Regression of carbon monoxideinduced cardiomegaly PHILLIP E. STYKA AND DAVID G. PENNEY Department of Biological Sciences, University of Illinois, Chicago, Illinois 60680; and Department of Physiology, Wayne State University Medical School, Detroit, Michigan 48201
STYKA, P. E., AND D. G. PENNEY. Regression of carbon monoxide-induced cardiomegaly. Am. J. Physiol. 235(5): H516-H522, 1978 or Am. J. Physiol.: 4(5): Heart Circ. Physiol. H516-H522, 1978. -To characterize regression of cardiomegaly male rats, 60 days of age, were caused to inhale CO at two concentrations over a period of 6 wk; 1) 400 ppm CO giving 35% carboxyhemoglobin (HbCO) (expt 1 ), and 2) 500 ppm progressively increased to 1,100 ppm CO, giving a final HbCO concentration of 58% (expt 2). Rats were killed at various daily intervals upon removal from CO. Hb concentration was elevated 47% and 71% in expt 1 and expt 2, respectively, O-3 days after cessation of CO exposure. Hct was elevated in a similar fashion. The ratio of heart weight (HW) to body weight (BW) was increased from 2.65 in controls to 3.52 in expt 1 and to 4.01 in expt 2. Forty-one to forty-eight days after termination of CO inhalation there were no significant differences in Hb concentration among treated and control groups. HW/BW in expt I was similar to the controls, but HW/BW in expt 2 remained significantly elevated. HW, weight of the left ventricle (LV) plus interventricular septum (S), and weight of th e right ventricle (RV), predicted on the basis of BW, declined to control values within 24-36 days in expt 1. In expt 2, however, HW and weights of LV + S and RV fell to a nadir about 10% above control weights in the same time period. Myocardial lactate dehydrogenase, M subunit (M LDH), was elevated 12-14% and 5-6% above controls in LV, S, and RV in expt 1 and expt 2, respectively. These values declined sharply in the first 3 days when CO exposure was terminated and was followed by a more gradual decline thereafter. Control values were reached in LV, S, and RV within 3 wk in expt 1. In expt 2, however, %M LDH did not return to control values 44 days after termination of CO exposure.
lactate dehydrogenase; moglobin; hematocrit
heart
weight;
carboxyhemoglobin;
he-
INHALATION of high sublethal levels of carbon monoxide in the rat produced rapid increases in hemoglobin (Hb) concentration, heart weight (HW), and alterations in myocardial lactate dehydrogenase (LDH) isozyme pattern (15). Furthermore, cardiomegaly induced by CO is approximately equal in both ventricles (11) and the degree of compensatory cardiac growth is age related (16). In the adult male rat a CO concentration in air of 500 ppm produces an approximately 40% increase in HW over several wk (11, 15). At 200 ppm CO, HW increases 12% over a period of 5 wk (15). Consid eration of the physiology of chronic CO poisoning suggests that the work overload driving cardiomegaly is of the volume type, at least at CO
CONTINUOUS
H516
This is provided by concentrations up to 500 ppm. initial sustained increases in stroke volume and cardiac output (13), and by later elevation of blood volume as polycythemia develops (20). The hematologic changes observed during 500 ppm CO inhalation are completely reversible 30 days after exposure (12). When work overloading is relieved, regression of cardiomegaly occurs. Weight regression of cardiac enlargement has been found to be complete in rats following altitude exposure in 13 days (17), following abdominal aortic constriction in 15 days (7), following hyperthyroidism in 13 days (2, 19), and following nutritional anemia in 16 days (2, 6). However, some investigators have obtained data suggesting that regression of cardiomegaly may fall short of complete return to normal heart mass, composition, and function. During regression of pressure overload-induced cardiomegaly caused by banding .of the ascending aorta for 28 days, left ventricle (LV) DNA and hydroxyproline content were found to be elevated during the 4 wk after debanding, although this was not the case in hearts banded for only 10 days (4). In the same study, LV-to-body weight (BW) ratios in hearts banded for a longer duration failed to return to control values. Other investigators mechanical performance in (7) have fo und altered hearts recovering from abdominal aortic constriction. In contrast, in a volume work-overloading stress such as nutritional anemia, myocardial DNA content is reported to have remained elevated after return to a control diet (2). Reversibility of the changes which occur in heart during cardiomegaly may depend upon 1) type of work overload imposed, 2) degree of cardiomegaly attained, and 3) duration of the cardiomegaly. The purpose of the present investigation was to characterize regression from CO-induced enlargement and to explore the nature of the regression itself (i.e., complete vs. incomplete regression) by varying the severity of the stress. This was done by examining both hematologic and cardiac morphologic parameters as well as alterations in myocardial LDH isozyme composition, which was previously shown to be a sensitive index to cardiomegaly produced by a number of work overloads (10, 14, 22). METHODS
Animal treatment. Male Charles River derived rats, 60 days of age, of approximately 250 g body wt were used. Three groups were set up: 1) controls not exposed
0363-6135/78/0000-OOOO$Ol.
25 Copyright
0 1978 the Americgn
Physiological
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REGRESSION
OF CO-INDUCED
H517
CARDIOMEGALY
for 6 to CO, 2) rats exposed to 400 ppm CO continually wk (expt L), and 3) rats exposed to a CO concentration which was gradually increased from 500 ppm to 1,100 ppm over a period of 6 wk. The latter treatment regimen was necessary because rats do not tolerate immediate exposure to 1,100 ppm CO. The rats inhaled CO in large plastic bags according to a method previously described (11). The CO bags were opened every other day for 5-10 min for animal care. Both control and treated rats were maintained under 12 h-12 h (8 A.M.-~ P.M .) light-dark cycle and room temperature of
21-23°C. Purnia rat chow and water were supplied to both control and treated rats ad libitum. Tissue preparation. In control and treated rats chosen at random at intervals following termination of 6 wk exposure to CO, blood was drawn from the tail by a scissor cut to provide samples for determination of Hb (5), hemato crit (Hct) (microhematocrit method), and carboxyhemoglobin (HbCO) (3). The rats were weighed to the nearest gram and killed by decapitation. Hearts were immediately excised and washed in ice-cold 0.9% saline. They were trimmed of fat and major vessels,
FIG. 1. Heart weight/body weight x upon termination of carbon monoxide sure. Open circles are data from treated filled circles, data from combined control
I
3.80
-
3.70
-
3.60
EXPERIMENT
0 2
6
10
14
18
22 TIME
26
30
34
1
38
42
464
(days)
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1000 exporats; rats.
H518
P. E. STYKA
blotted, and weighed to the nearest 0.1 mg. Then the heart was dissected into LV, interventricular septum (S), and right ventricle free wall (RV). Following weighing, each portion was frozen to dry ice temperature with Wollenberger tongs. Samples were stored at -20°C until assay for lactate dehydrogenase (LDH) isozyme composition. No differences in LDH were noted due to storage. LDH assay. Heart samples were homogenized in 0.05 TABLE 1. Hematologic changes termination of CO exposure
after
O-3 days Group
Expt
1
Hb, g/100 ml
64.2* 29
22.04* 20.31
2
Combined
81.8” k1.9
25.76* kO.54
(5)
(5)
controls
Values are comparing
60 R
Hb, g/100 ml
35*
45.8 kO.8
15.15 20.19
57.9”
means + SE; numbers of animals used expt 1 or expt 2 and combined controls.
are
given
- 2950
0
(4)
(4)
HW = 0.84 (BW + 146) + 1312 log (SW + 146)
(3)
14.93 20.14
LV + S = 0.424 (BW + 203) + 1336 log (BW + 203)
(4)
- 3170
*p I
in parentheses.
D. G. PENNEY
M phosphate buffer (pH 7.4) using glass-Teflon tissue grinders. An LDH-containing supernatant fraction was obtained by centrifugation at 12,000 x g at 4°C for 15 min. Separation and quantitation of LDH isozymes was carried out using 7% polyacrylamide slab gel electrophoresis as previously described (15). Samples underwent electrophoresis for 5 h at 16.5”C, and the gels were stained for 90 min at 37°C. The gels were scanned in a linear transport attached to a Gilford spectrophotometer, and staining density was recorded on a Honeywell recorder. The recorded peaks were cut from the chart paper, and the paper was weighed to determine staining density. Percent M subunit lactate dehydrogenase (%M LDH) was computed on the basis of the staining density of each isozyme and its fractional M subunit content. ’ StatisticaL anaZysis. Predictions of HW and weight of the LV + S and RV were made from BW using multiple regression equations.
0
(3)
47.0 50.4
(2)
0 (4)
14.62 20.62
(3)
0
HbCO, %
(4)
40.7” 51.7
(2)
(5)
(5)
0.05
Hct, %
(4)
15.04 kO.35
45.7 21.0
days
HbCO %
(6)
(7) Expt
41-48
Hct %
AND
RV = 0.21 (BW + 48) + 132 log (BW + 48) - 228 HW
0
55 50
EXPERIMENT
2
45 40
35 30 25 20
FIG. 2. Percent difference from predicted heart weight upon termination of carbon monoxide exposure. Symbols as in Fig. 1.
EXPERIMENT
0
0
I I I I I PI 0 2
6
10
1
14
18
0 I I I I I I I I I I I I I0 22
TIME
26
30
34
38
42
464;
(days)
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REGRESSION
OF CO-INDUCED
H519
CARDIOMEGALY
These equations were generated using the IBM computer facility at the University of Illinois, Chicago Circle, based on data from 289 normal male rats in the BW range, 20-500 g. Although confidence limits were not computed for differences from predicted weights of the regressing hearts in the present study (Figs. 2-4), the close approximation to zero by controls indicates the validity of using the three regression equations with the present data. Student’s t test was used for determination of statistical significance. Differences that resulted in probability values smaller than 0.05 were considered significant. Lines were drawn through the data points in Figs. l5 by eye.
as a percent difference from the predicted value for a normal rat of identical BW, regression is incomplete after 44 days in expt 2, while in expt 1 regression appears to be complete in 34-36 days. Note that at termination of CO exposure HWIBW is approximately 3.54 in expt 1 and 4.51 in expt 2, whereas control rats of similar BW have HW/BW of 2.66 (Fig. 1). Similarly, at this time, HW in expt 1 is approximately 30% above the predicted value, and in expt 2 HW is 50% above the predicted value (Fig. 2). Plots of LV + S and RV weights as percent differences from values predicted for male rats of identical BW (Figs. 3,4) reveal the same pattern as HW/BW and HW. In expt 1 regression appears to be complete within 26-30 days, but in expt 2, neither LV + S nor RV wt return to predicted values within 44 days after termiNote that following 6 wk nation of CO exposure. exposure to CO the LV + S weight was approximately 28% above predicted in expt 1, whereas in expt 2, it was about 38% above predicted. At the same time, RV wt in expt 1 was about 32% above the predicted value, but in expt 2, it was 50% above predicted. Regression of elevated M LDH in LV, S, and RV to control values following termination of CO exposure was biphasic, with a large decrease in percent M subunit occurring during the first 3 days, and a slower decline following thereafter (Fig. 5). In expt 1 %M LDH in the three heart portions appeared to return completely to control values within 2 wk, whereas in expt
RESULTS
Hb concentration increased from 15.04 g/100 ml in the controls to 22.04 and 25.7 g/100 ml in expt 1 and expt 2, respectively over 6 wk of CO exposure (Table 1). Hct increased from 45.7% in the control group to 64.2% and 81.8%, respectively, in expt 1 and expt 2. Forty-one to forty-eight days after termination of CO exposure, Hb in expt 1 and expt 2 and Hct in expt 1 had returned to the control value. Hct in expt 2 was significantly lower than the control value at this time. Following termination of CO exposure, relative HW declined rapidly in both expt 1 and 2 (Figs. 1, 2). However, whether HW is expressed as a ratio to BW or
LV+S EXPERIMENT
2
: FIG. 3. Percent difference from predicted left ventricle plus interventricular septum weight upon termination of carbon monoxide exposure. Symbols as in Fig. 1.
EXPERIMENT
0
IIIIIlll
2
6
10
14
III1 18
22
TIME
II
II
26
30
1
Illllll~ 34
38
42
464
(days)
Downloaded from www.physiology.org/journal/ajpheart at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
P. E. STYKA
H520
AND
D. G. PENNEY
RV EXPERIMENT
2
FIG. 4. Percent difference from predicted tricle weight upon termination of carbon exposure. Symbols as in Fig. 1.
El!
0
EXPERIMENT
right venmonoxide
1
a
111~11111 0 2 6
10
14
18
IllIll 22 TIME
26
30
~I~~~~~~~ 34 38
42
464
(dayc,)
2, return to control values appeared to be incomplete after 46 days. Note that %M LDH in LV, S, and RV upon termination of CO exposure are 27% in elx;pt 1 and 34% in expt 2, whereas control values for these three heart portions are approximately 22%. Control %M LDH values declined slightly as the animals became older. DISCUSSION
Results of the present study using CO-stimulated volume work overloading of the heart suggest that some residuum of the change in myocardial mass and LDH isozyme composition remains after the stress is relieved, at least over the time period used here. The severity of the stress is also shown to be an important factor, that is, that moderate work overloading is reversible, whereas extreme work overloading is not, or at least requires a much longer recovery period. It is not clear, however, whether chronic exposure to very high CO concentrations as in expt 2, generating extreme Hct values (e.g. 81.8%) offers an additional pressure overload stress not observed at 500 ppm CO (13) and below.
Our findings are consistent with those of Cutilletta et al. (4) (i.e., that some degree of irreversibility develops), who used banding of the ascending aorta to increase pressure work by the heart. They found significantly higher LV DNA content and ratio of LV to BW 4 wk after debanding in rats banded for 28 days, while in rats banded for only 10 days there were no differences from controls. Heart DNA content has likewise been found to remain elevated after regression of nutritional anemia-induced cardiomegaly (2). This implies that as a result of severe work overloading, hyperplasia of myocardial cells takes place. However, since the potential for cardiac muscle cell mitosis ceases after the 3rd or 4th wk of postnatal life (9, 21), hyperplasia is confined to non-muscle cells, such as fibroblasts. This notion is supported by the work of Cutilletta et al. (4), in which both hydroxyproline content and concentraare markedly elevated in hearts tion (i.e., collagen) from aortic-banded rats. Furthermore, neither concentration nor content of hydroxyproline returned to control levels during the 4 wk after debanding. This suggests that stimulation of fibroblast proliferation and deposition of collagen as a response to work overloading may be irreversible, representing a “permanent” lesion
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REGRESSION
OF CO-INDUCED
H521
CARDIOMEGALY
I
I
30
20 TIME FIG. 5. Percent M subunit lactate dehydrogenase tion of carbon monoxide exposure in left ventricle,
upon terminainterventricular
in myocardial composition. Such lesions could contribute to the reported prolongation of contraction in hearts due to abdominal aortic constriction (7). It should be pointed out, however, that measurement of hydroxyproline concentration in the hearts of the two groups of CO-exposed rats presented here (data not shown), and in hearts of rats treated to endurance exercise and sideropenic anemia (l), indicate no increase in hydroxyproline concentration. Thus, myocardial components other than collagen persist. LDH is made up of two subunits, H and M, aggregating to give five isozymes in most mammalian tissues.
I 40
(days) septum, and right ventricle. Squares are data from expt 1; triangles, from expt 2; and circles, from combined control rats.
In heart it is known to be a sensitive index to cardiomegaly induced by a number of work overloads (10, 14, 22). In sideropenic anemia (14), simulated altitude (lo), aortic constriction (22), and endurance exercise (22), the percent of myocardial M subunit increases, and alterations in %M LDH are brought about solely through changes in the concentration of the M subunit, while H remains constant. Higher LDH activity is seen in these conditions, and the specific activity of LDH is related to the amount of M subunit present (22). The specific activity of LDH determined spectrophotometrically by standard enzymatic techniques in the present
Downloaded from www.physiology.org/journal/ajpheart at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
H522
P. E. STYKA
study (data not shown) was elevated in LV and RV of rats in expt 2 at termination of CO exposure. It returned to control values 44 days later. The decrease in observed LDH activity during regression of cardiomegaly in the present study appears to be a function of the declining %M LDH. We believe that elevated %M LDH in expt 1 and 2 during CO exposure and its persistence in expt 2 following termination of CO inhalation may be related to membrane changes in heart mitochondria as monitored by cytochrome c concentration (18). Regression of HW to control values in expt 1, 24-36 days was longer than has been reported following
AND
D. G. PENNEY
altitude exposure (13 days) (17), abdominal aortic constriction (15 days) (7), hyperthyroidism (13 days) (2, 19), and nutritional anemia (16 days) (2, 6). The reason for this difference is not clear. However, it could not have resulted from retention of HbCO after termination of exposure, because rats exposed to 500 ppm CO lose nearly all their HbCO within 2-3 h after transfer to room air (8). This investigation Grant HL-16367. Received
21 December
was supported
1977; accepted
in part by Public
Health
Service
in final form 11 June 1978.
REFERENCES 1. BARTISOVA, D., M. CHVAPIL, B. KORECKY, 0. POUPA, K. RAKUSAN, Z. TUREK, AND M. VIZEK. The growth of the muscular and collagenous parts of the rat heart in various forms of cardiomegaly. J. PhysioZ. London 200: 285-295, 1969. 2. BEZNAK, M., B. KORECKY, AND G. THOMAS. Regression of cardiac hypertrophies of various origin. Can. J. PhysioZ. PharmacoZ. 47: 579486. 1969. 3. COMMINS, B., AND P. LAWTHER. A sensitive method for determination of COHb in a finger prick sample of blood. Brit. J. Ind. Med. 22: 139-143, 1965. 4. CUTILLETTA, A. F., R. T. DOWELL, M. RUDNIK, R. A., ARCILLA, AND R. ZAK. Regression of myocardial hypertrophy. I. Experimental model, changes in heart weight, nucleic acids, and collagen. J. MOL. CeZZuZar CardioZ. 7: 767-781, 1975. 5. DRABKIN, D. L., AND J. H. AUSTIN. Spectrophotometric studies. II. Preparations from washed blood cells, nitric oxide hemoglobin, and sulfhemoglobin. J. BioZ. Chem. 112: 51-65, 1935-36. 6. GOODMAN, J. R., J. B. WARSHAW, AND P. R. DALLMAN. Cardiac hypertrophy in rats with iron and copper deficiency: quantitative contribution of mitochondrial enlargement. Pediat. Res. 4: 244-256, 1970. 7. JOUANNOT, P., AND P. Y. HATT. Rat myocardial mechanics during pressure-induced hypertrophy development and reversal. Am. J. Physiol. 229: 355-364, 1975. 8. MONTGOMERY, M. R., AND R. J. RUBIN. The effect of carbon monoxide inhalation on in vivo drug metabolism in the rat. J. PharmacoZ. Exptl. Therap. 179: 465-471, 1971. 9. NEFFGEN J., AND B. KORECKY. Cellular hyperplasia and hypertrophy in cardiomegalies induced by anemia in young and adult rats. CircuZation Res. 30: 104-113, 1972. 10. PENNEY, D. G. Lactate dehydrogenase subunit and activity changes in hypertrophied heart of the hypoxically-exposed rat. Biochim. Biophys. Acta 358: 21-24, 1974. 11. PENNEY, D. G., M. BENJAMIN, AND E. DUNHAM. Effect of carbon monoxide on cardiac weight as compared with altitude effects. J. AppZ. Physiol. 37: 80-84, 1974.
12. PENNEY, D. G., AND P. A. BISHOP. Hematologic changes in the rat during and after exposure to carbon monoxide. J. Toxicol. Environ. Health. In press. 13. PENNEY, D. G., P. BISHOP, P. E. STYKA, AND P. SODT. Blood and heart weight changes during and after carbon monoxide exposure (Abstract). Federation Proc. 36: 518, 1977. 14. PENNEY, D. G., L. B. BUGAISKY, AND J. R. MIESZALA. Lactate dehydrogenase and pyruvate kinase in rat heart during sideropenic anemia. Biochim. Biophys. Acta 334: 24-30, 1974. 15. PENNEY, D. G., E. DUNHAM, AND M. BENJAMIN. Chronic carbon monoxide exposure: time course of hemoglobin, heart weight, and lactate dehydrogenase isozyme changes. Toxicol. AppZ. Pharmacol. 28: 493-497, 1974. 16. PENNEY, D. G., J. SAKAI, AND K. COOK. Heart growth: interacting effects of carbon monoxide and age. Growth 38: 321-328, 1974. 17. SIZEMORE, D. A., T. W. MCINTYRE, E. J. VAN LIERE, AND M. F. WILSON. Regression of altitude-produced cardiac hypertrophy. J. AppZ. PhysioZ. 35: 518-521, 1973. 18. STYKA, P. E., AND D. G. PENNEY. The perinatal rat: body weight, hematocrit and regional changes in heart weight and lactate dehydrogenase isozyme composition and activity. Growth 41: 325-336, 1977. 19. VAN LIERE, E. J., AND D. A. SIZEMORE. Regression of cardiac hypertrophy following experimental hyperthyroidism in rats. Proc. Sot. Exptl. BioZ. Med. 136: 645-648, 1971. 20. WILKS, S. S., J. F. TOMASHEFSKI, AND R. T. CLARK, JR. Physiological effects of chronic exposure to carbon monoxide. J. AppZ. PhysioZ. 14: 305-310, 1959. 21. WINICK, M., AND A. NOBLE. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. DeveZop. BioZ. 12: 451-466, 1965. 22. YORK, J. W., D. G. PENNEY, T. WEEKS, AND P. STAGNO. Lactate dehydrogenase changes following several cardiac hypertrophic stresses. J. AppZ. Physiol. 40: 923-926, 1976.
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STYKA, P. E., AND D. G. PENNEY. Regression of carbon monoxide-induced cardiomegaly. Am. J. Physiol. 235(5): H516-H522, 1978 or Am. J. Physiol.: 4(5): Heart Circ. Physiol. H516-H522, 1978. -To characterize regression of cardiomegaly male rats, 60 days of age, were caused to inhale CO at two concentrations over a period of 6 wk; 1) 400 ppm CO giving 35% carboxyhemoglobin (HbCO) (expt 1 ), and 2) 500 ppm progressively increased to 1,100 ppm CO, giving a final HbCO concentration of 58% (expt 2). Rats were killed at various daily intervals upon removal from CO. Hb concentration was elevated 47% and 71% in expt 1 and expt 2, respectively, O-3 days after cessation of CO exposure. Hct was elevated in a similar fashion. The ratio of heart weight (HW) to body weight (BW) was increased from 2.65 in controls to 3.52 in expt 1 and to 4.01 in expt 2. Forty-one to forty-eight days after termination of CO inhalation there were no significant differences in Hb concentration among treated and control groups. HW/BW in expt I was similar to the controls, but HW/BW in expt 2 remained significantly elevated. HW, weight of the left ventricle (LV) plus interventricular septum (S), and weight of th e right ventricle (RV), predicted on the basis of BW, declined to control values within 24-36 days in expt 1. In expt 2, however, HW and weights of LV + S and RV fell to a nadir about 10% above control weights in the same time period. Myocardial lactate dehydrogenase, M subunit (M LDH), was elevated 12-14% and 5-6% above controls in LV, S, and RV in expt 1 and expt 2, respectively. These values declined sharply in the first 3 days when CO exposure was terminated and was followed by a more gradual decline thereafter. Control values were reached in LV, S, and RV within 3 wk in expt 1. In expt 2, however, %M LDH did not return to control values 44 days after termination of CO exposure.
lactate dehydrogenase; moglobin; hematocrit
heart
weight;
carboxyhemoglobin;
he-
INHALATION of high sublethal levels of carbon monoxide in the rat produced rapid increases in hemoglobin (Hb) concentration, heart weight (HW), and alterations in myocardial lactate dehydrogenase (LDH) isozyme pattern (15). Furthermore, cardiomegaly induced by CO is approximately equal in both ventricles (11) and the degree of compensatory cardiac growth is age related (16). In the adult male rat a CO concentration in air of 500 ppm produces an approximately 40% increase in HW over several wk (11, 15). At 200 ppm CO, HW increases 12% over a period of 5 wk (15). Consid eration of the physiology of chronic CO poisoning suggests that the work overload driving cardiomegaly is of the volume type, at least at CO
CONTINUOUS
H516
This is provided by concentrations up to 500 ppm. initial sustained increases in stroke volume and cardiac output (13), and by later elevation of blood volume as polycythemia develops (20). The hematologic changes observed during 500 ppm CO inhalation are completely reversible 30 days after exposure (12). When work overloading is relieved, regression of cardiomegaly occurs. Weight regression of cardiac enlargement has been found to be complete in rats following altitude exposure in 13 days (17), following abdominal aortic constriction in 15 days (7), following hyperthyroidism in 13 days (2, 19), and following nutritional anemia in 16 days (2, 6). However, some investigators have obtained data suggesting that regression of cardiomegaly may fall short of complete return to normal heart mass, composition, and function. During regression of pressure overload-induced cardiomegaly caused by banding .of the ascending aorta for 28 days, left ventricle (LV) DNA and hydroxyproline content were found to be elevated during the 4 wk after debanding, although this was not the case in hearts banded for only 10 days (4). In the same study, LV-to-body weight (BW) ratios in hearts banded for a longer duration failed to return to control values. Other investigators mechanical performance in (7) have fo und altered hearts recovering from abdominal aortic constriction. In contrast, in a volume work-overloading stress such as nutritional anemia, myocardial DNA content is reported to have remained elevated after return to a control diet (2). Reversibility of the changes which occur in heart during cardiomegaly may depend upon 1) type of work overload imposed, 2) degree of cardiomegaly attained, and 3) duration of the cardiomegaly. The purpose of the present investigation was to characterize regression from CO-induced enlargement and to explore the nature of the regression itself (i.e., complete vs. incomplete regression) by varying the severity of the stress. This was done by examining both hematologic and cardiac morphologic parameters as well as alterations in myocardial LDH isozyme composition, which was previously shown to be a sensitive index to cardiomegaly produced by a number of work overloads (10, 14, 22). METHODS
Animal treatment. Male Charles River derived rats, 60 days of age, of approximately 250 g body wt were used. Three groups were set up: 1) controls not exposed
0363-6135/78/0000-OOOO$Ol.
25 Copyright
0 1978 the Americgn
Physiological
Downloaded from www.physiology.org/journal/ajpheart at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
Society
REGRESSION
OF CO-INDUCED
H517
CARDIOMEGALY
for 6 to CO, 2) rats exposed to 400 ppm CO continually wk (expt L), and 3) rats exposed to a CO concentration which was gradually increased from 500 ppm to 1,100 ppm over a period of 6 wk. The latter treatment regimen was necessary because rats do not tolerate immediate exposure to 1,100 ppm CO. The rats inhaled CO in large plastic bags according to a method previously described (11). The CO bags were opened every other day for 5-10 min for animal care. Both control and treated rats were maintained under 12 h-12 h (8 A.M.-~ P.M .) light-dark cycle and room temperature of
21-23°C. Purnia rat chow and water were supplied to both control and treated rats ad libitum. Tissue preparation. In control and treated rats chosen at random at intervals following termination of 6 wk exposure to CO, blood was drawn from the tail by a scissor cut to provide samples for determination of Hb (5), hemato crit (Hct) (microhematocrit method), and carboxyhemoglobin (HbCO) (3). The rats were weighed to the nearest gram and killed by decapitation. Hearts were immediately excised and washed in ice-cold 0.9% saline. They were trimmed of fat and major vessels,
FIG. 1. Heart weight/body weight x upon termination of carbon monoxide sure. Open circles are data from treated filled circles, data from combined control
I
3.80
-
3.70
-
3.60
EXPERIMENT
0 2
6
10
14
18
22 TIME
26
30
34
1
38
42
464
(days)
Downloaded from www.physiology.org/journal/ajpheart at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
1000 exporats; rats.
H518
P. E. STYKA
blotted, and weighed to the nearest 0.1 mg. Then the heart was dissected into LV, interventricular septum (S), and right ventricle free wall (RV). Following weighing, each portion was frozen to dry ice temperature with Wollenberger tongs. Samples were stored at -20°C until assay for lactate dehydrogenase (LDH) isozyme composition. No differences in LDH were noted due to storage. LDH assay. Heart samples were homogenized in 0.05 TABLE 1. Hematologic changes termination of CO exposure
after
O-3 days Group
Expt
1
Hb, g/100 ml
64.2* 29
22.04* 20.31
2
Combined
81.8” k1.9
25.76* kO.54
(5)
(5)
controls
Values are comparing
60 R
Hb, g/100 ml
35*
45.8 kO.8
15.15 20.19
57.9”
means + SE; numbers of animals used expt 1 or expt 2 and combined controls.
are
given
- 2950
0
(4)
(4)
HW = 0.84 (BW + 146) + 1312 log (SW + 146)
(3)
14.93 20.14
LV + S = 0.424 (BW + 203) + 1336 log (BW + 203)
(4)
- 3170
*p I
in parentheses.
D. G. PENNEY
M phosphate buffer (pH 7.4) using glass-Teflon tissue grinders. An LDH-containing supernatant fraction was obtained by centrifugation at 12,000 x g at 4°C for 15 min. Separation and quantitation of LDH isozymes was carried out using 7% polyacrylamide slab gel electrophoresis as previously described (15). Samples underwent electrophoresis for 5 h at 16.5”C, and the gels were stained for 90 min at 37°C. The gels were scanned in a linear transport attached to a Gilford spectrophotometer, and staining density was recorded on a Honeywell recorder. The recorded peaks were cut from the chart paper, and the paper was weighed to determine staining density. Percent M subunit lactate dehydrogenase (%M LDH) was computed on the basis of the staining density of each isozyme and its fractional M subunit content. ’ StatisticaL anaZysis. Predictions of HW and weight of the LV + S and RV were made from BW using multiple regression equations.
0
(3)
47.0 50.4
(2)
0 (4)
14.62 20.62
(3)
0
HbCO, %
(4)
40.7” 51.7
(2)
(5)
(5)
0.05
Hct, %
(4)
15.04 kO.35
45.7 21.0
days
HbCO %
(6)
(7) Expt
41-48
Hct %
AND
RV = 0.21 (BW + 48) + 132 log (BW + 48) - 228 HW
0
55 50
EXPERIMENT
2
45 40
35 30 25 20
FIG. 2. Percent difference from predicted heart weight upon termination of carbon monoxide exposure. Symbols as in Fig. 1.
EXPERIMENT
0
0
I I I I I PI 0 2
6
10
1
14
18
0 I I I I I I I I I I I I I0 22
TIME
26
30
34
38
42
464;
(days)
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REGRESSION
OF CO-INDUCED
H519
CARDIOMEGALY
These equations were generated using the IBM computer facility at the University of Illinois, Chicago Circle, based on data from 289 normal male rats in the BW range, 20-500 g. Although confidence limits were not computed for differences from predicted weights of the regressing hearts in the present study (Figs. 2-4), the close approximation to zero by controls indicates the validity of using the three regression equations with the present data. Student’s t test was used for determination of statistical significance. Differences that resulted in probability values smaller than 0.05 were considered significant. Lines were drawn through the data points in Figs. l5 by eye.
as a percent difference from the predicted value for a normal rat of identical BW, regression is incomplete after 44 days in expt 2, while in expt 1 regression appears to be complete in 34-36 days. Note that at termination of CO exposure HWIBW is approximately 3.54 in expt 1 and 4.51 in expt 2, whereas control rats of similar BW have HW/BW of 2.66 (Fig. 1). Similarly, at this time, HW in expt 1 is approximately 30% above the predicted value, and in expt 2 HW is 50% above the predicted value (Fig. 2). Plots of LV + S and RV weights as percent differences from values predicted for male rats of identical BW (Figs. 3,4) reveal the same pattern as HW/BW and HW. In expt 1 regression appears to be complete within 26-30 days, but in expt 2, neither LV + S nor RV wt return to predicted values within 44 days after termiNote that following 6 wk nation of CO exposure. exposure to CO the LV + S weight was approximately 28% above predicted in expt 1, whereas in expt 2, it was about 38% above predicted. At the same time, RV wt in expt 1 was about 32% above the predicted value, but in expt 2, it was 50% above predicted. Regression of elevated M LDH in LV, S, and RV to control values following termination of CO exposure was biphasic, with a large decrease in percent M subunit occurring during the first 3 days, and a slower decline following thereafter (Fig. 5). In expt 1 %M LDH in the three heart portions appeared to return completely to control values within 2 wk, whereas in expt
RESULTS
Hb concentration increased from 15.04 g/100 ml in the controls to 22.04 and 25.7 g/100 ml in expt 1 and expt 2, respectively over 6 wk of CO exposure (Table 1). Hct increased from 45.7% in the control group to 64.2% and 81.8%, respectively, in expt 1 and expt 2. Forty-one to forty-eight days after termination of CO exposure, Hb in expt 1 and expt 2 and Hct in expt 1 had returned to the control value. Hct in expt 2 was significantly lower than the control value at this time. Following termination of CO exposure, relative HW declined rapidly in both expt 1 and 2 (Figs. 1, 2). However, whether HW is expressed as a ratio to BW or
LV+S EXPERIMENT
2
: FIG. 3. Percent difference from predicted left ventricle plus interventricular septum weight upon termination of carbon monoxide exposure. Symbols as in Fig. 1.
EXPERIMENT
0
IIIIIlll
2
6
10
14
III1 18
22
TIME
II
II
26
30
1
Illllll~ 34
38
42
464
(days)
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P. E. STYKA
H520
AND
D. G. PENNEY
RV EXPERIMENT
2
FIG. 4. Percent difference from predicted tricle weight upon termination of carbon exposure. Symbols as in Fig. 1.
El!
0
EXPERIMENT
right venmonoxide
1
a
111~11111 0 2 6
10
14
18
IllIll 22 TIME
26
30
~I~~~~~~~ 34 38
42
464
(dayc,)
2, return to control values appeared to be incomplete after 46 days. Note that %M LDH in LV, S, and RV upon termination of CO exposure are 27% in elx;pt 1 and 34% in expt 2, whereas control values for these three heart portions are approximately 22%. Control %M LDH values declined slightly as the animals became older. DISCUSSION
Results of the present study using CO-stimulated volume work overloading of the heart suggest that some residuum of the change in myocardial mass and LDH isozyme composition remains after the stress is relieved, at least over the time period used here. The severity of the stress is also shown to be an important factor, that is, that moderate work overloading is reversible, whereas extreme work overloading is not, or at least requires a much longer recovery period. It is not clear, however, whether chronic exposure to very high CO concentrations as in expt 2, generating extreme Hct values (e.g. 81.8%) offers an additional pressure overload stress not observed at 500 ppm CO (13) and below.
Our findings are consistent with those of Cutilletta et al. (4) (i.e., that some degree of irreversibility develops), who used banding of the ascending aorta to increase pressure work by the heart. They found significantly higher LV DNA content and ratio of LV to BW 4 wk after debanding in rats banded for 28 days, while in rats banded for only 10 days there were no differences from controls. Heart DNA content has likewise been found to remain elevated after regression of nutritional anemia-induced cardiomegaly (2). This implies that as a result of severe work overloading, hyperplasia of myocardial cells takes place. However, since the potential for cardiac muscle cell mitosis ceases after the 3rd or 4th wk of postnatal life (9, 21), hyperplasia is confined to non-muscle cells, such as fibroblasts. This notion is supported by the work of Cutilletta et al. (4), in which both hydroxyproline content and concentraare markedly elevated in hearts tion (i.e., collagen) from aortic-banded rats. Furthermore, neither concentration nor content of hydroxyproline returned to control levels during the 4 wk after debanding. This suggests that stimulation of fibroblast proliferation and deposition of collagen as a response to work overloading may be irreversible, representing a “permanent” lesion
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REGRESSION
OF CO-INDUCED
H521
CARDIOMEGALY
I
I
30
20 TIME FIG. 5. Percent M subunit lactate dehydrogenase tion of carbon monoxide exposure in left ventricle,
upon terminainterventricular
in myocardial composition. Such lesions could contribute to the reported prolongation of contraction in hearts due to abdominal aortic constriction (7). It should be pointed out, however, that measurement of hydroxyproline concentration in the hearts of the two groups of CO-exposed rats presented here (data not shown), and in hearts of rats treated to endurance exercise and sideropenic anemia (l), indicate no increase in hydroxyproline concentration. Thus, myocardial components other than collagen persist. LDH is made up of two subunits, H and M, aggregating to give five isozymes in most mammalian tissues.
I 40
(days) septum, and right ventricle. Squares are data from expt 1; triangles, from expt 2; and circles, from combined control rats.
In heart it is known to be a sensitive index to cardiomegaly induced by a number of work overloads (10, 14, 22). In sideropenic anemia (14), simulated altitude (lo), aortic constriction (22), and endurance exercise (22), the percent of myocardial M subunit increases, and alterations in %M LDH are brought about solely through changes in the concentration of the M subunit, while H remains constant. Higher LDH activity is seen in these conditions, and the specific activity of LDH is related to the amount of M subunit present (22). The specific activity of LDH determined spectrophotometrically by standard enzymatic techniques in the present
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H522
P. E. STYKA
study (data not shown) was elevated in LV and RV of rats in expt 2 at termination of CO exposure. It returned to control values 44 days later. The decrease in observed LDH activity during regression of cardiomegaly in the present study appears to be a function of the declining %M LDH. We believe that elevated %M LDH in expt 1 and 2 during CO exposure and its persistence in expt 2 following termination of CO inhalation may be related to membrane changes in heart mitochondria as monitored by cytochrome c concentration (18). Regression of HW to control values in expt 1, 24-36 days was longer than has been reported following
AND
D. G. PENNEY
altitude exposure (13 days) (17), abdominal aortic constriction (15 days) (7), hyperthyroidism (13 days) (2, 19), and nutritional anemia (16 days) (2, 6). The reason for this difference is not clear. However, it could not have resulted from retention of HbCO after termination of exposure, because rats exposed to 500 ppm CO lose nearly all their HbCO within 2-3 h after transfer to room air (8). This investigation Grant HL-16367. Received
21 December
was supported
1977; accepted
in part by Public
Health
Service
in final form 11 June 1978.
REFERENCES 1. BARTISOVA, D., M. CHVAPIL, B. KORECKY, 0. POUPA, K. RAKUSAN, Z. TUREK, AND M. VIZEK. The growth of the muscular and collagenous parts of the rat heart in various forms of cardiomegaly. J. PhysioZ. London 200: 285-295, 1969. 2. BEZNAK, M., B. KORECKY, AND G. THOMAS. Regression of cardiac hypertrophies of various origin. Can. J. PhysioZ. PharmacoZ. 47: 579486. 1969. 3. COMMINS, B., AND P. LAWTHER. A sensitive method for determination of COHb in a finger prick sample of blood. Brit. J. Ind. Med. 22: 139-143, 1965. 4. CUTILLETTA, A. F., R. T. DOWELL, M. RUDNIK, R. A., ARCILLA, AND R. ZAK. Regression of myocardial hypertrophy. I. Experimental model, changes in heart weight, nucleic acids, and collagen. J. MOL. CeZZuZar CardioZ. 7: 767-781, 1975. 5. DRABKIN, D. L., AND J. H. AUSTIN. Spectrophotometric studies. II. Preparations from washed blood cells, nitric oxide hemoglobin, and sulfhemoglobin. J. BioZ. Chem. 112: 51-65, 1935-36. 6. GOODMAN, J. R., J. B. WARSHAW, AND P. R. DALLMAN. Cardiac hypertrophy in rats with iron and copper deficiency: quantitative contribution of mitochondrial enlargement. Pediat. Res. 4: 244-256, 1970. 7. JOUANNOT, P., AND P. Y. HATT. Rat myocardial mechanics during pressure-induced hypertrophy development and reversal. Am. J. Physiol. 229: 355-364, 1975. 8. MONTGOMERY, M. R., AND R. J. RUBIN. The effect of carbon monoxide inhalation on in vivo drug metabolism in the rat. J. PharmacoZ. Exptl. Therap. 179: 465-471, 1971. 9. NEFFGEN J., AND B. KORECKY. Cellular hyperplasia and hypertrophy in cardiomegalies induced by anemia in young and adult rats. CircuZation Res. 30: 104-113, 1972. 10. PENNEY, D. G. Lactate dehydrogenase subunit and activity changes in hypertrophied heart of the hypoxically-exposed rat. Biochim. Biophys. Acta 358: 21-24, 1974. 11. PENNEY, D. G., M. BENJAMIN, AND E. DUNHAM. Effect of carbon monoxide on cardiac weight as compared with altitude effects. J. AppZ. Physiol. 37: 80-84, 1974.
12. PENNEY, D. G., AND P. A. BISHOP. Hematologic changes in the rat during and after exposure to carbon monoxide. J. Toxicol. Environ. Health. In press. 13. PENNEY, D. G., P. BISHOP, P. E. STYKA, AND P. SODT. Blood and heart weight changes during and after carbon monoxide exposure (Abstract). Federation Proc. 36: 518, 1977. 14. PENNEY, D. G., L. B. BUGAISKY, AND J. R. MIESZALA. Lactate dehydrogenase and pyruvate kinase in rat heart during sideropenic anemia. Biochim. Biophys. Acta 334: 24-30, 1974. 15. PENNEY, D. G., E. DUNHAM, AND M. BENJAMIN. Chronic carbon monoxide exposure: time course of hemoglobin, heart weight, and lactate dehydrogenase isozyme changes. Toxicol. AppZ. Pharmacol. 28: 493-497, 1974. 16. PENNEY, D. G., J. SAKAI, AND K. COOK. Heart growth: interacting effects of carbon monoxide and age. Growth 38: 321-328, 1974. 17. SIZEMORE, D. A., T. W. MCINTYRE, E. J. VAN LIERE, AND M. F. WILSON. Regression of altitude-produced cardiac hypertrophy. J. AppZ. PhysioZ. 35: 518-521, 1973. 18. STYKA, P. E., AND D. G. PENNEY. The perinatal rat: body weight, hematocrit and regional changes in heart weight and lactate dehydrogenase isozyme composition and activity. Growth 41: 325-336, 1977. 19. VAN LIERE, E. J., AND D. A. SIZEMORE. Regression of cardiac hypertrophy following experimental hyperthyroidism in rats. Proc. Sot. Exptl. BioZ. Med. 136: 645-648, 1971. 20. WILKS, S. S., J. F. TOMASHEFSKI, AND R. T. CLARK, JR. Physiological effects of chronic exposure to carbon monoxide. J. AppZ. PhysioZ. 14: 305-310, 1959. 21. WINICK, M., AND A. NOBLE. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. DeveZop. BioZ. 12: 451-466, 1965. 22. YORK, J. W., D. G. PENNEY, T. WEEKS, AND P. STAGNO. Lactate dehydrogenase changes following several cardiac hypertrophic stresses. J. AppZ. Physiol. 40: 923-926, 1976.
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