Norepinephrine Turnover in the Heart and Spleen of the Cardiomyopathic Syrian Hamster

Norepinephrine Turnover in the Heart and Spleen of the Cardiomyopathic Syrian Hamster By Michael J. Sole, Chi-Man Lo, Cleve W. Laird, Edmund H. Sonnenblick, and Richard J. Wurtman ABSTRACT Although a reduction in myocardial norepinephrine stores in cardiac hypertrophy and congestive failure is well documented, norepinephrine turnover has been inadequately studied in such hearts. We compared norepinephrine turnover in control and cardiomyopathic hamsters by following the decline in specific activity of myocardial norepinephrine after labeling with an intraperitoneal tracer dose of 3 H-norepinephrine. Adult myocardial norepinephrine concentrations were not attained until 4 weeks of age in both strains. There was no difference in the rate constant (K) for myocardial norepinephrine turnover (0.107 ± 0.004 hours" 1 vs. 0.100 ± 0.005 hours" 1 ) in the two strains of hamsters during the neonatal period. In young control hamsters, K fell to 0.064 ± 0.004 hours" 1, but that for age-matched hamsters with mild cardiac hypertrophy was 0.102 ± 0.001 hours" 1 (P < 0.001). There was little change in K as control hamsters aged. With the development of more severe hypertrophy in cardiomyopathic hamsters, cardiac norepinephrine decreased and resting K rapidly increased to approach the value obtained when hamsters were subjected to immobilization stress (0.302 ± 0.013 hours" 1 ). The maximum achievable K remained the same for both control and dystrophic hamsters even during terminal disease. Prolonged immobilization led to a reduction in cardiac norepinephrine in both strains. Ganglionic blockade of failing hamsters completely restored the levels of both cardiac norepinephrine and K to control values. Splenic noradrenergic nerves showed no change in K, norepinephrine content, or maximum K during cardiac decompensation. We conclude that, in the late stages of hamster cardiomyopathy, there is a progressive and possibly specific increase in cardiac sympathetic tone which leads to a concomitant decrease in cardiac norepinephrine. With the loss of sympathetic reserve, congestive failure supervenes. The normal heart is richly supplied with noradrenergic sympathetic nerves. This innervation contributes little to intrinsic myocardial function (1) but is a most important mechanism for the elevation of cardiac output in response to a physiological stress such as exercise (2). In the absence of a catecholamine stimulus, cardiac output can be increased solely by the Frank-Starling mechanism (3). Dilated or noncompliant hearts, however, cannot take further advantage of the length-tension relationship and thus depend on sympathetic support to maintain cardiac output (4). From the Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. This investigation was supported by U. S. Public Health Service Grants AM-11709 and HL-14117 from the National Institutes of Health. This paper won the Young Investigators' Award given by the Canadian Cardiovascular Society. Dr. Sole was a Hunter Fellow of the Ontario Heart Foundation. Please address reprint requests to Dr. Michael J. Sole, Clinical Sciences Division, Medical Sciences Building, University of Toronto, Toronto, Canada M5S 1A8. Received May 1, 1975. Accepted for publication October 2, 1975. There is considerable evidence for sympathetic dysfunction in the hearts of humans with congestive heart failure and animals with experimentally induced cardiac disease. Several investigators have demonstrated a decrease in the norepinephrine concentration (5-8) and the norepinephrine storage capacity (5) of enlarged hearts. Covell et al. (9) have shown that the failing heart does not respond adequately to stimulation of its sympathetic nerves. Diminished levels of tyrosine hydroxylase, felt to be the rate-limiting enzyme for norepinephrine biosynthesis, have also been found in such hearts (7, 10). In contrast to the abundant measurements of norepinephrine levels in the failing and hypertrophied heart, very little information is available concerning its turnover and synthesis rates in such hearts. Spann et al. (11) have demonstrated that the cardiac norepinephrine turnover time is identical in normal guinea pigs and guinea pigs with heart failure induced by supravalvular aortic constriction. These guinea pigs were models of acute heart failure, since the aortic band was placed only 10 days prior to assay; hence, their autonomic function might not be similar to that associated 855

856 SOLE. LO. LAIRD. SONNENBLICK. WURTMAN with the chronic form of cardiac hypertrophy secondary to human heart disease. Fischer et al. (12), in experiments with rat hearts hypertrophied secondary to banding of the abdominal aorta, found no change in norepinephrine turnover time in mildly enlarged hearts and somewhat of an increase in moderately enlarged ones. This model of aortic coarctation, although classic in biochemical studies of pressure-overload hypertrophy, exhibits a marked rostral-caudal difference in blood pressure which in turn may contribute significantly to the observed cardiac sympathetic response. Angelakos et al. (8) have performed preliminary studies on norepinephrine metabolism in the hearts of cardiomyopathic hamsters, but they measured labeled cardiac norepinephrine at only a single interval after the administration of the tracer. Thus, their data were insufficient to allow calculation of turnover rates. Surgical models of heart failure requiring thoracotomy and vascular banding can exhibit artifactual changes in sympathetic nervous function secondary to the operative procedure rather than the myocardial dysfunction. The cardiomyopathic hamster provides a natural model of chronic congestive heart disease (13). We therefore decided to examine norepinephrine turnover in the cardiac and splenic sympathetic nerves of these animals and to relate our findings to the course of their heart disease. Methods Cardiomyopathic (Bio 14.6) and control (Bio 2.4) Syrian hamsters of both sexes 8-340 days of age were used in these experiments. The hamsters were age and sex matched. All hamsters were allowed at least 2 weeks to acclimatize to our laboratory animal facility following delivery from the breeder (Bio Research Inst., Cambridge, Massachusetts). Hamsters were fed a standard laboratory diet. A 12-hour light, 12-hour dark cycle was maintained in the animal housing area. Each hamster was given 4 MC/100 g of 7-( 3 H)-dZnorepinephrine ( 3 H-NE) (New England Nuclear Corp., 7.2 c/mmole) in 150 ^liters of 10" 4 N acetic acid by intraperitoneal injection. Preliminary studies demonstrated that less than 0.5% of the radioactivity was detectable in the peritoneal cavity 20 minutes after the injection. The rate of change of the specific activity of norepinephrine was measured between 2 and 26 hours after labeling. At appropriate times after the injection, the hamsters were killed by decapitation, and their hearts and spleens were removed and rinsed. The ventricles were dissected free of the atria and great vessels, and the organs were then immediately frozen on Dry Ice. The tissues were weighed and then homogenized in 6 volumes of iced 0.4N perchloric acid containing 0.4% ethylenediaminetetraacetic acid (EDTA) and 3 mg/100 ml of sodium metabisulfite. The homogenate was centrifuged at 30,000 g for 30 minutes. Catecholamines were absorbed from the supernatant fluid onto an alumina column at ph 8.6 and eluted with 4 ml of 0.2N acetic acid. A 500 jiliter sample of the alumina eluate was added to 13 ml of Bray's solution and counted in a Packard liquid scintillation counter. Counting efficiency was calculated by the subsequent addition of 3 H-toluene as an internal standard. Norepinephrine was measured in the alumina eluate by conversion to its trihydroxyindole derivative by the modification of O'Hanlon et al. (14) of the method of Laverty and Taylor. Fluorescence was read in an Aminco-Bowman spectrophotofluorometer. Norepinephrine recovery varied between 86% and 94%. Under steady-state conditions, the rates of synthesis and removal of norepinephrine are equal. Thus, the overall rate of norepinephrine synthesis can be expressed as K[NE] (15), where K is the fraction of labeled norepinephrine lost per unit time (the turnover rate constant) and [NE] is the steady-state level of norepinephrine. K[NE] is the norepinephrine turnover rate. The turnover rate constant was calculated from the rate of decline of the logarithm of the specific activity of the labeled norepinephrine stores (regression coefficient) (15). Analysis of variance was used for calculating the standard error of the regression coefficient, the significance of the regression coefficients, and the difference between them. A minimum of 12 hamsters (from at least six litters) in each of the control and cardiomyopathic groups was used for each turnover study. Initial studies using 30-45 hamsters of each strain and 3-7 time points showed that first-order kinetics were obeyed for both the heart and the spleen over the time period examined (2-26 hours after injection). Since altered circulatory dynamics in congestive heart failure could affect the uptake of the administered norepinephrine, we did not consider a comparison of the absolute uptake of tracer to be informative. The Syrian hamster hibernates; thus, we were unable to use cold as a cardiovascular stress. Therefore, the hamsters were stressed by taped immobilization to a board. Each stress experiment consisted of three groups of (six or more) hamsters for each of the strains. All of the hamsters were given 3 H-NE intraperitoneally as described earlier. Two hours after the injection, one group of hamsters was killed, a second group was immobilized, and a third was left undisturbed. Eight hours after the injection, the second and third groups were killed. Norepinephrine content and turnover were calculated as described earlier. The first group served as an initial point for both turnover studies. Chlorisondamine (Ecolid chloride), a ganglionic blocker which does not enter the central nervous system, was used to inhibit peripheral sympathetic activity in some of our studies; 1 10 mg/kg was given by intraperitoneal injection every 6 hours for 24 hours to the treated hamsters. hamsters received the distilled water vehicle. Hamsters were alternated in a set and timed order between groups or strains or both at every stage of these experiments. Results are expressed as means ± SE. 'Chlorisondamine was generously provided by the CIBA Pharmaceutical Co., Summit, N. J.

NOREPINEPHRINE TURNOVER IN HAMSTERS 857 Results The disease course of our cardiomyopathic hamsters can be divided into several stages as previously reported (13). During the first or prenecrotic stage, the hamsters show no clinical or histological evidence of disease. Between 40 and 70 days of age (necrotic stage), areas of focal myolysis and cellular infiltrates can be seen throughout the myocardium. Healing occurs over the subsequent 40 days. The heart hypertrophies and dilates gradually over the next 250 days. Little, if any, active myolytic disease is in evidence at this time. Hamsters with cardiac enlargement were classified as having early hypertrophy (150-180 days), moderate hypertrophy (250-300 days), severe hypertrophy (older than 300 days), and congestive failure (older than 300 days with hepatic congestion, ascites, subcutaneous edema, and at times pulmonary edema). 600 - cardiomyopathic ^ control BODY AND VENTRICULAR WEIGHTS Body weight did not differ significantly between the two groups of hamsters except between failing hamsters (older than 300 days) and their agematched controls. The former had body weights of 160.3 ± 13.7 g and the latter 108.4 ± 2.7 g (P < 0.01). Hamsters with failure were separated from those of the same age with only hypertrophy on the basis of body weight and appearance at autopsy (the selection was made prior to any biochemical measurements). Ventricular wet weights are illustrated in Figure 1. The differences became significant at the stage of early hypertrophy (150-180 days). Ventricular weights of cardiomyopathic hamsters 300-340 days old with hypertrophy or hypertrophy and failure did not differ significantly. CARDIAC NOREPINEPHRINE CONCENTRATION AND CONTENT Cardiac norepinephrine concentration (Fig. 2) and content (Fig. 3) were measured at each stage of the disease process. In both control and diseased hamsters, cardiac norepinephrine concentration did not reach adult levels until approximately 1 month following birth. There was no significant difference in norepinephrine concentration between the two groups at 8 days of age. The cardiomyopathic hamsters had a slightly but significantly greater concentration of cardiac norepinephrine than did the control hamsters by 2.5 weeks of age. This increase persisted through the stage of moderate hypertrophy. During the stage of late hypertrophy and failure, the concentration of ventricular norepinephrine decreased to approximately 37% of that of matched controls and to 31% of the norepinephrine concentration found prior to the onset of hypertrophy. The decrease in norepi- 60 120 180 240 300 360 Age (days) FIGURE 1 Ventricular wet weights of cardiomyopathic and control hamsters of various ages. Each value is the mean ± SE for at least 12 hamsters. nephrine concentration in hamsters 300-340 days old with hypertrophy with or without failure was significant compared with that of cardiomyopathic hamsters at earlier stages of the disease. Norepinephrine content per heart (right and left ventricle) does not reflect dilution of sympathetic nerve endings by the hypertrophied myocardium. Using this mode of expressing our data, basically the same pattern of norepinephrine stores was seen. 1. MOO ^ J ri fnyopoimc * la 8 18-20 45-65 80-180 S 200-1 * L -'- 1rh Age (doys) FIGURE 2 250-300 300-340 300-340 (ftyparfttcay) (Wurtl Cardiac norepinephrine concentration of cardiomyopathic and control hamsters of various ages. Each value is the mean ± SE for at least 12 hamsters. Double asterisks = significantly different from control at P < 0.01, and triple asterisks = significantly different from control at P < 0.001

858 SOLE. LO. LAIRD. SONNENBLICK. WURTMAN [ j control myopothic 8 18-20 45-65 150-180 250-300 300-340 300-340 (hypertrophy) (foihjre) Age(days) FIGURE 3 Cardiac norepinephrine content of cardiomyopathic and control hamsters of various ages. Each value is the mean ± SE for at least 12 hamsters. Single asterisk = significantly different from control at P < 0.05, double asterisks - significantly different from control at P < 0.02, and.triple asterisks = significantly different from control at P < 0.001. At the age of 45-65 days, there appeared to be a small but significant relative increase in total ventricular norepinephrine stores in the cardiomyopathic hamsters. With the progression of hypertrophy, norepinephrine stores eventually decreased. The cardiac norepinephrine content at 300-340 days in hamsters with hypertrophy with or without failure was significantly less than that in hamsters during the earlier stages of the disease. Norepinephrine content in hamsters with failure was approximately 64% of that in matched controls and approximately 53% of the peak norepinephrine content found in younger cardiomyopathic hamsters. TABLE 1 Norepinephrine (NE) Turnover in the Ventricles of and Cardiomyopathic Hamsters Stage and age (days) Prenecrotic (8-20) Necrotic (45-65) Early hypertrophy (150-180) Moderate hypertrophy (250-300) Severe hypertrophy (300-340) Congestive failure (300-340) 0.107 0.072 0.064 0.064 0.092 0.084 Rate constant (K) (hours" 1 ) ± 0.004 ± 0.006 ± 0.004 ± 0.005 ± 0.02 ± 0.03 0.100 ± 0.108 ± 0.102 ± 0.101 ± 0.205 ± 0.235 ± 0.005 0.007* O.OOlt 0.007* 0.02* 0.04$ VENTRICULAR NOREPINEPHRINE TURNOVER The results of the norepinephrine turnover studies in ventricles of resting hamsters are summarized in Table 1. As can be seen from the study illustrated in Figure 4, the decline of specific activity followed first-order kinetics over the time measured. The cardiac norepinephrine turnover rate constant was identical for both groups of hamsters in the neonatal period. This rate decreased with the attainment of adult levels of ventricular norepinephrine in normal hamsters but remained fixed at neonatal levels in cardiomyopathic ones. With the onset of the late stages of the cardiomyopathy, the cardiac norepinephrine turnover rate constant increased greatly. The norepinephrine turnover rate per gram of ventricle was identical in both groups of very young hamsters. At the necrotic stage and prior to the onset of cardiac hypertrophy, there was a 70% increase in the turnover per gram of myopathic heart compared with the level in control hearts (Table 1). This increase persisted until the stage of failure when the high rate constant was offset by the decrease in norepinephrine concentration and turnover fell sharply. When norepinephrine turnover per total ventricular mass was examined (Table 1), we found a persistent 75-90% increase in the turnover rate of cardiomyopathic hamsters compared with that of controls beginning at 45-65 days of age. STRESS TURNOVER To determine whether the maximum achievable rate constant was affected by the disease process, 6.5 9.7 10.8 10.8 7.5 8.3 Half-life (hours) 6.9 6.4* 6.8t 6.8* 3.4* 2.9! NE turnover rate (ng/heart hour"') 6.3 26.6 29.5 32.2 44.9 38.8 6.4 47.2* 56.8* 56.2t 86.4* 68.9* NE turnover rate (ng/g heart hour" ) 81.1 98.6 78.4 68.0 104.2 108.1 83.3 169.1* 136.0* 138.9* 177.6* 113.8 All values are means ± SE. Half-life = 0.693/K, and the norepinephrine turnover rate is the product of K and norepinephrine content or concentration. * Differs from control at P < 0.001. t Differs from control at P < 0.01. t Differs from control at P < 0.02.

859 NOREPINEPHRINE TURNOVER IN HAMSTERS 100 90 - OL(IO) (10) > cardiomyopothic hearts control hearts 80.(9) 70 mycpoitlic 60 50 40 30 early moderate Stage of severe Hyperfrophic failure Disease FIGURE E. 20 Calculated cardiac norepinephrine turnover rate per total ventricular mass in control and cardiomyopathic hamsters at rest and during immobilization stress. The short bar rising from the base represents the resting turnover rate, and the total bar represents the turnover during immobilization stress. The hatched area represents the sympathetic reserve the amount by which resting norepinephrine turnover can be increased during stress. 10 9 10 14 18 22 26 Number of Hours After Injection FIGURE 4 Decline of specific activity of cardiac norepinephrine over time in hamsters 250-300 days old. Each value is the mean ± SE. Individual data points were used to calculate the regression coefficient and the intercept of the best-fit straight line. The number of hearts assayed is given in parentheses. hamsters were stressed as described in Methods. Cardiomyopathic hamsters and matched controls were examined at each stage of life from early hypertrophy to failure. Neither age nor stage of the disease had any bearing on the maximum achievable rate constant for cardiac norepinephrine turnover. This maximum rate constant was identical for both cardiomyopathic and control hamsters (0.302 ± 0.013). We did not find significant differences in norepinephrine stores between resting hamsters and those subjected to 6 hours of immobilization stress, but a gradual decline may not be manifested during such a brief period. Thus, to determine whether a chronic elevation in turnover rate could result in a reduction of cardiac norepinephrine, we immobilized a group of hamsters for 24 hours. We observed (Table 2) a 40% decrease in cardiac norepinephrine in the stressed control hamsters and a 47% decrease in those with the cardiomyopathy. CARDIAC RESERVE FOR NOREPINEPHRINE TURNOVER We examined cardiac sympathetic reserve by comparing resting and stressed turnover rates. Figure 5 illustrates the norepinephrine turnover TABLE 2 Steady-State Norepinephrine Levels after 24 Hours of Stress (300-Day-Old Hamsters) NE/heart (ng) Group NE/g heart (ng/g) Rest Stress Rest Stress 488.5 ± 43.3 322.0 ± 51.8 293.1 ± 40.2* 171.6 ± 29.1* 1229.0 ± 104.3 571.3 ± 90.8 824. 7 ± 79.7t 331. 2 ± 52.5t All values are means ± SE. * P < 0.02. fp < 0.05.

860 SOLE. LO. LAIRD. SONNENBLICK. WURTMAN rate per heart per hour in both resting and stressed states. The resting turnover rate was 21-30% of that during stress in control hamsters at all ages. Cardiomyopathic hamsters with early and moderate hypertrophy had resting turnover rates 33-34% of those achievable with stress. Hamsters with severe hypertrophy had resting rates 68% of those obtained under stress, and hamsters with failure had resting rates 78% of those obtained from their stressed colleagues. The identical turnover reserve data apply, of course, not only for turnover rate per hour but also for the rate constant alone or for turnover rate per gram of heart. GANGLIONIC BLOCKADE We wished to determine whether our observed high turnover rates were indeed indicative of an actual increase in sympathetic tone. Therefore, we examined cardiac norepinephrine turnover following peripheral ganglionic blockade with chlorisondamine (Table 3). After ganglionic blockade, both failing and control hearts exhibited identically low rate constants for norepinephrine turnover. In addition, there was a complete restoration of cardiac catecholamine stores in the failing hamsters. SPLENIC NOREPINEPHRINE TURNOVER In all hamsters but sucklings, splenic norepinephrine turnover was also studied (Table 4). We found a great deal of variability in splenic weight and norepinephrine content as well as considerable scatter in calculated specific activity. We could not definitely account for the great differences in splenic weights, but we believe it was due to variations in blood content. As we recorded our findings per total spleen rather than per gram of organ, our findings were not affected by this TABLE 3 Effect of Chlorisondamine on Cardiac Norepinephrine Metabolism NE/heart (ng) NE/g heart (ng/g) Rate constant (K) (hours- 1 ) Half-life (hours) Untreated Treated Untreated variability in organ weight. The rate constant for splenic norepinephrine turnover appeared to be consistently higher in cardiomyopathic hamsters throughout life and bore no relationship to the disease state. The maximum achievable rate constant for splenic norepinephrine turnover was the same for both control and failing hamsters. In contrast to data obtained for the heart, no loss of turnover reserve was seen in severely diseased hamsters. Discussion The present observations characterize norepinephrine turnover in hearts of control and cardiomyopathic hamsters from infancy to old age and during resting and stress states. Our turnover data should not be accepted as a literal numerical measure of norepinephrine synthesis rate or sympathetic tone. The experimental rate constant is a weighed mean of the rate constants of possibly several norepinephrine pools some of these pools may bear no relationship to actual sympathetic activity (15). Norepinephrine turnover under steady-state conditions can provide, however, a valid comparative assessment of the norepinephrine synthesis rate (15). The rate constant itself is independent of catecholamine content and, providing it can be shown to vary appropriately with sympathetic tone, can be thought of as a functional index of mean neuronal activity. Cardiac norepinephrine stores required approximately 4 weeks following birth to attain adult concentrations in both groups of hamsters. A similar delay in the maturation of sympathetic innervation has been found in other mammalian hearts (16, 17). Our infant control hamsters had Cardiomyopathic Treated 695.0 ± 20.3 720.6 ± 75.6 474.0 ± 35.1* 764.3 ± 34.5f 1714.4 ± 69.4 1698.3 ± 124.2 1035.6 ± 86.7* 1945.6 ± 143.6t 0.052 ± 0.007 0.032 ± 0.007:): 0.187 ± 0.015* 0.043 ± 0.0071 13.4 21.6 3.7 16.2 All values are means ± SE. * P < 0.001 for cardiomyopathic untreated vs. control untreated. tp < 0.001 for cardiomyopathic treated vs. cardiomyopathic untreated. X P < 0.05 for control treated vs. control untreated.

NOREPINEPHRINE TURNOVER IN HAMSTERS 861 TABLE 4 Splenic Weight, Norepinephrine (NE) Content and Norepinephrine Turnover Spleen weight (mg) NE/spleen (ng) Rate constant (hours" 1 ) Half-life (hours) Age (days) 45-65 150-180 250-300 300-340 (hypertrophy) 320-340 (failure) 300-340 + hypertrophy and failure (stressed)^: 114.0 ± 113.4 ± 213.0 ± 203.0 ± 83.5 ± 66.9 ± 3.6 9.8 20.0 54.8 3.6 4.0 202.1 258.7 194.8 189.8 241.4 128.5 ± 7.6* ± 23.4* ± 12.0 ± 39.8 ± 44.6f ± 16.2* 105.4 124.9 81.2 134.7 84.6 72.6 ± 4.3 ± 8.1 ± 9.3 ± 24.6 ± 4.6 ± 4.2 107.5 199.8 84.2 107.9 146.4 133.2 ± 4.0 ± 8.5* ± 7.2 ± 15.1 ± 11.0* ± 10.2* 0.075 0.087 0.062 0.050 0.055 0.263 ± 0.012 ± 0.022 ± 0.017 ± 0.100 ± 0.057 ± 0.126 0.125 0.107 0.133 0.099 0.097 0.283 ± 0.013t ± 0.008 ± 0.016t ± 0.089 ± 0.067 ± 0.074 9.2 8.0 11.2 13.8 12.7 2.6 5.5t 6.5 5.2t 7.0 7.1 2.4 All values are means ±SE. Half-life = 0.693/K. * Differs from control at P < 0.001. t Differs from control at P < 0.01. ^Hamsters were stressed by immobilization for 6 hours. higher cardiac norepinephrine turnover rate constants than did their adult cohorts. This difference could be an artifact secondary to the handling of suckling hamsters. However, infant hamsters were injected with extreme care and neither they nor their mothers appeared to be disturbed by the brief injection process. Whether a similar decrease in the turnover rate constant accompanies the maturation of cardiac norepinephrine stores in other species has not been determined. In contrast to our findings in control hamsters, no fall in the relatively high turnover rate constant of infancy accompanied the maturation of hamsters destined to develop the cardiomyopathy. Our observation that the ventricular norepinephrine stores of young cardiomyopathic hamsters were increased relative to those of controls supports similar findings by Angelakos et al. (8). This increase of both stores and rate constant led to a considerable elevation of cardiac norepinephrine turnover by the necrotic stage. As the myocardial cells of unhypertrophied cardiomyopathic hamster hearts have been reported to be exquisitely sensitive to catecholamines (18, 19), this relatively high turnover rate may be of importance in the genesis of the disease. That cardiac norepinephrine stores decrease during congestive failure is well established (5-8), but how this change mirrors neural performance has not been clarified. A measurement of the maximum cardiac norepinephrine turnover rate constant under steady-state conditions may be a reflection of the maximum cardiac sympathetic tone. We found no impairment of the maximum turnover rate constant even during terminal disease. Our control hamsters and those with early heart disease could increase cardiac norepinephrine turnover three- to fivefold in response to a cardiac stress. During the late stages of the dystrophy, however, norepinephrine turnover rate in the resting ventricle rapidly approached the maximum achievable under stress, leaving failing hamsters with little, if any, reserve. This high basal turnover rate constant did not compensate for a concomitant reduction in the steady-state concentration of norepinephrine; thus, a fall in norepinephrine turnover per gram of heart accompanied the onset of congestive failure. Angelakos et al. (8) have examined the noradrenergic nerve endings of the dystrophic hamster heart by the formaldehyde fluorescence histochemical technique of Falk and Hillarp. There was an increase in the fluorescence intensity of the nerve terminals of the cardiomyopathic hearts compared with that of the controls in all but the final stages of the disease. Distinct focal areas, perhaps regions of necrosis, were observed where no catecholaminergic fibers were present. There is a distinct possibility, then, that our observations were really a manifestation of catecholamine loss secondary to a degenerative process at the nerve terminal rather than the result of an increase in sympathetic tone. With immobilization stress, we could maintain an

862 SOLE. LO. LAIRD. SONNENBLICK. WURTMAN elevated cardiac norepinephrine turnover rate constant similar to that found in failing hamsters in our control hamsters. Under these conditions, the steady-state content of cardiac norepinephrine fell dramatically, closely simulating the picture of the cardiomyopathy. Conversely, peripheral ganglionic blockade of failing hamsters completely restored both the norepinephrine turnover rate constant and the norepinephrine stores of cardiomyopathic hearts to control values. Cardiac decompensation did not affect the rate constant, norepinephrine stores, or sympathetic reserve of the spleen. We conclude, therefore, that there is a progressive and possibly specific increase in cardiac sympathetic tone in the later stages of hamster cardiomyopathy. With a worsening of the disease state, the input-output relationships of the post-ganglionic cardiac sympathetic nerves approach those achieved with severe stress. Eventually, norepinephrine biosynthesis can no longer compensate for the mounting demand, and steady-state levels of norepinephrine fall. Ultimately, cardiac norepinephrine turnover per unit of myocardial mass declines. With the loss of sympathetic reserve, congestive failure supervenes. Acknowledgment We are grateful to Dr. William Rand for his helpful discussions pertinent to the statistical calculations. References 1. SPANN JF, SONNENBLICK EH, COOPER T, CHIDSEY CA, WILLMAN VL, BRAUNWALD E: Cardiac norepinephrine stores and the contractile state of heart muscle. Circ Res 29:317-325, 1966 2. RUSHMER RF: Cardiovascular responses during exertion. In Cardiovascular Dynamics, 3rd ed. Philadelphia, W.B. Saunders, 1970, pp 220-243 3. DONALD DE, MILBURN SE, SHEPHERD JT: Effects of cardiac denervation on the maximal capacity for exercise in the racing greyhound. J Appl Physiol 19:849-852, 1964 4. GAFFNEY TE, BRAUNWALD E: Importance of the adrenergic nervous system in the support of circulatory function in patients with congestive heart failure. Am J Med 34:320-324, 1963 5. CHIDSEY CA, BRAUNWALD E: Sympathetic activity and neurotransmitter depletion in congestive heart failure. Pharmacol Rev 18:685-700, 1966 6. YAMAZAKI N, OGAWA K: Cardiac catecholamine metabolism in heart failure. Jap Circ J 35:965-971, 1971 7. DEQUATTRO V, NAGATSU T, MENDEZ A, VERSKA J: Determinants of cardiac noradrenaline depletion in human congestive failure. Cardiovasc Res 7:344-350, 1973 8. ANGELAKOS ET, CARBALLO LC, DANIELS JB, KING MP, BAJUSZ E: Adrenergic neurohumours in the heart of hamsters with hereditary myopathy during cardiac hypertrophy and failure. In Myocardiology: Recent Advances in Studies of Cardiac Structure and Metabolism, vol 1, edited by E Bajusz and G Rona. Baltimore, University Park Press, 1972, pp 262-278 9. COVELL JW, CHIDSEY CA, BRAUNWALD E: Reduction of the cardiac response to postganglionic sympathetic nerve stimulation in experimental heart failure. Circ Res 19:51-56, 1966 10. POOL PE, COVELL JW, LEVITT M, GIBB J, BRAUNWALD E: Reduction of cardiac tyrosine hydroxylase activity in experimental congestive heart failure. Circ Res 20:349-353, 1967 11. SPANN JF, CHIDSEY CA, POOL PE, BRAUNWALD E: Mechanism of norepinephrine depletion in experimental heart failure produced by aortic constriction in the guinea pig. Circ Res 17:312-321, 1965 12. FISCHER JE, HORST WD, KOPIN IJ: Norepinephrine metabolism in hypertrophied rat hearts. Nature (Lond) 207:951-953, 1965 13. GERTZ EW: Cardiomyopathic Syrian hamster: Possible model of human disease. In Pathology of the Syrian Hamster: Progress in Experimental Tumor Research, vol 16, edited by F Homburger. Basel, S. Karger, 1972, pp 242-260 14. O'HANLON JF, CAMPUZANO HC, HORVATH SM: Fluorometric assay for subnanogram concentrations of adrenaline and noradrenaline in plasma. Anal Biochem 34:568-581, 1970 15. WEINER N: Critical assessment of methods for the determination of monoamine synthesis turnover rates in vivo. In Neuropsychopharmacology of Monoamines and Their Regulatory Enzymes, edited by E Usdin. New York, Raven Press, 1974, pp 143-159 16. FRIEDMAN WF: Intrinsic physiologic properties of the developing heart. Prog Cardiovasc Dis 15:87-111, 1972 17. BOERTH RC: Postnatal development of myocardial adrenergic mechanisms in the cat (abstr). Circulation 48(suppl IV):IV-36, 1973 18. BAJUSZ E, BAKER JR, NIXON CW: Effects of catecholamines upon cardiac and skeletal muscles of dystrophic hamsters (abstr). Fed Proc 25:475, 1966 19. FLECKENSTEIN A, JANKE J, DORING HJ, LEDER O: Myocardial fiber necrosis due to intracellular Ca overload: A new principle in cardiac pathophysiology. In Myocardial Biology: Recent Advances in Studies on Cardiac Structure and Metabolism, vol 4, edited by NS Dhalla. Baltimore, University Park Press, 1974, pp 563-580