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The Relationship Between Atrial Granularity and Circulating Atrial Natriuretic Peptide in Hamsters With Congestive Heart Failure
^ Proceedings Vol. 61
ROCHESTER, MINNESOTA
JULY 1986
The Relationship Between Atrial Granularity and Circulating Atrial Natriuretic Peptide in Hamsters With Congestive Heart Failure
BROOKS S. EDWARDS, M.D., Research Fellow in Cardiovascular Diseases; DOUGLAS M. ACKERMANN, M.D., Resident in Pathology*; THOMAS R. SCHWAB, M.D., Division of Nephrology and Internal Medicine; DENISE M. HEUBLEIN, C.L.T., Department of Physiology and Biophysics; WILLIAM D. EDWARDS, M.D., Section of Medical Pathology, Department of Pathology; LESTER E. WOLD, M.D., Section of Surgical Pathology, Department of Pathology; JOHN C. BURNETT, Jr., M.D., Division of Cardiovascular Diseases and Internal Medicine
The BIO 14.6 strain of hamster is a model of familial cardiomyopathy complicated by congestive heart failure, sodium retention, and edema. In previous studies, bioassay techniques have demonstrated that the cardiac content of atrial natriuretic peptide
This investigation was supported in part by the American
Recent studies h a v e d e m o n s t r a t e d t h a t t h e heart,
Heart Association Grant-in-Aid 83-964 and by the Hearst and Rappaport Foundations Dr B. S Edwards is supported by Training Grant HL-07111 from the National Institutes of
e n d o c r i n e g l a n d - T h e
in a d d i t i o n to its role a s a p u m p , functions a s a n i n d i v i d u a l m y o c a r d i a l cells . , ,. , . ,
Health, Public Health Service. *Mayo Graduate School of Medicine, Rochester, Minnesota,
°f t h e cardiac a t r i a possess n u m e r o u s secretory g r a n u l e s . These g r a n u l e s c o n t a i n a peptide hormone k n o w n a s atrial natriuretic peptide (ANP). 1
Address reprint requests to Dr. J. C. Burnett, Jr., Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic,
A t r i a l d i s t e n t i o n h a s b e e n s h o w n to s t i m u l a t e r e l e a s e of t h i s h o r m o n e . I m p o r t a n t p h y s i o l o g i c
Rochester, MN 55905. Mayo Clin Proc 61:517-521, 1986
actions of A N P include t h e following: (1) increase 517
518
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
in glomerular filtration rate, (2) promotion of excretion of sodium and water, (3) inhibition of release of renin and synthesis of aldosterone, and (4) venodilatation. This hormone may play a fundamental role in the regulation of extracellular volume. The existence of a hormone of cardiac origin provides a new mechanism by which one could explain some of the observations of Henry, Gauer, and Reeves.2 These investigators demon strated that balloon distention of the cardiac atrium results in pronounced diuresis. ANP is a humoral mediator that may be partially responsi ble for this phenomenon. Morphologic studies by Kisch 3 and later by Jamieson and Palade 4 demon strated that the cardiac atria but not the ventricles contain specific secretory granules. Alterations in sodium and water balance have reportedly been associated with alterations in atrial granularity, as determined by histomorphometric techniques. 5 Using a bioassay to determine the ANP content within the atria, Chimoskey and associates 6 re cently reported that ANP is reduced in a strain of hamsters in which hereditary dilated cardiomyopathy and congestive heart failure develop. On the basis of this observation, these investigators concluded that cardiomyopathic hamsters are "deficient" in ANP and that the sodium retention associated with heart failure may be mediated by such a deficiency. Rector and colleagues 7 deter mined the ANP content in the atria of rats with chronic congestive heart failure, which had been produced by experimental myocardial infarction. These experimental rats had reduced ANP activ ity within the atria as compared with shamoperated rats. Although they suggested that an ANP deficiency may account for the sodium reten tion observed in conjunction with congestive heart failure, they cautioned that the atrial con tent may not reflect the circulating levels of ANP and that the importance of their findings must await concomitant assessment of both atrial con tent and circulating ANP. Our group and others recently reported mea surement of circulating ANP by using a specific radioimmunoassay. 8,9 Those studies demon strated that circulating ANP is substantially ele vated in humans with chronic congestive heart failure. To date, however, no study has correlated atrial granularity with circulating levels of ANP. We hypothesize that a reduction in cardiac ANP content reflects a decrease in storage of this hor mone secondary to stimulation of secretion of ANP.
Mayo Clin Proc, July 1986, Vol 61
Therefore, the objective of this investigation was to examine the relationship between immunoreactive ANP in the atria and circulating ANP in cardiomyopathic hamsters as compared with strain-, age-, and sex-matched control hamsters. METHODS Studies were performed in six male cardiomyo pathic BIO 14.6 hamsters and six control male FiB Syrian hamsters (Bio Breeders, Inc., Watertown, Massachusetts). Each hamster was 231 days of age at the time of study. The animals were anesthetized with thiobutabarbital (100 mg/kg administered intraperitoneally). Blood was col lected for determination of circulating ANP, and the animals were killed immediately by en bloc removal of the heart, lungs, and liver. All tissue was immediately fixed in 10% buffered formalin. Blood was collected in chilled ethylenediaminetetraacetic acid tubes and centrifuged at 4°C and 2,500 rpm for 10 minutes. Plasma was de canted and stored at -20°C. Extracted plasma ANP was determined by radioimmunoassay with use of a specific antibody (Peninsula Laborato ries, Belmont, California). 8 All 12 formalin-fixed hamster hearts were as signed random numbers and inspected as un knowns. Each heart was weighed and evaluated for the presence or absence of chamber dilatation and hypertrophy. The corresponding livers were inspected grossly for indications of chronic pas sive congestion. Each specimen was classified as demonstrating normal or cardiomyopathic fea tures. After the investigators were "unblinded," it was confirmed that all specimens had been cor rectly grouped. The base of the heart, including both atria, was amputated at the level of the aortic valve, dehy drated, and embedded in paraffin so as to include both atria in a single section. Tissue sections were cut at 5 μτα and mounted on glass slides. Every 10th slide was stained with hematoxylin and eosin to ensure proper orientation. Four appropriate slides were selected from each block. Sections were deparaffinized and hydrated. Two-stage immunoperoxidase staining was done by modifying the protocol of Chapeau and asso ciates. 10 Slides were placed in a 3% hydrogen peroxide solution for 5 minutes, followed by 30 minutes of exposure to an egg albumen suspen sion to minimize nonspecific binding. Two slides
Mayo Clin Proc, July 1986, Vol 61
from each animal were exposed to a 1:800 dilution of rabbit anti-cr-ANP serum (RAS8798, Peninsula Laboratories), and the remaining two slides were exposed to a 1:800 dilution of a nonspecific rabbit serum. Both sets of slides were incubated for 46 hours at 4°C and then for 2 hours at 22°C. After incubation, the tissue was washed in phosphatebuffered saline and then exposed for 20 minutes at room temperature to goat antirabbit IgG covalently linked to horseradish peroxidase diluted 1:100. The peroxidase was visualized in a medium containing 3 mg of 3,3'-diaminobenzidine per 10 ml of 0.05 M tris HC1 (pH 7.6) and 0.1% hydrogen peroxide for 15 minutes at room temperature. Nuclear detail was enhanced by counterstaining all sections with hematoxylin. All slides were reviewed by two independent observers under blind conditions. After review of specimens from all 12 animals, each hamster was assigned a granularity score that ranged from 1 to 5—grade 1 denoting the least granularity and grade 5 denoting the most dense granulation (Fig. 1). Each slide was scored on the basis of the degree of granularity in comparison with the others. All revealed some degree of granularity, whereas all slides stained with nonspecific rabbit serum were "negative." Both right and left atria were appraised. The findings of the two observers were averaged to produce the granularity score. RESULTS Gross inspection of the organs from all the cardiomyopathic hamsters revealed four-chamber cardiac dilatations (mean heart weight, 505 ± 69 mg) and evidence of passive liver congestion (mean liver weight, 3.8 ± 0.12 g). In the control animals, no gross abnormalities were apparent (mean heart weight, 385 ± 55 mg; mean liver weight, 4.3 ± 0.14 g). No statistically significant differences in cardiac weight were noted between the two groups of animals. The liver weight, however, was significantly less in the cardiomyopathic hamsters than in the control animals (Table 1). Evaluation of immunohistochemical preparations of atria revealed the mean ANP granularity score for the cardiomyopathic hamsters to be 2.1 ± 0.3. In the normal hamsters, the mean granularity score was 3.5 ± 0.5, significantly greater than that for the cardiomyopathic hamsters (P<0.05). When specimens were classified as demonstrating high (grades 4 and 5), intermediate (grade 3), or reduced
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
519
(grades 1 and 2) granularity, interobserver disagreement occurred in only two cases; in these cases, the observers differed by only a single category. Other than granular content, no histologic characteristics distinguished cardiomyopathic specimens from control specimens. Determination of immunoreactive plasma ANP by radioimmunoassay demonstrated significantly higher levels in the cardiomyopathic hamsters than in the age-matched control animals: 185.5 + 27.2 pg/ml versus 77.7 ± 10.8 pg/ml (P<0.005). DISCUSSION These studies demonstrate for the first time the relationship between ANP granularity in the atria and the circulating concentration of ANP. Although this study supports the observation that the cardiac content of ANP is reduced in cardiomyopathic hamsters, it does not support the hypothesis that congestive heart failure results from or is associated with an absolute deficiency of ANP. Coincident with reduced atrial granularity, we observed an increase in circulating immunoreactive ANP. These two observations provide insight into the dynamics of storage and release of ANP. The best explanation for the reduction in atrial granularity and the elevation of circulating levels of ANP is a stimulated system in which the atrial peptide is synthesized and released into the circulation and the storage of ANP is minimal. Recent morphologic studies have reported certain regions of the heart to be more richly granular than others. 1 ° The more granular regions might be assumed to participate more directly in volume regulation; however, we would propose an alternative interpretation. Regions such as the atrial appendage that are known to be rich in ANP granules may be reserve storage depots for ANP, which would be recruited only during states of volume expansion in which atrial dimensions or pressure would increase. The current study suggests cautious interpretation of previous investigations in which atrial and not plasma ANP was measured. 6,7 A reduced atrial content may suggest, as in our current study, an elevated circulating content of ANP. In congestive heart failure of ischemic or idiopathic cause, plasma ANP is elevated. 89 The current study not only extends those earlier observations to include hereditary cardiomyopathy, in
520
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
Mayo Clin Proc, July 1986, Vol 61
UP?
;
.
*
&
*
'
$
#
&
■
Fig. 1. Representative examples of immunohistochemical staining in normal and cardiomyopathic hamsters. Brown granules in perinuclear region represent immunoreactive atrial natriuretic peptide. (x250.) See text for discussion of staining procedure and granularity score. Upper Left, Atrial section from normal hamster; representative granularity score is 5. Upper Right, "Negative" control of atrial section; no nonspecific staining is present. Lower Left, Atrial section from cardiomyopathic hamster; representative granularity score is 2. Lower Right, "Negative" control of atrial section; no nonspecific staining is present.
which plasma ANP is likewise increased, but for the first time also demonstrates an important link between cardiac content and circulating plasma levels of ANP. There is little evidence to support the hypothesis that the sodium retention associated with congestive heart failure results from an absolute deficiency of ANP. Rather, the elevated circulating levels of ANP in congestive
heart failure support the interpretation that sodium retention may, in part, be mediated by a reduced renal response to increased circulating levels of ANP. 11
ACKNOWLEDGMENT We thank Lori J. Meyer for secretarial assistance.
Mayo Clin Proc, July 1986, Vol 61
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
521
Table 1.—Individual Heart and Liver Weights, Atrial Granularity Score, and Plasma Content of Atrial Natriuretic Peptide (ANP) in Normal and Cardiomyopathic Hamsters Liver Mean atrial Heart weight weight granularity Plasma ANP (mg) Hamster no. score (pg/ml) (g) Normal 350 1 5.0 4.0 87.0 260 2 4.0 4.0 78.0 350 3 4.2 4.5 48.0 350 4 4.2 4.0 79.8 650 5 4.4 1.0 52.2 350 6 4.2 3.5 121.2 385 ± 55 Mean ± SE 4.3 ±0.14 3.5 ± 0.5 77.7 ± 10.8 Cardiomyopathic 350 7 3.5 2.5 186.6 8 650 4.0 2.0 82.8 350 9 4.2 2.5 154.2 650 10 3.8 3.0 243.0 350 11 3.5 1.5 270.6 680 12 4.0 1.0 175.8 505 ± 69 Mean ± SE 3.8 ± 0.12* 2.1 ± 0.3* 185.5 ± 27.2t Significantly different from normal (Student's unpaired t test): *P<0.05. tP<0.005.
REFERENCES
1. De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H: A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sei 28:89-94, 1981 Henry JP, Gauer OH, Reeves JL: Evidence of the atrial location of receptors influencing urine flow. Circ Res 4:8590, 1956 3. Kisch B: Electron microscopy of the atrium of the heart. I. Guinea pig. Exp Med Surg 14:99-112, 1956 4. Jamieson JD, Palade GE: Specific granules in atrial muscle cells. J Cell Biol 23:151-172, 1964 De Bold AJ: Heart atria granularity effects of changes in water-electrolyte balance. Proc Soc Exp Biol Med 161:508511, 1979 Chimoskey JE, Spielman WS, Brandt MA, Heidemann SR: Cardiac atria of BIO 14.6 hamsters are deficient in natriuretic factor. Science 223:820-822, 1984
Rector TS, Carlyle PF, Cohn JN: Reduced atrial natriuretic factor after ligation of the left coronary artery in rats. Am Heart J 110:1197-1199, 1985 Burnett JC Jr, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, Opgenorth TJ, Reeder GS: Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 231:1145-1147, 1986 Shenker Y, Sider RS, Ostafin EA, Grekin RJ: Plasma levels of immunoreactive atrial natriuretic factor in healthy subjects and in patients with edema. J Clin Invest 76:1684-1687, 1985 10. Chapeau C, Gutkowska J, Schiller PW, Milne RW, Thibault G, Garcia R, Genest J, Cantin M: Localization of immunoreactive synthetic atrial natriuretic factor (ANF) in the heart of various animal species. J Histochem Cytochem 33:541-550, 1985 11. Scriven TA, Burnett JC Jr: Effects of synthetic atrial natriuretic peptide on renal function and renin release in acute experimental heart failure. Circulation 72:892-897, 1985
ROCHESTER, MINNESOTA
JULY 1986
The Relationship Between Atrial Granularity and Circulating Atrial Natriuretic Peptide in Hamsters With Congestive Heart Failure
BROOKS S. EDWARDS, M.D., Research Fellow in Cardiovascular Diseases; DOUGLAS M. ACKERMANN, M.D., Resident in Pathology*; THOMAS R. SCHWAB, M.D., Division of Nephrology and Internal Medicine; DENISE M. HEUBLEIN, C.L.T., Department of Physiology and Biophysics; WILLIAM D. EDWARDS, M.D., Section of Medical Pathology, Department of Pathology; LESTER E. WOLD, M.D., Section of Surgical Pathology, Department of Pathology; JOHN C. BURNETT, Jr., M.D., Division of Cardiovascular Diseases and Internal Medicine
The BIO 14.6 strain of hamster is a model of familial cardiomyopathy complicated by congestive heart failure, sodium retention, and edema. In previous studies, bioassay techniques have demonstrated that the cardiac content of atrial natriuretic peptide
This investigation was supported in part by the American
Recent studies h a v e d e m o n s t r a t e d t h a t t h e heart,
Heart Association Grant-in-Aid 83-964 and by the Hearst and Rappaport Foundations Dr B. S Edwards is supported by Training Grant HL-07111 from the National Institutes of
e n d o c r i n e g l a n d - T h e
in a d d i t i o n to its role a s a p u m p , functions a s a n i n d i v i d u a l m y o c a r d i a l cells . , ,. , . ,
Health, Public Health Service. *Mayo Graduate School of Medicine, Rochester, Minnesota,
°f t h e cardiac a t r i a possess n u m e r o u s secretory g r a n u l e s . These g r a n u l e s c o n t a i n a peptide hormone k n o w n a s atrial natriuretic peptide (ANP). 1
Address reprint requests to Dr. J. C. Burnett, Jr., Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic,
A t r i a l d i s t e n t i o n h a s b e e n s h o w n to s t i m u l a t e r e l e a s e of t h i s h o r m o n e . I m p o r t a n t p h y s i o l o g i c
Rochester, MN 55905. Mayo Clin Proc 61:517-521, 1986
actions of A N P include t h e following: (1) increase 517
518
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
in glomerular filtration rate, (2) promotion of excretion of sodium and water, (3) inhibition of release of renin and synthesis of aldosterone, and (4) venodilatation. This hormone may play a fundamental role in the regulation of extracellular volume. The existence of a hormone of cardiac origin provides a new mechanism by which one could explain some of the observations of Henry, Gauer, and Reeves.2 These investigators demon strated that balloon distention of the cardiac atrium results in pronounced diuresis. ANP is a humoral mediator that may be partially responsi ble for this phenomenon. Morphologic studies by Kisch 3 and later by Jamieson and Palade 4 demon strated that the cardiac atria but not the ventricles contain specific secretory granules. Alterations in sodium and water balance have reportedly been associated with alterations in atrial granularity, as determined by histomorphometric techniques. 5 Using a bioassay to determine the ANP content within the atria, Chimoskey and associates 6 re cently reported that ANP is reduced in a strain of hamsters in which hereditary dilated cardiomyopathy and congestive heart failure develop. On the basis of this observation, these investigators concluded that cardiomyopathic hamsters are "deficient" in ANP and that the sodium retention associated with heart failure may be mediated by such a deficiency. Rector and colleagues 7 deter mined the ANP content in the atria of rats with chronic congestive heart failure, which had been produced by experimental myocardial infarction. These experimental rats had reduced ANP activ ity within the atria as compared with shamoperated rats. Although they suggested that an ANP deficiency may account for the sodium reten tion observed in conjunction with congestive heart failure, they cautioned that the atrial con tent may not reflect the circulating levels of ANP and that the importance of their findings must await concomitant assessment of both atrial con tent and circulating ANP. Our group and others recently reported mea surement of circulating ANP by using a specific radioimmunoassay. 8,9 Those studies demon strated that circulating ANP is substantially ele vated in humans with chronic congestive heart failure. To date, however, no study has correlated atrial granularity with circulating levels of ANP. We hypothesize that a reduction in cardiac ANP content reflects a decrease in storage of this hor mone secondary to stimulation of secretion of ANP.
Mayo Clin Proc, July 1986, Vol 61
Therefore, the objective of this investigation was to examine the relationship between immunoreactive ANP in the atria and circulating ANP in cardiomyopathic hamsters as compared with strain-, age-, and sex-matched control hamsters. METHODS Studies were performed in six male cardiomyo pathic BIO 14.6 hamsters and six control male FiB Syrian hamsters (Bio Breeders, Inc., Watertown, Massachusetts). Each hamster was 231 days of age at the time of study. The animals were anesthetized with thiobutabarbital (100 mg/kg administered intraperitoneally). Blood was col lected for determination of circulating ANP, and the animals were killed immediately by en bloc removal of the heart, lungs, and liver. All tissue was immediately fixed in 10% buffered formalin. Blood was collected in chilled ethylenediaminetetraacetic acid tubes and centrifuged at 4°C and 2,500 rpm for 10 minutes. Plasma was de canted and stored at -20°C. Extracted plasma ANP was determined by radioimmunoassay with use of a specific antibody (Peninsula Laborato ries, Belmont, California). 8 All 12 formalin-fixed hamster hearts were as signed random numbers and inspected as un knowns. Each heart was weighed and evaluated for the presence or absence of chamber dilatation and hypertrophy. The corresponding livers were inspected grossly for indications of chronic pas sive congestion. Each specimen was classified as demonstrating normal or cardiomyopathic fea tures. After the investigators were "unblinded," it was confirmed that all specimens had been cor rectly grouped. The base of the heart, including both atria, was amputated at the level of the aortic valve, dehy drated, and embedded in paraffin so as to include both atria in a single section. Tissue sections were cut at 5 μτα and mounted on glass slides. Every 10th slide was stained with hematoxylin and eosin to ensure proper orientation. Four appropriate slides were selected from each block. Sections were deparaffinized and hydrated. Two-stage immunoperoxidase staining was done by modifying the protocol of Chapeau and asso ciates. 10 Slides were placed in a 3% hydrogen peroxide solution for 5 minutes, followed by 30 minutes of exposure to an egg albumen suspen sion to minimize nonspecific binding. Two slides
Mayo Clin Proc, July 1986, Vol 61
from each animal were exposed to a 1:800 dilution of rabbit anti-cr-ANP serum (RAS8798, Peninsula Laboratories), and the remaining two slides were exposed to a 1:800 dilution of a nonspecific rabbit serum. Both sets of slides were incubated for 46 hours at 4°C and then for 2 hours at 22°C. After incubation, the tissue was washed in phosphatebuffered saline and then exposed for 20 minutes at room temperature to goat antirabbit IgG covalently linked to horseradish peroxidase diluted 1:100. The peroxidase was visualized in a medium containing 3 mg of 3,3'-diaminobenzidine per 10 ml of 0.05 M tris HC1 (pH 7.6) and 0.1% hydrogen peroxide for 15 minutes at room temperature. Nuclear detail was enhanced by counterstaining all sections with hematoxylin. All slides were reviewed by two independent observers under blind conditions. After review of specimens from all 12 animals, each hamster was assigned a granularity score that ranged from 1 to 5—grade 1 denoting the least granularity and grade 5 denoting the most dense granulation (Fig. 1). Each slide was scored on the basis of the degree of granularity in comparison with the others. All revealed some degree of granularity, whereas all slides stained with nonspecific rabbit serum were "negative." Both right and left atria were appraised. The findings of the two observers were averaged to produce the granularity score. RESULTS Gross inspection of the organs from all the cardiomyopathic hamsters revealed four-chamber cardiac dilatations (mean heart weight, 505 ± 69 mg) and evidence of passive liver congestion (mean liver weight, 3.8 ± 0.12 g). In the control animals, no gross abnormalities were apparent (mean heart weight, 385 ± 55 mg; mean liver weight, 4.3 ± 0.14 g). No statistically significant differences in cardiac weight were noted between the two groups of animals. The liver weight, however, was significantly less in the cardiomyopathic hamsters than in the control animals (Table 1). Evaluation of immunohistochemical preparations of atria revealed the mean ANP granularity score for the cardiomyopathic hamsters to be 2.1 ± 0.3. In the normal hamsters, the mean granularity score was 3.5 ± 0.5, significantly greater than that for the cardiomyopathic hamsters (P<0.05). When specimens were classified as demonstrating high (grades 4 and 5), intermediate (grade 3), or reduced
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
519
(grades 1 and 2) granularity, interobserver disagreement occurred in only two cases; in these cases, the observers differed by only a single category. Other than granular content, no histologic characteristics distinguished cardiomyopathic specimens from control specimens. Determination of immunoreactive plasma ANP by radioimmunoassay demonstrated significantly higher levels in the cardiomyopathic hamsters than in the age-matched control animals: 185.5 + 27.2 pg/ml versus 77.7 ± 10.8 pg/ml (P<0.005). DISCUSSION These studies demonstrate for the first time the relationship between ANP granularity in the atria and the circulating concentration of ANP. Although this study supports the observation that the cardiac content of ANP is reduced in cardiomyopathic hamsters, it does not support the hypothesis that congestive heart failure results from or is associated with an absolute deficiency of ANP. Coincident with reduced atrial granularity, we observed an increase in circulating immunoreactive ANP. These two observations provide insight into the dynamics of storage and release of ANP. The best explanation for the reduction in atrial granularity and the elevation of circulating levels of ANP is a stimulated system in which the atrial peptide is synthesized and released into the circulation and the storage of ANP is minimal. Recent morphologic studies have reported certain regions of the heart to be more richly granular than others. 1 ° The more granular regions might be assumed to participate more directly in volume regulation; however, we would propose an alternative interpretation. Regions such as the atrial appendage that are known to be rich in ANP granules may be reserve storage depots for ANP, which would be recruited only during states of volume expansion in which atrial dimensions or pressure would increase. The current study suggests cautious interpretation of previous investigations in which atrial and not plasma ANP was measured. 6,7 A reduced atrial content may suggest, as in our current study, an elevated circulating content of ANP. In congestive heart failure of ischemic or idiopathic cause, plasma ANP is elevated. 89 The current study not only extends those earlier observations to include hereditary cardiomyopathy, in
520
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
Mayo Clin Proc, July 1986, Vol 61
UP?
;
.
*
&
*
'
$
#
&
■
Fig. 1. Representative examples of immunohistochemical staining in normal and cardiomyopathic hamsters. Brown granules in perinuclear region represent immunoreactive atrial natriuretic peptide. (x250.) See text for discussion of staining procedure and granularity score. Upper Left, Atrial section from normal hamster; representative granularity score is 5. Upper Right, "Negative" control of atrial section; no nonspecific staining is present. Lower Left, Atrial section from cardiomyopathic hamster; representative granularity score is 2. Lower Right, "Negative" control of atrial section; no nonspecific staining is present.
which plasma ANP is likewise increased, but for the first time also demonstrates an important link between cardiac content and circulating plasma levels of ANP. There is little evidence to support the hypothesis that the sodium retention associated with congestive heart failure results from an absolute deficiency of ANP. Rather, the elevated circulating levels of ANP in congestive
heart failure support the interpretation that sodium retention may, in part, be mediated by a reduced renal response to increased circulating levels of ANP. 11
ACKNOWLEDGMENT We thank Lori J. Meyer for secretarial assistance.
Mayo Clin Proc, July 1986, Vol 61
ATRIAL NATRIURETIC PEPTIDE IN HEART FAILURE
521
Table 1.—Individual Heart and Liver Weights, Atrial Granularity Score, and Plasma Content of Atrial Natriuretic Peptide (ANP) in Normal and Cardiomyopathic Hamsters Liver Mean atrial Heart weight weight granularity Plasma ANP (mg) Hamster no. score (pg/ml) (g) Normal 350 1 5.0 4.0 87.0 260 2 4.0 4.0 78.0 350 3 4.2 4.5 48.0 350 4 4.2 4.0 79.8 650 5 4.4 1.0 52.2 350 6 4.2 3.5 121.2 385 ± 55 Mean ± SE 4.3 ±0.14 3.5 ± 0.5 77.7 ± 10.8 Cardiomyopathic 350 7 3.5 2.5 186.6 8 650 4.0 2.0 82.8 350 9 4.2 2.5 154.2 650 10 3.8 3.0 243.0 350 11 3.5 1.5 270.6 680 12 4.0 1.0 175.8 505 ± 69 Mean ± SE 3.8 ± 0.12* 2.1 ± 0.3* 185.5 ± 27.2t Significantly different from normal (Student's unpaired t test): *P<0.05. tP<0.005.
REFERENCES
1. De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H: A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sei 28:89-94, 1981 Henry JP, Gauer OH, Reeves JL: Evidence of the atrial location of receptors influencing urine flow. Circ Res 4:8590, 1956 3. Kisch B: Electron microscopy of the atrium of the heart. I. Guinea pig. Exp Med Surg 14:99-112, 1956 4. Jamieson JD, Palade GE: Specific granules in atrial muscle cells. J Cell Biol 23:151-172, 1964 De Bold AJ: Heart atria granularity effects of changes in water-electrolyte balance. Proc Soc Exp Biol Med 161:508511, 1979 Chimoskey JE, Spielman WS, Brandt MA, Heidemann SR: Cardiac atria of BIO 14.6 hamsters are deficient in natriuretic factor. Science 223:820-822, 1984
Rector TS, Carlyle PF, Cohn JN: Reduced atrial natriuretic factor after ligation of the left coronary artery in rats. Am Heart J 110:1197-1199, 1985 Burnett JC Jr, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, Opgenorth TJ, Reeder GS: Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 231:1145-1147, 1986 Shenker Y, Sider RS, Ostafin EA, Grekin RJ: Plasma levels of immunoreactive atrial natriuretic factor in healthy subjects and in patients with edema. J Clin Invest 76:1684-1687, 1985 10. Chapeau C, Gutkowska J, Schiller PW, Milne RW, Thibault G, Garcia R, Genest J, Cantin M: Localization of immunoreactive synthetic atrial natriuretic factor (ANF) in the heart of various animal species. J Histochem Cytochem 33:541-550, 1985 11. Scriven TA, Burnett JC Jr: Effects of synthetic atrial natriuretic peptide on renal function and renin release in acute experimental heart failure. Circulation 72:892-897, 1985