isovolumic performance and during ejection were less in hearts perfused with the mixture at 20 cm atria1 pressure, and these functions remained less during ejection in the recovery period. Cardiac output also was markedly lower in the mixture group than in the control group at 20 cm atria1 pressure and a significant difference remained during the recovery period. This was the only group in which maximum dP/dt declined significantly (P
Studies with creatinim, guanidinosuccinic acid and methyl guanidine All the above studies included urea in the perfusion medium. To determine the role of urea in the depressant effects of the mixtures, a combination of MG, GSA and creatinine were studied. Figure 4 demonstrates, that except for the isovolumic maximum dP/dt at 5 cm atria1 pressure, there were no differences between control hearts and those perfused with this mixture. There were also no effects of the mixture upon CO x PLVSP, oxygen consumption, or the lactate/pyruvate ratios. Residual glycogen levels were the same in control and uremic groups in each series. In order to insure that any alterations observed in performance were not due to differences in the preload, end-diastolic pressures were carefully measured in each experiment for each level of performance during both isovolumic contraction and during ejection. There were no significant differences between the end-diastolic pressures except in the urea, MG, GSA, creatinine experiments. During ejection
UREMIC 120
1
I
AGENTS
IN CARDIAC
297
FUNCTION
I 250
s
IIO-
-
,\
F
\
80
81 5
I 5
I 20
II 5
I
I 5
I 5
I 20
I I 5
Atrial pressure (cm)
FIGURE 4. The effects of creatinine 0.88 mM, guanidinosuccinic acid 0.31 rnM and methyl guanidine 0.46 nm on cardiac dynamics. The format is the same as in previous figures. (0) Control, 6; (0) uremic mixture, 7.
the left ventricular end-diastolic pressures at 20 cm atria1 pressure were 13.9 & 0.7 mmHg in controls and 17.9 & 0.6 in experimental hearts (P
4. Discussion
The analysis of experiments in the working rat heart apparatus in terms of classical muscle mechanics is difficult, because ventricular volumes are not known. However, certain inferences can be drawn from hearts of the same weight when the filling pressures are known, especially when isovolumic beats are analyzed. In the current experiments, there was evidence of decreased contractility in the group with all four compounds. These hearts had depressed external performance at 20 cm atria1 pressure even though end-diastolic pressures were significantly higher than in controls. This and the depressed responses to isovolumic clamping (for pressure and maximum dP/dt) also suggest decreased contractility. Whether or not one accepts the supposition that myocardial contractility was depressed by the mixtures of
298
J. SCHEUER
AND
S. W. STEZOSKI
uremic compounds, it is evident that the function of the heart as a pump and particularly its response to increasing atria1 pressure (Starling effect) was attenuated by these compounds. Thus in contrast to our earlier studies, in which normal or increased intrinsic function was found in hearts from uremic animals, this study demonstrates that the products of uremia can be deleterious to normal hearts. Urea alone produced some depressant effect at the 20 cm atria1 pressure. The possibility was considered that this effect was due to an increase in the osmolarity of the perfusion medium. Urea and glycerol at concentrations that raise osmolarity to 500 mosmol or more decrease contractility in atria1 muscle [16], but sucrose, mannitol and urea in osmolar concentrations up to 400 mosmol/L have all been observed to enhance contractile performance of ventricular myocardium [8, 191. In our experiments osmolarity would have been increased only by 20 mosmol. Also the dose response studies with urea demonstrated similar degrees of depression of cardiac output when urea was present in concentrations of 2.5 or 10 mM. The finding of a slight depressant effect of urea even at a concentration of 2.5 mM and lack of a distinct dose depression relationship mitigates against an osmolar cause for the effects of urea. Any of the compounds tested in combination with urea caused exaggeration of the depressant effects, yet these compounds in the concentrations used would not add significantly to the osmolarity. Thus increased osmolarity does not appear to be responsible for the findings. The lack of a dose depression relationship with urea remains unexplained. If the effect of urea was inhibition of an enzyme activity or a change in membrane permeability a dose depression relationship would be expected. The possibility was considered that the depressant effects seen in experiments with urea were due to breakdown products. Ammonium and cyanate accumulate when high concentrations of urea are permitted to remain in solution for long periods [IO]. The concentrations of urea used in the present experiment were very low. Also the solutions were made fresh each day making this an unlikely cause of the depressed cardiac responses. Furthermore, there was no significant difference between the first experiments conducted with fresh solutions each morning and those performed several hours later. The concentrations of the uremic compounds employed were as high or higher than are generally found in severe uremia. These concentrations were used purposefully in order to determine if any effect on cardiac function could be detected. Even though the degree of depression seemed to be related to the number of compounds included in the mixture, urea was obligitory for the mixture to be depressant. This was demonstrated by the similar function in control and experimental hearts when all the compounds except urea were included. It is possible that if the concentrations of the compounds in combination with urea were lowered, or if the urea concentration alone was decreased the additive deleterious effects might not have been seen.
UREMICAGENTSIN
CARDIAC FUNCTION
299
The mechanism of the depression appeared to be partially related to a defect in coronary flow and oxygen delivery. In hearts perfused with any combination of compounds with urea, coronary flow rates and responses to increasing atria1 pressure tended to be less than in control hearts. In absence of urea coronary flow rates and responses were the same as in controls. Oxygen consumption also tended to be lower when urea was present, although these differences were seldom statistically significant. On the other hand, in the group perfused with all four compounds, oxygen extraction, lactate production and effluent lactate/pyruvate ratios were all significantly higher in experimental hearts than in controls, suggesting that oxygen delivery was limited by inadequate coronary flow, glycolytic metabolism was increased and the cytoplasmic NAD+/NADH was shifted to a more reduced state. In these studies the hearts were only exposed to uremic compounds for a short period. The results suggests that high concentrations of compounds known to be present in the blood of uremic subjects can cause acute depression of cardiac function when elevated levels of urea are also present. The effects of lower concentrations of the compounds or of more prolonged exposure, as might be seen in chronic uremia, are important variables to be considered before translating these data to clinical states,
Acknowledgements We are grateful for the technical assistance of Ms. Patricia Pisanelli and for the secretarial assistance of Miss Carol Gundlach and Mrs Donna Costabile. Dr Scheuer is a recipient of the National Institutes of Health Career Development Award HL-15867. This work was supported by the U.S. Public Health Services Research Grant HL-14032-01.
REFERENCES 1. COHEN, B. D., STEIN, I. M., & KORNHAUSER,R. S. Guanidine retention and the urea cycle. Proceedings of the 4th International Congress on .Nejhrology, Stockholm. Vol. 2, pp. 255262 (1969). 2. DEL GRECO, F., SIMON, N. M., ROGUSICA, J., & WALKER, C. Hemodynamic studies in chronic uremia. Circulation 40,87-95 (1969). 3. GIOVANNETTI, S., BUGINI, M., BALESTRI, P. L., NAVALESI, R., GUGNONI, P., DE MATEIS, A., FERRO-MILONE, P., & PEFLFETTI, C. Uraemic symptoms in dogs chronically intoxicated with methyl-guanidine. Proceedings of the 4th International Congress on Jvephrology, Stockholm. Vol. 2, pp. 253-254 (1969). 4. Goss, J. E., ALXREY, A. C., VOGEL, J. H. K., & HOLMES, J. H. Hemodynamic changes during hemodialysis. Transactions of the American Society of Artijcial Internal Organs 13, 68-74 ( 1967). 5. HICK, J. M., YOUNG, D. S. & WOOTTON, I. D. P. Abnormal blood constituents in acute renal failure. Clinica Chin&a Acta 7,623-633 (1962). 6. HOHORST,H. J. L-(+)-Lactate. Determination with lactic dehydrogenase and DPN. In
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7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
17. 18. 19.
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Methods of Enzymatic Analysis, (H. U. Bergmeyer, Ed.) pp. 266-270. New York: Academic Press ( 1963). KNOWLAN, D. M. PIATNEK, D. A. & OLSON, R. E. Myocardial metabolism and cardiac output in acute uremia. Clinical Research9, 141 (1960) (Abstract). KOCH-WESER, J. Influence of osmolarity of perfusate on contractility of mammalian myocardium. AmericanJournal of Physiology 204,957-962 (1963). LANGENDOW, R., & PIRANI, M. D. The heart of uremia. An electrocardiographic and pathologic study. American HeartJournal 33,282-307 (1947). MARIER, J. R. & ROSE, D. Determination of cyanate, and a study of its accumulation in aqueous solutions of urea. Anabtical Biochemistry 7,304-314 (1964). MERRILL, J. P. The treatment of renal failure. New York: Grune & Stratton. p. 67 (1955). NEELY, J. R., LIEBERMEISTER, H., BATTERSBY, E. J., & MORGAN, H. E. Effect of pressure development on oxygen consumption by isolated rat heart. American Journal of Physiolosy 212,804814 (1967). PENPARGKUL, S. & SCHEUER, J. The effect of uremia upon the performance of the rat heart. Cardiovascular Research 6, 702-708 (1972). IIAAB, W. Cardiotoxic substances in the blood and heart muscle in uremia (their nature & action). TheJournal of Laboratory and Clinical Medicine 29,7 15-734 (1944). SCHEUER, J. & STEZOSKI,S. W. Effect of physical training on the mechanical and metabolic response of the rat heart to hypoxia. Circulation Research 30,418-429 (1972). SCHMIDT, E., WILKES, B., & HOLLAND, W. C. Effects of various glycerol or urea concentrations and incubation times on atria1 contractions and ultrastructure. Journal of Molecular d Cellular Cardiology4, 113-120 ( 1972). &GAL, S. A., BLAIR, A. E., & WYNGAARDEN, J. B. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. Journal of Laboratory and Clinical Medicine 48, 137-143 (1956). SNEDECOR, G. W. Statistical Methods (5th ed.) Iowa: Iowa State University Press (1956). WILDENTHAL, K., MIERZWWC, D. S. & MITCHELL, J. H. Acute effects of increased serum osmolality on left ventricular performance. American Journal of Physiology 216, 898-904 (1969).
and Cell&r
The Effects
Cardiology (1973) 5, 287-300
of Uremic Compounds Function and Metabolism
JAMES SCHEUER Mpardial
AND
S. WILLIAM
on Cardiac
STEZOSKI
Metabolism Luboratol-y, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152 13, U.S.A. (Received 14 November 1972, and accepted 26 January
1973)
J. SCHEUER AND S. W. STEZOSIU.The Effects of Uremic Compounds on Cardiac Function and Metabolism. Journal of Molecular and Cellular Cardiology (1973) 5, 287-300. In order to study the effects of some compounds known to be elevated in the uremic syndrome, hearts of normal rats were perfused in an isolated working heart apparatus. High concentrations of urea, creatinine, methyl guanidine, and guanidinosuccinic acid were used alone or in combination. Urea at 20, 10, and 2.5 mM all decreased the cardiac output response to increasing atria1 pressure. A combination of urea 20 rnq creatinine 0.88 mM, and guanidinosuccinic acid 0.31 rnM caused a depression in coronary flow, cardiac output and maximum left ventricular dP/dt. These three agents with methyl guanidine 0.46 mM caused depressions in cardiac output, coronary flow, left ventricular pressure and dP/dt, and was associated with a rise in end-diastolic pressure. With these four agents, myocardial oxygen extraction, lactate production and lactate pyruvate ratios were increased. Hearts perfused with methyl guanidine, guanidinosuccinic acid, and creatinine but without urea had the same metabolic and dynamic performance as control hearts. Thus acute exposure to high concentrations of uremic compounds depresses cardiac pumping function in the rat heart. The depressant effect is related to the number of agents present, but is not seen when urea is absent. Depression appears to be partially due to a limitation in oxygen delivery and to the resultant hypoxia. KEY
WORDS:
Cardiac function;
Lactate;
Myocardial
mechanism;
Myocardial
metabo-
lism.
1. Introduction
Altered cardiac function has been reported in uremic patients. These include both decreased or increased hemodynamic function [Z, 4, 7,11,14]. Histological changes in the heart have also been described which are presumably characteristic of uremia [9]. However, it is not known whether these effects are primarily due to the accumulation of the retained products of protein breakdown such as urea, creatinine or guanidine, or are secondary to related factors such as anemia, hypertension, hypervolemia or electrolyte disorders. Recent studies from our laboratory demonstrated that hearts of rats made moderately uremic had increased ventricular performance when studied in the isolated heart apparatus [13]. Hearts from rats with profound, acute uremia had normal cardiac function when measured in the same manner. The previous investigation evaluated the responses of hearts from uremic rats
288
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when they were perfused in the absence of the products of uremia. Plasma levels of a variety of compounds increase to abnormal levels in the blood of uremic subjects [5]. Methyl guanidine and guanidino-succinic acid are thought to be particularly toxic [I, 31. The purpose of the present investigation was to study the effects of some of these compounds on the function and metabolism of hearts from normal rats.
2. Methods The hearts of male Wistar rats weighing between 320 and 400 g were studied in an isolated working rat heart apparatus as described in its modified form by Scheuer and Stezoski [ 251. In this apparatus, the heart is perfused through the left atrium, and it ejects fluid from the left ventricle into the aorta. The overflow of the aorta drips into a calibrated aortic chamber that is used to measure aortic flow. The effluent fluid that drips out of the right ventricle is the coronary flow, and this is also measured in a calibrated chamber. In the present experiments, the perfusion medium contained 143 mM-sodium, 123 mu-chloride, 25 mM-bicarbonate, 6 mM-potassium, 1.2 mM-magnesium, 1.2 mhl-phosphate, 2.0 mM-calcium, 0.5 mM-disodium EDTA, and 5 mr+glucose. The gas bubbled through the solution was 95% 0~5% COz. This maintained the oxygen tension of the perfusate above 600 mmHg and the pH close to 7.4. The pH was monitored in each perfusion reservoir and kept the same on each side (control and uremic) by adjusting the rate of gassing. Special adjustments were not necessary with the addition of the small amount of compounds used in these experiments. To maintain cardiac output during exposure to “uremic toxins,” the height of the aortic column was 62.5 cm, which was lower than we originally used in this apparatus [13]. Also, there were two parallel systems either of which could be switched into the atria1 cannula for an abrupt change from normal to ?iremic” medium. In both systems the apparatus was in a waterjacket so that perfusion was at 37 “C. Left ventricular pressures were monitored through a flanged 17-cm polyethylene-90 catheter which pierced the left ventricular wall. The catheter was attached to a Statham P23Gb strain gauge. The system had a frequency response of 21 Hz. The rate of left ventricular pressure rise (dP/dt) was recorded with a resistancecapacitance differentiating channel on an Electronics for Medicine photographic recorder. The time constant for the differentiating circuit was 0.5 msec, and the response was linear within 5% from 1 to 57 Hz. Although the frequency response of the recording system may be borderline for the measurement of dP/dt at rapid heart rates, such a limitation would tend to mask any potential differences between control and uremic perfusions. The system was sensitive enough to demonstrate significant differences in this apparatus in our previous studies [13,15].
UREMIC
AGENTS
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Oxygen tension in the influent and effluent perfusion media was monitored as described previously [ 151. To avoid a change in heart rate, hearts were paced through a platinum wire attached to a Grass SD 5 stimulator. The active lead was placed on the right atrium and the ground lead was placed on the aortic cannula. The constant pacing rate was 330 beatslmin, which is the minimal rate necessary to constantly take over the pacemaker role in perfused rat hearts. The square wave stimulus had an amplitude of5 V and a duration of 4 msec. Thii apparatus permits assessment of performance at a controlled, paced ventricular rate with a constant aortic diastolic pressure. Coronary flow, cardiac output, myocardial oxygen extraction and oxygen consumption, left ventricular pressure, and the rate of left ventricular pressure rise (dP/dt) can all be measured. Since there is an air trap in the aortic tubing system, the aortic column contains an inertial component that permits left ventricular systolic pressure transiently to exceed that hydrostatic pressure which would be imposed by the height of the aortic column. Since pressures upon which analyses are based are recorded from the left ventricle, these provide an accurate reflection of forces that the left ventricle perceives. In the current experiments, the purpose was to determine if high levels of uremic products would affect cardiac function. The experimental side of the perfusion apparatus contained medium without any uremic products (control hearts) or medium containing urea 20 mu (120 mg/lOOml), creatinine 0.88 mM (10 mg/ 100 ml), methyl guanidine (MG) 0.46 mu (5 mg/lOO ml) or guanidinosuccinic acid (GSA) 0.31 mu (5.5 mg/lOO ml).* These agents were used alone or in combination. All solutions were made up freshly each day. The protocol was to perfuse the hearts in a retrograde manner through the aorta for a lo-minute period while the atria1 cannula and ventricular catheters were placed. Antegrade perfusion through the left atrium was begun for an initial IOminute control period at 5 cm atria1 pressure.t Then the experimental side was abruptly switched into the perfusion cannula at 5 cm atria1 pressure. With the experimental perfusion medium still being used, the atria1 pressure was raised to 20 cm in order to evaluate the Starling response of the heart. Finally control perfusion conditions were returned (normal medium at 5 cm atria1 pressure) for a recovery period. Each step was maintained for 5 min and all measurements were made during the final minute at any one level. In order to determine performance during isovolumic contraction, the tubing just above the aortic cannula was clamped. At least five beats were recorded once the heart had reached a steady state of isovolumic performance. At the end of the experiment hearts were removed and a portion of the ventricles * All of these compounds were purchased from Sigma Chemical Company, St Louis, Missouri. 7 Pressure units are cm of perfusion medium.
290
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was then taken for determining the dry weight. For lactate and pyruvate determinations, per&sate was delivered into tubes containing 1 ml of cold 6% perchloric acid, and the substances were determined enzymically [S, 171. In these experiments cardiac work was not calculated but an approximate correlate was determined by multiplying cardiac output and peak left ventricular systolic pressure (CO x PLVSP) . Control hearts and hearts exposed to uremic products were perfused on the same day in random order. Each set of “uremic” conditions was compared with its own set of control hearts. Results of dynamic performance are presented as percentage of performance prior to imposing the experimental conditions. Initial values are given in Table 1. Statistical significance was determined by analysis of variance, and when paired data were compared interaction was employed [l8]. All flow-related data are expressed per gram dry heart weight. 3. Results
Table 1 shows the body weights and heart weights in each group and the baseline values for dynamic performance for the hearts used in these experiments. Baseline values were obtained in the minute prior to switching to “uremic” perfusion. There was some variation between groups with regard to cardiac output, CO x PLVSP and maximum dP/dt. The greater initial performance in some studies, such as the control and matched urea, GSA, creatinine group, was probably related to the lesser heart weight in this series of experiments. It has been noted previously that performance per gram of heart measured in this apparatus tends to be inversely related to the heart weight [12]. I n any case in terms of dynamic performance each experimental group was well matched with its control group at the start of experimental perfusion. Studies with urea
Figure 1 shows the results of perfusion with 20 nm-urea. Performance was generally similar to hearts perfused under control conditions. However, coronary flow remained elevated in control hearts after return to the original perfusion conditions at the end of the experiment. Hearts perfused with urea returned close to the initial value for coronary flow. The cardiac output response to increased atria1 pressure (20 cm) was slightly but significantly depressed in hearts perfused with urea, and these hearts failed to demonstrate a rise in maximum dP/dt with an increasing atria1 pressure. A significant rise in dP/dt (P< 0.05) was present in control hearts. The responses to isovolumic clamping were similar in control hearts and those perfused with urea and there was no difference in the CO x PLVSP response in the two groups. Table 2 shows that oxygen consumption, lactate production and the effluent lactatelpyruvate ratios were similar in hearts perfused with urea and in controls.
361 f 15 376 & 8
6
7
382 f 8
11
222 * 3
228 f 8
228 & 4
225 f 5
199&5
200 f 7
220 f 6 216 & 5
51 f2
52 f 1
51 f2
52 &2
51 f2
50 f2
55 *4 52 f2
Dry heart Coronary weight flow (ml glmin-l) k>
Results are mean + S.E.Flow related data are per gram dry heart weight PLVSP = peak left ventricular pressure CO x PLVSP = cardiac output x PLVSP Max dP/dt = maximum rate of left ventricular pressure rise
Control GSA, MG Creatinine
385 f 8
10
338 f 5
8
Control Urea, GSA, MG Creatinine
344&8
7
Control Urea, GSA Creatinine
385 f 14 386 f 16
8 11
Body weight k)
Control Urea
Number of hearts
159 f6
157 f 8
154 f6
146f7
203 f 9
197 * 10
149 f4 138 f 13
Cardiac output (ml grmir-1)
80 f 3
82 rt 3
88 f 2
84 &2
80 f 2
84 f 4
80 & 2 75 f2
PLVSP (mmHg)
1. Weight relationships and initial control dynamic values for perfused hearts
Conditions
TABLE
12 650 f 671
12 870 f 1000
13 460 f 476
11900*630
16 530 f 910
16 680 f 1070
11830 &300 11300 f460
co x PLVSP (ml x mmHg grmin-1)
3700 f 132
3520 f 196
3224 f 70
3414 f 168
3858 f 130
3954 f 206
3690 f 108 3580 f 172
Max dP/dt (mmHg/sec)
292
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';i 1202 b IIOloo95-
AND
S. W. STEZOSKI
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FIGURE 1. The effects of urea 20 rn~ ( 120 mg/ 100 ml) on cardiac dynamics. Perfusion during the initial and the final periods were in the absence of urea. Perfusion during the second and third periods was with urea for the experimental group. CF = coronary flow; CO = cardiac output; LVSP = left ventricular pressure; max dP/dt = maximum rate of left ventricular pressure rise. Panel A indicates LVSP and dP/dt during isovolumic beats, and panel B shows these variables during ejecting beats. Results are mean f S.E. as percent of the initial 5 cm value. The number of hearts are shown in the parenthesis. *indicates P-c 0.05. (0) Control, 8; (0) urea, 11. Atria1 pressures are in cm of perfusion medium, ventricular pressures are in mmHg.
In order to test whether the response to 20 m-urea represented a toxic effect, hearts were perfused with lower concentrations of this agent. In general the hearts performed similarly to the hearts perfused with 20 mM-urea. At 20 cm atria1 pressure, performance in the presence of urea always tended to be less than in controls. The changes in cardiac output and CO x PLVSP at 20 cm atria1 pressure are shown in Table 3.
Studies with urea, creatinine and guanidinosuccinic acid (GSA)
Figure 2 shows the effects of adding creatinine and GSA to urea. More marked differences in performance were noted with this mixture. Coronary flow fell with
UREMIC
TABLE
2.
AGENTS
IN CARDIAC
293
FUNCTION
Metabolic values for perfused hearts
Control Urea Control Urea, GSA Creatinine Control Urea, GSA, MG Creatinine Control MG, GSA Crealinine
Control Urea Control Urea, GSA Creatinine Control Urea, GSA, MG Creatinine control MG, GSA Creatinine
G5CM 0.84 & 0.03 0.78 f 0.03 0.81 & 0.03
Oxygen consumption ml g-lmin-l 5CM 20CM 0.86 f 0.05 1.12 & 0.10 0.79 * 0.03 1.02 f 0.08 0.84 & 0.02 1.10 & 0.08
0.81 f 0.04 0.80 * 0.03
0.80 & 0.03 0.81 f 0.03
0.91 f 0.06 0.92 5 0.04
0.80 & 0.03 0.84 f 0.02
0.80 f 0.04 0.85 f 0.02
0.79 f 0.05 0.82 5 0.03
0.81 f 0.03 0.93 f 0.02
0.75 f 0.04 0.81 f 0.02
0.82 f 0.02
0.81 & 0.03 1.04 f 0.05 Lactate production ymol g-l min-l ___.__ 5CM 20CM 3.1 f 0.4 6.7 f 0.5 3.2 + 0.7 7.7 -j= 0.6 2.4 f 0.3 7.6 * 0.8
0.80 & 0.03
C4CM 3.2 f 0.5 3.6 f 0.6 2.9 * 0.4
.__.-~~
~-
R-5CM 0.94 & 0.08 0.82 & 0.04 0.90 f 0.04
R-5CM 3.2 5 0.64.6 f 0.6 2.7 & 0.4
2.7 f 0.3 3.2 +- 0.7
2.7 f 0.4 2.0 + 0.5
9.0 + 1.0 6.6 & 0.4
3.3 f 0.6 2.5 & 0.6
3.6 f 0.6 3.2 & 0.7
4.0 & 0.8* 2.0 f 0.3
9.8 f 0.9* 6.5 f 0.8
5.8 & 1.3* 2.5 f 0.5
2.6 f 0.5
3.4 f 0.8 7.6 f 0.8 3.6 Lactate/Pyruvate 5CM 20CM ~-- -Rx6 7.2 f 1.2 13.4 f 0.7 6.9 7.0 & 1.1 15.7 f 1.0 9.4 5.7 f 0.7 15.5 & 2.0 6.4
& 0.8
C-SCM -__ Control f 7.1 f 0.9 Urea & 6.6 f 1.0 Control 6.1 f 0.7 f Urea, GSA Creatinine 4.8 f 0.7 6.0 i 0.6 15.9 f 2.0 6.9 & Control 7.0 f 1.2 5.2 f 0.3 15.4 * 1.3 5.7 f Urea, GSA, MG Creatinine 7.1 f 1.2 9.8 f 1.27 25.1 f 2.5t 12.1 & Control 5.8 f 0.9 5.1 & 0.6 13.8 -& 1.2 5.9 & MG, GSA Creatinine 4.9 f 0.9 8.5 f 1.5 16.4 f 1.4 8.2 f Results are mean f S.E.QOs and lactate are per gram dry weight. C = initial control period; R = recovery period; 5 CM and 20 CM indicate atria1 pressures. * P< 0.05. t P< 0.01.
1.1 1.0 0.7 0.9 1.2 2.5* 0.7 1.8
294
J. SCHFXJER
TABLE
3.
Performance
Concentration b-4
AND
S. W. STEZOSKI
at 20 cm atria1 pressure-urea Number of hearts
0
9
2.5 10
6 8
20
9
Cardiac .~ -~ .-@--266 f 217 f 200 f 213 f
perfusions output
co -
12 12* 15t 14t
x PLVSP
~-~~ -~-~~ (%I ~~~~ 295 + 25
211 f21* 232 121:: 237 & 23*
Results for urea at 20 cm atria1 pressure are mean -J= S.E. as y0 of initial values recorded at 5 cm atria1 pressure with urea absent. CO x PLVSP = the product of cardiac output and peak left ventricular pressure. * PcO.05 t P< 0.01 $ P< 0.2
the switch from initial conditions (5 cm, no uremic compounds) to perfusion with the mixture (P < 0.05). Coronary flow also failed to rise with increasing atria1 pressure when exposed to this mixture. At 20 cm atria1 pressure coronary flow
115 I IOE
Atrial pressure (cm)
FIGURE 2. The effects of urea 20 mu, creatmine 0.88 mM, and guanidinosuccinic acid 0.31 m~ on cardiac dynamics. The experimental design and format are the same as in Figure 1. **indicates PcO.01. (a) Control, 7; (0) uremicmixture, 8.
UREMIC
AGENTS
IN CARDIAC
FUNCTION
295
was significantly higher in control hearts than in those perfused with the mixture. Cardiac output was also depressed by the mixture at 5 cm atria1 pressure, and at 5 cm, 20 cm and during recovery cardiac outputs were lower in the experimental than in the control hearts. Maximum dP/dt failed to rise with increasing atria1 pressure in hearts perfused with the mixture, and isovolumic maximum dP/dt was lower during the recovery period in the experimental group. That this combination was more depressant than urea alone is shown by the CO xPLVSP value, at 20 cm atria1 pressure. CO xPLVSP rose to 265% of the initial value in controls and only 174% in experimental hearts (P
Studies with urea, creatinitu, guanidinosuccinic acid and methyl guanidine (MG)
Figure 3 shows the results of perfusions with an urea, MG, GSA, and creatinine mixture. The performance with this mixture generally showed the most marked
Atrial pressure km)
FIGURE 3. The effects of urea 20 mu, creatinine 0.88 mM, guanidinosuccinic acid 0.31 mly and methyl guanidine 0.46 mM on cardiac dynamics. The format is the same as in Figures 1 and 2. (a) Control, 10; (0) mixture, 11.
296
J. SCHEUER
AND
S. W. STEZOSKI
differences from control perfusions. Coronary flow was significantly lower with the mixture at 20 cm atria1 pressure and during the recovery period. Peak left ventricular pressure and maximum dP/dt during both isovolumic performance and during ejection were less in hearts perfused with the mixture at 20 cm atria1 pressure, and these functions remained less during ejection in the recovery period. Cardiac output also was markedly lower in the mixture group than in the control group at 20 cm atria1 pressure and a significant difference remained during the recovery period. This was the only group in which maximum dP/dt declined significantly (P
Studies with creatinim, guanidinosuccinic acid and methyl guanidine All the above studies included urea in the perfusion medium. To determine the role of urea in the depressant effects of the mixtures, a combination of MG, GSA and creatinine were studied. Figure 4 demonstrates, that except for the isovolumic maximum dP/dt at 5 cm atria1 pressure, there were no differences between control hearts and those perfused with this mixture. There were also no effects of the mixture upon CO x PLVSP, oxygen consumption, or the lactate/pyruvate ratios. Residual glycogen levels were the same in control and uremic groups in each series. In order to insure that any alterations observed in performance were not due to differences in the preload, end-diastolic pressures were carefully measured in each experiment for each level of performance during both isovolumic contraction and during ejection. There were no significant differences between the end-diastolic pressures except in the urea, MG, GSA, creatinine experiments. During ejection
UREMIC 120
1
I
AGENTS
IN CARDIAC
297
FUNCTION
I 250
s
IIO-
-
,\
F
\
80
81 5
I 5
I 20
II 5
I
I 5
I 5
I 20
I I 5
Atrial pressure (cm)
FIGURE 4. The effects of creatinine 0.88 mM, guanidinosuccinic acid 0.31 rnM and methyl guanidine 0.46 nm on cardiac dynamics. The format is the same as in previous figures. (0) Control, 6; (0) uremic mixture, 7.
the left ventricular end-diastolic pressures at 20 cm atria1 pressure were 13.9 & 0.7 mmHg in controls and 17.9 & 0.6 in experimental hearts (P
4. Discussion
The analysis of experiments in the working rat heart apparatus in terms of classical muscle mechanics is difficult, because ventricular volumes are not known. However, certain inferences can be drawn from hearts of the same weight when the filling pressures are known, especially when isovolumic beats are analyzed. In the current experiments, there was evidence of decreased contractility in the group with all four compounds. These hearts had depressed external performance at 20 cm atria1 pressure even though end-diastolic pressures were significantly higher than in controls. This and the depressed responses to isovolumic clamping (for pressure and maximum dP/dt) also suggest decreased contractility. Whether or not one accepts the supposition that myocardial contractility was depressed by the mixtures of
298
J. SCHEUER
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uremic compounds, it is evident that the function of the heart as a pump and particularly its response to increasing atria1 pressure (Starling effect) was attenuated by these compounds. Thus in contrast to our earlier studies, in which normal or increased intrinsic function was found in hearts from uremic animals, this study demonstrates that the products of uremia can be deleterious to normal hearts. Urea alone produced some depressant effect at the 20 cm atria1 pressure. The possibility was considered that this effect was due to an increase in the osmolarity of the perfusion medium. Urea and glycerol at concentrations that raise osmolarity to 500 mosmol or more decrease contractility in atria1 muscle [16], but sucrose, mannitol and urea in osmolar concentrations up to 400 mosmol/L have all been observed to enhance contractile performance of ventricular myocardium [8, 191. In our experiments osmolarity would have been increased only by 20 mosmol. Also the dose response studies with urea demonstrated similar degrees of depression of cardiac output when urea was present in concentrations of 2.5 or 10 mM. The finding of a slight depressant effect of urea even at a concentration of 2.5 mM and lack of a distinct dose depression relationship mitigates against an osmolar cause for the effects of urea. Any of the compounds tested in combination with urea caused exaggeration of the depressant effects, yet these compounds in the concentrations used would not add significantly to the osmolarity. Thus increased osmolarity does not appear to be responsible for the findings. The lack of a dose depression relationship with urea remains unexplained. If the effect of urea was inhibition of an enzyme activity or a change in membrane permeability a dose depression relationship would be expected. The possibility was considered that the depressant effects seen in experiments with urea were due to breakdown products. Ammonium and cyanate accumulate when high concentrations of urea are permitted to remain in solution for long periods [IO]. The concentrations of urea used in the present experiment were very low. Also the solutions were made fresh each day making this an unlikely cause of the depressed cardiac responses. Furthermore, there was no significant difference between the first experiments conducted with fresh solutions each morning and those performed several hours later. The concentrations of the uremic compounds employed were as high or higher than are generally found in severe uremia. These concentrations were used purposefully in order to determine if any effect on cardiac function could be detected. Even though the degree of depression seemed to be related to the number of compounds included in the mixture, urea was obligitory for the mixture to be depressant. This was demonstrated by the similar function in control and experimental hearts when all the compounds except urea were included. It is possible that if the concentrations of the compounds in combination with urea were lowered, or if the urea concentration alone was decreased the additive deleterious effects might not have been seen.
UREMICAGENTSIN
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299
The mechanism of the depression appeared to be partially related to a defect in coronary flow and oxygen delivery. In hearts perfused with any combination of compounds with urea, coronary flow rates and responses to increasing atria1 pressure tended to be less than in control hearts. In absence of urea coronary flow rates and responses were the same as in controls. Oxygen consumption also tended to be lower when urea was present, although these differences were seldom statistically significant. On the other hand, in the group perfused with all four compounds, oxygen extraction, lactate production and effluent lactate/pyruvate ratios were all significantly higher in experimental hearts than in controls, suggesting that oxygen delivery was limited by inadequate coronary flow, glycolytic metabolism was increased and the cytoplasmic NAD+/NADH was shifted to a more reduced state. In these studies the hearts were only exposed to uremic compounds for a short period. The results suggests that high concentrations of compounds known to be present in the blood of uremic subjects can cause acute depression of cardiac function when elevated levels of urea are also present. The effects of lower concentrations of the compounds or of more prolonged exposure, as might be seen in chronic uremia, are important variables to be considered before translating these data to clinical states,
Acknowledgements We are grateful for the technical assistance of Ms. Patricia Pisanelli and for the secretarial assistance of Miss Carol Gundlach and Mrs Donna Costabile. Dr Scheuer is a recipient of the National Institutes of Health Career Development Award HL-15867. This work was supported by the U.S. Public Health Services Research Grant HL-14032-01.
REFERENCES 1. COHEN, B. D., STEIN, I. M., & KORNHAUSER,R. S. Guanidine retention and the urea cycle. Proceedings of the 4th International Congress on .Nejhrology, Stockholm. Vol. 2, pp. 255262 (1969). 2. DEL GRECO, F., SIMON, N. M., ROGUSICA, J., & WALKER, C. Hemodynamic studies in chronic uremia. Circulation 40,87-95 (1969). 3. GIOVANNETTI, S., BUGINI, M., BALESTRI, P. L., NAVALESI, R., GUGNONI, P., DE MATEIS, A., FERRO-MILONE, P., & PEFLFETTI, C. Uraemic symptoms in dogs chronically intoxicated with methyl-guanidine. Proceedings of the 4th International Congress on Jvephrology, Stockholm. Vol. 2, pp. 253-254 (1969). 4. Goss, J. E., ALXREY, A. C., VOGEL, J. H. K., & HOLMES, J. H. Hemodynamic changes during hemodialysis. Transactions of the American Society of Artijcial Internal Organs 13, 68-74 ( 1967). 5. HICK, J. M., YOUNG, D. S. & WOOTTON, I. D. P. Abnormal blood constituents in acute renal failure. Clinica Chin&a Acta 7,623-633 (1962). 6. HOHORST,H. J. L-(+)-Lactate. Determination with lactic dehydrogenase and DPN. In
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7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
17. 18. 19.
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Methods of Enzymatic Analysis, (H. U. Bergmeyer, Ed.) pp. 266-270. New York: Academic Press ( 1963). KNOWLAN, D. M. PIATNEK, D. A. & OLSON, R. E. Myocardial metabolism and cardiac output in acute uremia. Clinical Research9, 141 (1960) (Abstract). KOCH-WESER, J. Influence of osmolarity of perfusate on contractility of mammalian myocardium. AmericanJournal of Physiology 204,957-962 (1963). LANGENDOW, R., & PIRANI, M. D. The heart of uremia. An electrocardiographic and pathologic study. American HeartJournal 33,282-307 (1947). MARIER, J. R. & ROSE, D. Determination of cyanate, and a study of its accumulation in aqueous solutions of urea. Anabtical Biochemistry 7,304-314 (1964). MERRILL, J. P. The treatment of renal failure. New York: Grune & Stratton. p. 67 (1955). NEELY, J. R., LIEBERMEISTER, H., BATTERSBY, E. J., & MORGAN, H. E. Effect of pressure development on oxygen consumption by isolated rat heart. American Journal of Physiolosy 212,804814 (1967). PENPARGKUL, S. & SCHEUER, J. The effect of uremia upon the performance of the rat heart. Cardiovascular Research 6, 702-708 (1972). IIAAB, W. Cardiotoxic substances in the blood and heart muscle in uremia (their nature & action). TheJournal of Laboratory and Clinical Medicine 29,7 15-734 (1944). SCHEUER, J. & STEZOSKI,S. W. Effect of physical training on the mechanical and metabolic response of the rat heart to hypoxia. Circulation Research 30,418-429 (1972). SCHMIDT, E., WILKES, B., & HOLLAND, W. C. Effects of various glycerol or urea concentrations and incubation times on atria1 contractions and ultrastructure. Journal of Molecular d Cellular Cardiology4, 113-120 ( 1972). &GAL, S. A., BLAIR, A. E., & WYNGAARDEN, J. B. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. Journal of Laboratory and Clinical Medicine 48, 137-143 (1956). SNEDECOR, G. W. Statistical Methods (5th ed.) Iowa: Iowa State University Press (1956). WILDENTHAL, K., MIERZWWC, D. S. & MITCHELL, J. H. Acute effects of increased serum osmolality on left ventricular performance. American Journal of Physiology 216, 898-904 (1969).