PETER BUTTRICK, CHARLES PERLA, ASHWANI MALHOTRA, DAVID GEENEN, MARIA LAHORRA, AND JAMES SCHEUER Division of Cardiology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York 10467
BUTTRICK,PETER,CHARLES DAVID
GEENEN,
MARIA
PERLA,ASHWANIMALHOTRA,
LAHORRA,
AND
JAMES
SCHEUER.
Ef-
fects of chronic dobutamine on cardiac mechanics and biochemistry after myocardial infarction in rats. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H473-H479,1991.-After myocardial infarction in rats, muscle performance in the remaining hypertrophied myocardium deteriorates and is associated with a decrease in myosin adenosinetriphosphatase (ATPase) activity and a shift to the V3 myosin heavy-chain isoform. We have previously shown in another model of hypertrophy, secondary to renovascular hypertension, that chronic intermittent adrenergic stimulation with dobutamine (Db) can prevent this biochemical adaptation. The present study was undertaken to assess the effects of chronic Db treatment on cardiac mass, function, metabolism, and myosin biochemistry in animals subjected to chronic myocardial infarction. Four groups of rats were studied: controls, animals treated with Db (2 mg/kg 2~ daily for 4 wk), animals subjected to myocardial infarction and killed after 4 wk (MI), and MI animals concurrently treated with Db for 4 wk (MI-Db). The two MI groups were subdivided into those with and without congestive heart failure (CHF). Heart weight was increased by 13% with Db, unchanged in the infarct groups without CHF, and increased by 9 and 22% in the infarct groups with CHF. Db did not have any additional effect on heart weight in these later groups. Infarct weight was greatest in the animals with CHF, and viable myocardium was equivalent in all infarct groups suggesting that CHF was associated with a greater degree of hypertrophy. Ventricular performance, as assessed in an isovolumic heart apparatus, was markedly depressed in both infarct groups with CHF and was not affected by Db. Db increased myosin ATPase activity in control and infarcted animals both with and without congestive heart failure. Myosin oxygen consumption and lactate production were not adversely affected by Db. cardiac
hypertrophy;
myosin
INFARCTION is associated with a spectrum of adaptations that allows the heart to maintain effective systolic performance. Acutely, these include enhanced sympathetic tone, activation of the renin-angiotensin system, and alterations in regional vascular capacitance. Chronically, the infarcted chamber tends to increase its end-diastolic volume to maintain an effective cardiac output. These physiological adaptations, although effectively enhancing systolic ejection, are limited by the mechanical capacity of the noninfarcted muscle and also by the increased energy costs of contraction at an increased end-diastolic wall stress in the presence of enMY~CARDIAL
hanced sympathetic tone. Pfeffer et al. (24) have shown that the ventricular dilatation after infarction in rats is associated with an increase in compliance, and the development of a significant increase in ventricular volume postmyocardial infarction is associated both with deteriorated muscle function (8, 10) and with a poor prognosis (7). Chronic administration of the sympathomimetic amine dobutamine induces a variety of potentially salutory physiological adaptations. These include maintenance of exercise capacity during a period of deconditioning in normal human volunteers (30), improvement in exercise capacity in patients with congestive heart failure ( F) associated with decreased mitochondrial swelling, the loss of electron-dense cytoplasmic inclusion bodies in endomyocardial biopsy specimens (19), and a diminished catecholamine response to submaximal exercise in dogs (20). In addition, we have previously shown that chronic dobutamine administration is associated with increased cardiac myosin adenosinetriphosphatase (ATPase) activity in normal rats and with prevention of the biochemical adaptations of cardiac myosin which accompany renovascular hypertension in this species (4). These patterns of response might benefit ventricular performance in conditions, such as chronic myocardial infarction, which are associated with limited cardiac reserve. Potentially limiting side effects, myocardial necrosis and adrenergic receptor downregulation, have not been observed with chronic intermittent dobutamine administration (4, 6), although it remains possible that the acute inotropic and chronotropic effects of t might increase myocardial oxygen consumption To explore this hypothesis, we studied the e long-term intermittent administration of dobutamine on cardiac biochemistry and physiology in rats subjected to ic myocardial infarctions both with and without The drug was administered twice daily as a subeous bolus at a dose that we have previously shown induces a prompt increase in change in pressure over change in time (dP/dt) without a marked rise in systolic blood pressure. METHODS
Animal models. emale Wistar rats (Charles River) weighing 200-250 g at the onset of study were employed and were allocated at the outset into four treatment
0363-6135/91 $1.50 Copyright 0 1991 the American Physiological Society
H473
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H474
DOBUTAMINE
AFTER
MYOCARDIAL
groups: shams; animals that received intermittent dobutamine treatment (Db); animals that were subjected to ligation of the left coronary artery (MI); and infarcted animals that were concurrently treated with dobutamine (MI-Db) starting on the day after surgery. Dobutamine was provided as a racemic mixture of (+) and (-) isomers by Eli Lilly (Indianapolis, IN) and was administered subcutaneously at a dose of 2 mg/kg twice daily 5 days/ wk for 4-6 wk. This dosing schedule was similar to that used in our previous studies and was chosen to approximate the intensity (both chronotropic and inotropic) of a 90-min bout of swimming (27). Sham animals received injections of saline twice daily. Myocardial infarction was created using previous described techniques (12). Briefly, animals were anesthetized, their hearts exteriorized through a left thoracotomy, the pericardium stripped, and a 7-O prolene suture placed around the left coronary artery 2-3 mm from the aorta. Sham animals underwent the same procedure except that the suture was passed under the coronary artery and then removed. There was a perioperative mortality of -4050% in the infarcted animals. On the day after surgery, electrocardiograms were obtained under light ether anesthesia to document the development of infarcts. Animals were then subgrouped according to the depth and persistence across the precordial leads of pathological Q waves (17). This provided a gross estimate of infarct size and assured that large and small infarcts were equally distributed in the MI and MI-Db groups. Animals were killed after 4-6 wk, and their hearts were removed for physiological and biochemical study. A portion of the left ventricle (-0.2 g) and the scar were saved and dried to constant weight to establish wet-to-dry weight ratios. In addition, the lungs were removed, wet weights were obtained, and the lungs were then dried to constant dry weight to determine lung water content. Animals with lung water content >2 SD above the mean were designated as having CHF. During the 4-6 wk of observation, 0 of 15 shams, 1 of 16 Db, 3 of 16 MI, and 5 of 19 MI-Db animals died. In a separate group of experiments, six sham and seven chronically infarcted animals were studied 3-4 wk after surgery to compare their acute hemodynamic responses to bolus injections of dobutamine. In these studies, animals were anesthetized with ketamine and xylazine, their left carotid arteries were cannulated, and a 3F ultraminiature pressure transducer was advanced retrograde across the aortic valve. The animals were then given bolus injections of dobutamine, and their heart rate, ventricular pressure, and dP/dt responses were measured. Heart perfusions. Hearts were removed from animals anesthetized with ether 18-24 h after the last dobutamine injection and were immediately placed in iced saline before attachment to the perfusion apparatus. The perfusate was a modified Krebs-Henseleit buffer at 37°C gassed with a 95% OZ-5% CO2 mixture and contained 15 mM glucose as substrate, 0.01 U/ml regular insulin (Lilly), and 2.0 mM calcium with 0.5 mM EDTA to yield 1.5 mM free calcium. Hearts were suspended by the aorta, and the coronaries were filled retrograde at an aortic pressure of 89 mmHg. A fluid-filled latex balloon at-
INFARCTION
IN
RATS
tached via PE-60 polyethylene tubing to a Statham P23d strain-gauge pressure transducer was inserted into the left ventricular cavity across the mitral valve and inflated so as to establish a spectrum of left ventricular enddiastolic pressures according to the methods established by Apstein et al. (2, 21). The left ventricle was vented to drain Thebesian flow. Pressure measurements were made on the flat portion of the balloon’s compliance curve. An additional catheter was placed in the pulmonary outflow tract, and the effluent was collected for measurement of oxygen tension and myocardial lactate production. Coronary flow in this isolated heart apparatus approximates the right ventricular effluent, which was measured directly. All hearts were paced via the right ventricle at a rate of 350 beats/min. Cardiac function was assessed at two end-diastolic pressures: first at 10 mmHg (moderate) and then at 20 mmHg (high), at a fixed aortic perfusion pressure. Studies were then repeated at the moderate preload to confirm the stability of the preparation. Whenever possible, individual hearts from all experimental and control groups were perfused on the same day. Analog data were digitized on an IBM 9000 laboratory computer, and measurements of dynamic pressure data were determined. At the end of each experiment, the atria and great vessels were dissected free, and the right ventricular free wall was removed. The left ventricle was then divided into infarcted and noninfarcted tissue, weighed, and subsequently saved for contractile protein analysis. Lactate content was determined in the buffer collected from the pulmonary artery outflow, which represents coronary effluent using a, commercially available kit (Behring Diagnostics, La Jolla, CA). Oxygen tension was also measured both in the aortic inflow and the pulmonary effluent using an oxygen electrode (Radiometer). Cardiac contractile proteins. Noninfarcted left ventricular tissue was stored at -80°C in 50% glycerol containing 50 mM KC1 and 10 mM KHPO, before preparation of the myosin extracts. Myosin was extracted and purified by previously described techniques (22, 23) and was shown by sodium dodecyl sulfate gel electrophoresis to be free of actin, troponin, and tropomyosin; no evidence of proteolytic degradation was found. Calcium myosin ATPase activities were assayed at 30°C in 0.3 M KCl, 50 mM tris(hydroxymethyl)aminomethane . HCl (pH 7.6), 10 mM CaC12, 5 mM ATP, and 5 mM sodium azide according to previously described techniques (22, 23). Results are expressed as micromoles of Pi per milligram per minute. Myosin isoenzymes were analyzed by electrophoresis of purified myosin on polyacrylamide gels under nondissociating conditions at 2OC as reported by d’Albis et al. (5). Two to 3 pugof myosin were layered on each gel and run at a constant voltage gradient of 14 V/cm for 20-22 h using a Pharmacia Apparatus (GE 2/4). Gels were stained with Coomassie blue, and densitometric scans were recorded at 605 nm on an E-C densitometer. The semiquantitative estimate of each isoenzyme was calculated from the height of each peak by integration (Hewlett-Packard Integrator, model 3390A, Palo Alto, CA). The relative contribution of V, was equally distributed
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DOBUTAMINE
AFTER
MYOCARDIAL
INFARCTION
IN
H475
RATS
between the V1 and V3 peaks to reflect the myosin heavychain isoform distribution. Statistical analysis. Results were submitted to analysis of variance. The mean square error within groups was then used in a Newman-Keuls multiple comparison test to evaluate differences between hearts from any two groups (32). Significance is reported at the 0.05 level.
chronic drug therapy, myocardial infarction, or CHF. In animals without MI, total heart and left ventricular mass were increased by treatment with dobutamine. MI in the presence of CHF significantly increased the weights of both ventricles. Scar weight was greatest in the animals with CHF, and because noninfarcted ventricular mass was equivalent in all infarcted groups the degree of hypertrophy of the noninfarcted muscle was probably greatest in these animals as well. Dobutamine did not RESULTS cause additive left ventricular hypertrophy in the inThe acute hemodynamic responses to bolus injections farcted animals and also did not appear to influence of dobutamine in sham and infarcted animals are shown infarct size. Of note, animals whose lung weights defined in Table 1. Marked increases in both heart rate and dP/ them as having CHF included those with 11 of the 12 dt were seen in both groups 5 min after drug administralargest infarcts as determined by scar weight. tion, whereas peak left ventricular pressure increased to Figure 1 shows data obtained from the isolated peronly a modest degree. The magnitude of these responses fused heart studies. Data are presented at both moderate in the sham and MI groups was statistically similar. End(10 mmHg) and high (20 mmHg) end-diastolic pressures. diastolic pressure was higher and peak systolic pressure Hearts from dobutamine-treated noninfarcted animals and dP/dt were slightly lower in the infarcted animals. did not show any change in peak left ventricular pressure Those animals with the largest infarcts had higher end- or in positive or negative dP/dt when contrasted with diastolic pressures and less marked increases in both left hearts from placebo-treated animals. Both Db and conventricular pressure and dP/dt in response to dobutatrol hearts from animals subjected to myocardial infarcmine injections, although the chronotropic response to tion not complicated by CHF displayed mechanical functhe drug did not appear to be affected by infarct size. tion similar to controls. The slight reduction in peak left The duration of the drug’s action was equivalent in sham ventricular pressure seen in the Db-MI group did not and MI animals. attain statistical significance. In contrast, those animals Table 2 shows heart and body weight data among the with CHF did have significant impairment in all three various experimental groups. All body weights were sim- measures of mechanical function. Left ventricular volilar and were not affected by the presence of either ume measurements were not systematically obtained and therefore pressure to volume comparisons between the TABLE 1. Acute hemodynamic responses groups cannot be made. to dobutamine administration Table 3 shows data for myocardial oxygen extraction, MVoZ, and lactate production from the isolated perfused Heart Rate, EDP, LVP, dP/dt, Groups hearts at the higher end-diastolic pressure. Control valbeats/min mmHg mmHg mmHg/s ues for 0, extraction, MOON, and lactate production were Sham 0.75 ml/ml 02, 1.37 ml 02*g-1.min-1, and 11.6 ~rnolg-‘. 5,724+514 Initial 220t17 9-+3 114&5 min-l, respectively. Data are presented as a percentage 15,579+808* 5 Min 320&14* 3-+1* 133t7* of control values and, in the case of MVO, and lactate 11,941+743* 60 Min 292t20” 7&2 134t6” MI production, are normalized for viable left ventricular 101t9 4,375+640 Initial 209k9 18+3”r weight. Oxygen extraction decreased in the infarcted 111t14 10,113+2,447* 5 Min 336t25” llt3*? hearts in proportion to the loss of myocardium so that 109+llt 8,503+2,405* 60 Min 301t33” 14tlOt the hearts from animals with CHF, which for the most Data are means t SE. Comparisons were made before, and 5 and 60 part sustained larger infarctions, tended to have lower min after injections of 2 mg/kg SC of dobutamine. EDP, end-diastolic pressure; LVP, left ventricular peak systolic pressure. * P c 0.05 vs. values irrespective of concurrent drug therapy. MVo2 was slightly increased in the animals with MI but was initial; “r P < 0.05 vs. sham. TABLE
2. Body and heart weights MI Weights
Sham
(14)
-CHF Body,
g
Dry heart, mg Dry LV, mg Scar, mg % LV scar, mg/mg Dry viable LV, mg Dry RV, mg Dry/wet (muscle) Dry/wet (scar) Wet lung, g
260&3 149t6 118*5
261&3 168t6* 130t5
31t2 0.16t0.01
38t2* 0.17t0.01
1.12t0.05
Data are means t SE; values in parentheses MI, myocardial infarct; CHF, congestive heart +CHF.
MI-Db
Db (14)
1.14t0.04
264-t4 140tll lllt8 15&2 14t2 96t8 29*3 0.14~0.01 O.lOtO.O1 0.94kO.06
(9)
+CHF
(5)
262-+4 182&17t$ 139+15?$ 36+4$ 28+5$ 103*17 43+4Q 0.15*0.01 0.09t0.01 2.31+0.14$
-CHF
260&2 159t6 122t6 22&4 17*3 102*6 31&4 0.15t0.01 O.lltO.O1 1.02t0.06
(8)
+CHF
(6)
259t4 162klO 116t7 17t2 24+5$ 90t6 46+3-t* 0.14t0.01 0.08t0.01 2.28+0.12$
are no. of animals. LV and RV, left and right ventricle, respectively. Db, dobutamine-treatment; failure; * P < 0.05, sham vs. Db; “f P < 0.05, sham vs. MI or Db vs. MI-Db; $ P < 0.05, -CHF
vs.
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DOBUTAMINE
H476
AFTER
MYOCARDIAL
PEAK LV PRESSURE
INFARCTION
IN
RATS
PEAK 0 dP/dT
PEAK 0 dP/dT
240
180 $ 160 E E 140 120 100
)------I o------(> M FIG. 1. Cardiac mechanical data obtained from isolated isovolumic perfused end-diastolic pressures. Values are means t SE of 5-14 studies. Abbreviations sham vs. MI or Db vs. MI-Db; O P c 0.05, CHF vs. no CHF.
TABLE
m -----a e-----o e-----a
Sham MI-CHF MI+CHF
hearts at moderate (M) or high (H) as indicated in Table 1. ‘P < 0.05,
3. Indexes of myocardial metabolism MI Sham
(14)
O2 extraction MVo, Lactate production
1.00t0.04 1.00~0.11 1.00~0.18
l.Ol-+-0.03 1.01t0.07
0.86t0.08
I~I SE and are expressed as a fraction * P < 0.05 vs. sham; t- P c 0.05, -CHF vs. +CHF. are
means
MI-Db
Db (14) -CHF
Data
Db MIDb -CHF MIDb+CHF
of sham
(9)
1.00t0.05 1.23t0.10
1.55t0.17* values;
not further increased in the animals with CHF, which probably reflected the small increases in coronary blood flow seen in these groups (19% in the MI and 12% in the MI-Db groups with CHF). Parallel changes in lactate production were seen. The metabolic responses of the animals treated with Db did not differ from their matched nontreated cohorts. Figure 2 shows the results of contractile protein analysis. Dobutamine increased calcium-activated myosin ATPase activity in control animals. This increase was not reflected by a measurable shift in isomyosin distribution probably because the %V1 myosin in the control animals was >95% and further increases in this isoform were beyond the limits of detection. Myocardial infarction markedly depressed ATPase activity and shifted the isomyosin distribution towards VS. These biochemical alterations occurred regardless of whether the infarction produced CHF or not. The effect of myocardial infarction on ATPase activity was completely prevented by dobutamine if the infarct was not associated with CHF and was partially prevented even when CHF was present. In the MI-Db group, calcium-activated myosin ATPase activity was moderately depressed in animals with CHF but was near normal in those without failure. Overall, the V, isomyosin content in the Db-MI group was 90% and was not statistically different from either controls
values
+CHF 0.69&0.05-j. 1.15t0.20
1.42t0.21 in parentheses
(5)
-CHF 1.00-c-0.08 1.26rtO.16
1.08t0.10 are no. of animals.
(8)
+CHF
(6)
0.73t0.10 1.38t0.11 1.94+0.28*? Abbreviations
as in Table
1.
or noninfarcted dobutamine-treated animals. A trend toward a depression in the V1 isoform in MI animals with CHF was also observed both in the presence and absence of dobutamine, but inadequate band separation on some of the pyrophosphate gels in these groups preeluded meaningful statistical comparisons. DISCUSSION The present study was conducted to assessthe utility of chronic intermittent dobutamine treatment on the evolution of myocardial infarction in the rat. This agent and mode of administration were studied because it is widely employed in the clinical management of CHF and it has been previously shown in rats to increase both myosin ATPase activity and %V1 myosin (4) and therefore might enhance the force-generating capacity and the velocities of contraction and relaxation in failing hearts with depressed myosin ATPase activities. A paradigm for this is the physiological cardiac hypertrophy seen after exercise conditioning in which myosin ATPase and the velocity of contraction are increased (26). Myocardial infarction was created using standard techniques and a spectrum of left ventricular damage was created, which is consistent with the experience of others (8, 10, 24). Infarct size was estimated by scar
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DOBUTAMINE
Ca++ -activated
AFTER
MYOCARDIAL
INFARCTION
IN
Myosin
H477
RATS
Myosin
Isoenzymes
ATPase
13 . 12 .
80
11 . + 2I 1.0 \F c .9 z =t .8
g
60 .... ... .... ... . . . . .. . ..... .. .... ... . . . . . .. . .. . .. . ..... .. .... ... . . . . .*.*.*.*. . . . . . ..a. . .. . .. . ...a . . .*.*.*.* . . . .*.*.-.*. . . . .. . .. . . ..-.*.-....... . ...... . ...... . . . . . . .. . .. . .. .
. . . . . . . . .
. . . . . . . . . .
. .. . .. . .. . .. .. .. .
*7 , 1
/ /
Sham
Db
+CHF
-CHF
MI FIG.
sham
2. Contractile protein vs. Db or MI vs. MI-Db;
+CHF
t-l
-CHF
anam
Db
MI
MIDb
MIDb
data. Values are means t SE of 4-14 studies. Abbreviations + P < 0.05, sham vs. MI or Db vs. MI-Db; o P < 0.05, CHF
weight, which most likely underestimates the mass of the ventricle that is infarcted (25). Pfeffer et al. (24), for example, reported the development of overt CHF in rats with an infarct size estimated by planimetry of >45%. Approximately one-third of the total animals infarcted in that study were in this group. We also found that CHF developed with a similar frequency, whereas scar weight was -25% of total left ventricular weight. This crude correlate of infarct size was used because of the need to carefully preserve the hearts for con tractile protein analysis. The infarct sizes in th e groups not treated and treated with dobutamine were comparable, and, strikthe mass of the hypertrophied, noninfarcted musWY, cle wa.s similar in all i.nfarcted groups. Because the mass of muscle lost in the animals that developed CHF was probably larger, a greater degree of ventricular hypertrophy in these animals is also likely. This is consistent with the careful morphometric studies of Anversa et al. (1). The apparent exaggerated hypertrophic response of these animals was inadequate in maintaining cardiac compensation, suggesting a limitation to the degree of adaptive hypertrophy available to overloaded muscle. The apparent increase in mass in the MI-CHF group might reflect the relatively small number of animals in this group, although it does not countermand this hypothesis. The increase in ventricular mass after myocardial infarction might have masked a dobutamine-induced hypertrophy in these animals. This has been previously suggested using other hemodynamic stimuli to ventricular hypertrophy (29, 31).
as in Table vs. no CHF.
1. * P < 0.05,
The major finding of the pharmacological portion of the study is that dobutamine treatment results in preservation of normal myosin ATPase activity and myosin heavy-chain isoform distribution in infarcted hearts without any apparent improvement in cardiac performante. In .farction resulted in a 43% decrease in V1 myosin and an -25% depression in calcium-activated myosin ATPase activity, regardless of whether or not the infarct was associated with heart failure. Concurrent treatment with dobutamine prevented the shift in isomyosin distribution and ameliorated the depression in ATPase activity associated with myocardial infarction. The fact that dobutamine totally corrected the ATPase activity in the absence of CHF but only partially corrected it in the presence of CHF suggeststhat the load on the remaining myocardium was greater with CHF and thus ,&myosin heavy-chain gene expression was more active in this setting and could not be overridden by adrenergic stimulation with dobutamine. Lactate production in the isovolumic perfusion apparatus at high end-diastolic pressure was increased in the MI group both with and without CHF, whereas in the MI-Db group normal effluent lactate concentrations were observed except in those animals with extensive infarction, in which lactate production was increased to a degree equivalent to that seen in the MI plus CHF group. This argues against myocardial ischemia during perfusion as an explanation for the lack of any dobutamine-associated improvement in performance. The data also suggest that shifts in the lactate dehydrogenase
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H478
DOBUTAMINE
AFTER
MYOCARDIAL
isoenzyme system might have been induced by the hemodynamic overload (15) and partially ameliorated by dobutamine administration, a situation which would be analogous to that seen with the myosin isoenzymes. The hemodynamic data obtained in the isovolumic perfusion apparatus is consistent with the work of others in that moderately sized infarctions were not associated with altered baseline hemodynamics. It was not until a substantial portion of the ventricle was scarred that depressed left ventricular performance was observed (3, 14). The failure of dobutamine to improve the pressuregenerating capacity of the myocardium is consistent with our previous work (4) and likely reflects a true disassociation between myosin enzymology and contractility, although this might reflect the insensitivity of the perfusion system to detect subtle changes in muscle performance. Because left ventricular end-diastolic volumes were not measured, however, any judgment about force generation must be made with caution. It is conceivable that dobutamine prevented ventricular dilatation postinfarction and thus the Db-MI groups were able to generate similar peak left ventricular pressures from a reduced left ventricular end-diastolic volume when contrasted with MI animals. This would represent a true increase in contractility. The relationship between adrenergic stimulation and isomyosin gene expression is not unique to the present study. Simpson and his colleagues (28), studying neonatal myocytes in culture, have shown that cul-adrenergic stimulation selectively enhances ,&myosin heavy-chain gene expression. More recently, Kawana et al. (16) have shown that ,6-adrenergic stimulation increases the %V1 myosin in denervated rabbit hearts, a result which is more concordant with our own data. A possible mechanism by which catecholamines (and specifically ,&agonists) might mediate these effects is the recent demonstration that cardiac myosin gene expression can be influenced by an adenosine 3’,5’-cyclic monophosphateresponsive element in the a-myosin heavy-chain gene promotor region (13). A potential criticism of this study might be that the infarcted animals with and without failure and the controls did not receive pharmacologically similar doses of the drug. The acute hemodynamic data (Table 1) make this unlikely as the heart rate response to injections and the duration of drug action were similar in sham and MI groups. What is likely, however, was that those animals with large infarcts were unable to generate equivalent peak left ventricular pressures and shortening velocities that might have altered the magnitude of the hemodynamic stimulus associated with drug administration. The lack of an apparent hemodynamic effect of dobutamine in the present study must also be viewed in light of the work of Pfeffer and co-workers (24) and others (9, 11) who have studied the effects of various pharmacological treatments on the evolution of myocardial infarction in the rat model. As described by these workers, the natural history of infarction is one of gradual ventricular dilatation (in proportion to infarct size) associated with a reduction in chamber stiffness and a decrease in maximal ventricular performance. These adaptations can be ameliorated by reducing preload and afterload with drugs
INFARCTION
IN
RATS
such as captopril. Gay et al. (11) have shown a modest improvement in in situ hemodynamics postmyocardial infarction with thyroxine treatment independent of the effects of thyroxine on isomyosin distribution, which may also reflect effects on the peripheral circulation. The present study did not assess ventricular performance in situ but the vasodilatory effect of dobutamine (18) raises the possibility that this agent might exert a beneficial effect on postmyocardial infarction ventricular remodeling independent of the direct cardiac effects of the drug. The authors thank Alwyn Murphy and Kirit Pate1 for technical assistance and Janice Brewton for secretarial help. This project was supported in part by National Heart, Lung, and Blood Institute Grant HL-15498. P. Buttrick is the recipient of a Clinical Investigator Award, HL-01552. Address for reprint requests: P. Buttrick, Div. of Cardiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 E. 210th St., Bronx, NY 10467. Received
10 April
1990; accepted
in final
form
18 September
1990.
REFERENCES 1. ANVERSA, P., C. BEGHI, Y. KIKKAA, AND G. OLIVETTI. Myocardial infarction in rats-infarct size, myocyte hypertrophy, and capillary growth. Circ. Res. 58: 26-37, 1986. 2. APSTEIN, C. S., M. MUELLER, AND W. B. HOOD, JR. Ventricular contracture and compliance changes with global ischemia and reperfusion and their effect on coronary resistance in the rat. Circ. Res. 41: 206-217, 1977. 3. BOGEN, D. K., S. A. RABINOWITZ, A. NEEDLEMAN, T. A. McMAHON, AND W. H. ABELMANN. An analysis of the mechanical disadvantage of myocardial infarction in the canine left ventricle. Circ. Res. 47: 728-741, 1980. 4. BUTTRICK, P. M., A. MALHOTRA, S. FACTOR, D. GEENEN, AND J. SCHEUER. Effects of chronic dobutamine administration on hearts of normal and hypertensive rats. Circ. Res. 63: 173-181, 1988. 5. D’ALBIS, A., C. PANTALONI, AND J. J. BEHCET. An electrophoretic study of native myosin isoenzymes and their subunit content. Eur. J. Biochem. 99: 261-272,1979. 6. DAVIDSON, W. R., JR., S. P. BANERJEE, AND C. S. LIANG. Dobutamine induced cardiac adaptations: comparison with exercise trained and sedentary rats. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H725-H730, 1986. 7. EPSTEIN, S. E., S. T. PALMERI, AND R. E. PATTERSON. Evaluation of patients after acute myocardial infarction: indications for cardiac catheterization and surgical intervention. N. Engl. J. Med. 307: 1487-1492, 1982. 8. FELLENIUS, E., C. A. HANSEN, 0. MJOS, AND J. R. NEELEY. Chronic infarction decreases maximum cardiac work and sensitivity of heart to extracellular calcium. Am. J. Physiol. 249 (Heart Circ. Physiol. 18): H80-H87, 1985. 9. FISHBEIN, M. L., L.-Q. LER, AND S. A. RUBIN. Long-term propran0101 administration alters myocyte and ventricular geometry after myocardial infarction in rat hearts. Circukztion 78: 369-375, 1988. 10. FLETCHER, P. J., J. M. PFEFFER, M. A. PFEFFER, AND E. BRAUNWALD. Left ventricular diastolic pressure-volume relations in rats with healed myocardial infarction. Effects on systolic function. Circ. Res. 49: 618-626, 1981. 11. GAY, R., T. A. GUSTAFSON, S. GOLDMAN, AND E. MORKIN. Effects of L-thyroxine in rats with chronic heart failure after myocardial infarction. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H341H346,1987. D. L., T. P. WHITE, AND R. M. LAMPMAN. Papillary 12. GEENEN, mechanics and cardiac morphology of infarcted rat hearts after training. J. Appl. Physiol. 63: 92-96, 1987. of 13. GUPTA, M. P., M. GUPTA, S. JAKOVAIC, AND R. ZAK. Activation rat cardiac myosin gene expression by CAMP (Abstract). Circukztion 80, SuppZ. II: 11-605, 1989. 14. HOOD, W. B., JR. Experimental myocardial infarction. III. Recovery of left ventricular function in the healing phase. Contribution
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DOBUTAMINE
15.
16.
17.
18.
19.
20.
21.
22.
23.
AFTER
MYOCARDIAL
of increased fiber shortening in noninfarcted myocardium. Am. Heart J. 79: 531-538, 1970. KAAJA, R., AND K. AVE. Myocardial LDH isoenzyme patterns in rats exposed to cold and/or hypobaric hypoxia. Acta Med. Stand. 668: 136-142,1982. KAWANA, M., N. ISHIZUKA, A. TAIRA, S. KIMATA, AND S. HOSADA. Effects of cardiac sympathetic nerve activity on myosin isoenzymes of rabbit heart (Abstract). Circukztion 80, Suppl. II: 11-462, 1989. KLONER, R. A., AND J. A. KLONER. The effect of early exercise on myocardial infarct scar formation. Am. Heart J. 51: 1009-1013, 1983. LEIER, C. V., P. HEBAN, P. Huss, C. A. BUSH, AND R. P. LEWIS. Comparative systemic and hemodynamic effects of dopamine and dobutamine in patients with cardiomyopathic heart failure. Circulation 58: 466-475, 1977. LIANG, C.-S., L. G. SHERMAN, J. U. DOHERTY, K. WELLINGTON, V. W. LEE, AND W. B. HOOD, JR. Sustained improvement of cardiac function in patients with congestive heart failure after short-term infusion of dobutamine. Circukztion 69: 113-119, 1984. LIANG, C.-S., R. R. TUTTLE, W. B. HOOD, JR., AND H. GAVRA~. Conditioning effects of chronic infusion of dobutamine. J. CZin. Inuest. 64: 613-619, 1979. LORELL, B. H., L. F. WEXLER, S. MONOMURA, E. WEINBERG, AND C. S. APSTEIN. The influence of pressure overload left ventricular hypertrophy on diastolic properties during hypoxia in isovolumitally contracting rat hearts. Circ. Res. 58: 653-663, 1986. MALHOTRA, A., S. PENPARGKUL, F. FEIN, E. H. SONNENBLICK, AND J. SCHEUER. The effect of streptozotocin-induced diabetes in rats on cardiac contractile proteins. Circ. Res. 49: 1243-1250, 1981. MALHOTRA, A., S. PENPARGKUL, T. SCHAIBLE, AND J. SCHEUER.
INFARCTION
24.
25.
26. 27.
28.
29.
30.
31.
32.
IN
RATS
H479
Contractile proteins and sarcoplasmic reticulum in physiological cardiac hypertrophy. Am. J. Physiol. 241 (Heart Circ. Physiol. 10): H263-H267,1981. PFEFFER, J. M., M. A. PFEFFER, AND E. BRAUNWALD. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ. Res. 57: 84-95, 1985. RUBIN, S. A., M. C. FISHBEIN, AND H. J. C. SWAN. Compensatory hypertrophy in the heart after myocardial infarction in the rat. J. Am. CoZZ. Cardiol. 1: 1435-1441, 1983. SCHAIBLE, T. F., AND J. SCHEUER. Cardiac adaptation to chronic exercise. Prog. Cardiovasc. Dis. 27: 297-324, 1985. SCHAIBLE, T. F., AND J. SCHEUER. Effects of physical training by running and swimming on ventricular performance of rat hearts. J. AppZ. Physiol. 46: 854-860, 1979. SIMPSON, P. C., C. S. LONG, L. E. WASPE, C. J. HENRICH, AND C. P. ORDAHL. Transcription of early developmental isogenes in cardiac myocyte hypertrophy. J. Mol. CeZZ. CardioZ. 21: 79-89,1989. SPANN, J. H., A. A. BOVE, AND G. NATARAJAN. Ventricular performance, pump function and compensatory mechanism in patients with aortic stenosis. CircuZation 62: 576-585, 1980. SULLIVAN, M. J., P. F. BINKLEY, D. V. UNVERFERTH, J.-H. REN, H. BOUDOULAS, T. M. BASHORE, J. MEROLA, AND C. V. LEIER. Prevention of bedrest-induced physical deconditioning by daily dobutamine infusions. J. CZin. Inuest. 76: 1632-1642, 1985. WILLIAMS, J. F., W. MATTHEW, D. L. HERN, R. D. POTTER, AND W. P. DEISS. Myocardial hydroxyproline and mechanical response to prolonged pressure loading followed by unloading in the cat. J. CZin. Inuest. 72: 1910, 1983. ZAR, J. H. BiostatisticaZ Analysis. Englewood Cliffs, NJ: PrenticeHall, 1974, p. 151-155.
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BUTTRICK,PETER,CHARLES DAVID
GEENEN,
MARIA
PERLA,ASHWANIMALHOTRA,
LAHORRA,
AND
JAMES
SCHEUER.
Ef-
fects of chronic dobutamine on cardiac mechanics and biochemistry after myocardial infarction in rats. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H473-H479,1991.-After myocardial infarction in rats, muscle performance in the remaining hypertrophied myocardium deteriorates and is associated with a decrease in myosin adenosinetriphosphatase (ATPase) activity and a shift to the V3 myosin heavy-chain isoform. We have previously shown in another model of hypertrophy, secondary to renovascular hypertension, that chronic intermittent adrenergic stimulation with dobutamine (Db) can prevent this biochemical adaptation. The present study was undertaken to assess the effects of chronic Db treatment on cardiac mass, function, metabolism, and myosin biochemistry in animals subjected to chronic myocardial infarction. Four groups of rats were studied: controls, animals treated with Db (2 mg/kg 2~ daily for 4 wk), animals subjected to myocardial infarction and killed after 4 wk (MI), and MI animals concurrently treated with Db for 4 wk (MI-Db). The two MI groups were subdivided into those with and without congestive heart failure (CHF). Heart weight was increased by 13% with Db, unchanged in the infarct groups without CHF, and increased by 9 and 22% in the infarct groups with CHF. Db did not have any additional effect on heart weight in these later groups. Infarct weight was greatest in the animals with CHF, and viable myocardium was equivalent in all infarct groups suggesting that CHF was associated with a greater degree of hypertrophy. Ventricular performance, as assessed in an isovolumic heart apparatus, was markedly depressed in both infarct groups with CHF and was not affected by Db. Db increased myosin ATPase activity in control and infarcted animals both with and without congestive heart failure. Myosin oxygen consumption and lactate production were not adversely affected by Db. cardiac
hypertrophy;
myosin
INFARCTION is associated with a spectrum of adaptations that allows the heart to maintain effective systolic performance. Acutely, these include enhanced sympathetic tone, activation of the renin-angiotensin system, and alterations in regional vascular capacitance. Chronically, the infarcted chamber tends to increase its end-diastolic volume to maintain an effective cardiac output. These physiological adaptations, although effectively enhancing systolic ejection, are limited by the mechanical capacity of the noninfarcted muscle and also by the increased energy costs of contraction at an increased end-diastolic wall stress in the presence of enMY~CARDIAL
hanced sympathetic tone. Pfeffer et al. (24) have shown that the ventricular dilatation after infarction in rats is associated with an increase in compliance, and the development of a significant increase in ventricular volume postmyocardial infarction is associated both with deteriorated muscle function (8, 10) and with a poor prognosis (7). Chronic administration of the sympathomimetic amine dobutamine induces a variety of potentially salutory physiological adaptations. These include maintenance of exercise capacity during a period of deconditioning in normal human volunteers (30), improvement in exercise capacity in patients with congestive heart failure ( F) associated with decreased mitochondrial swelling, the loss of electron-dense cytoplasmic inclusion bodies in endomyocardial biopsy specimens (19), and a diminished catecholamine response to submaximal exercise in dogs (20). In addition, we have previously shown that chronic dobutamine administration is associated with increased cardiac myosin adenosinetriphosphatase (ATPase) activity in normal rats and with prevention of the biochemical adaptations of cardiac myosin which accompany renovascular hypertension in this species (4). These patterns of response might benefit ventricular performance in conditions, such as chronic myocardial infarction, which are associated with limited cardiac reserve. Potentially limiting side effects, myocardial necrosis and adrenergic receptor downregulation, have not been observed with chronic intermittent dobutamine administration (4, 6), although it remains possible that the acute inotropic and chronotropic effects of t might increase myocardial oxygen consumption To explore this hypothesis, we studied the e long-term intermittent administration of dobutamine on cardiac biochemistry and physiology in rats subjected to ic myocardial infarctions both with and without The drug was administered twice daily as a subeous bolus at a dose that we have previously shown induces a prompt increase in change in pressure over change in time (dP/dt) without a marked rise in systolic blood pressure. METHODS
Animal models. emale Wistar rats (Charles River) weighing 200-250 g at the onset of study were employed and were allocated at the outset into four treatment
0363-6135/91 $1.50 Copyright 0 1991 the American Physiological Society
H473
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H474
DOBUTAMINE
AFTER
MYOCARDIAL
groups: shams; animals that received intermittent dobutamine treatment (Db); animals that were subjected to ligation of the left coronary artery (MI); and infarcted animals that were concurrently treated with dobutamine (MI-Db) starting on the day after surgery. Dobutamine was provided as a racemic mixture of (+) and (-) isomers by Eli Lilly (Indianapolis, IN) and was administered subcutaneously at a dose of 2 mg/kg twice daily 5 days/ wk for 4-6 wk. This dosing schedule was similar to that used in our previous studies and was chosen to approximate the intensity (both chronotropic and inotropic) of a 90-min bout of swimming (27). Sham animals received injections of saline twice daily. Myocardial infarction was created using previous described techniques (12). Briefly, animals were anesthetized, their hearts exteriorized through a left thoracotomy, the pericardium stripped, and a 7-O prolene suture placed around the left coronary artery 2-3 mm from the aorta. Sham animals underwent the same procedure except that the suture was passed under the coronary artery and then removed. There was a perioperative mortality of -4050% in the infarcted animals. On the day after surgery, electrocardiograms were obtained under light ether anesthesia to document the development of infarcts. Animals were then subgrouped according to the depth and persistence across the precordial leads of pathological Q waves (17). This provided a gross estimate of infarct size and assured that large and small infarcts were equally distributed in the MI and MI-Db groups. Animals were killed after 4-6 wk, and their hearts were removed for physiological and biochemical study. A portion of the left ventricle (-0.2 g) and the scar were saved and dried to constant weight to establish wet-to-dry weight ratios. In addition, the lungs were removed, wet weights were obtained, and the lungs were then dried to constant dry weight to determine lung water content. Animals with lung water content >2 SD above the mean were designated as having CHF. During the 4-6 wk of observation, 0 of 15 shams, 1 of 16 Db, 3 of 16 MI, and 5 of 19 MI-Db animals died. In a separate group of experiments, six sham and seven chronically infarcted animals were studied 3-4 wk after surgery to compare their acute hemodynamic responses to bolus injections of dobutamine. In these studies, animals were anesthetized with ketamine and xylazine, their left carotid arteries were cannulated, and a 3F ultraminiature pressure transducer was advanced retrograde across the aortic valve. The animals were then given bolus injections of dobutamine, and their heart rate, ventricular pressure, and dP/dt responses were measured. Heart perfusions. Hearts were removed from animals anesthetized with ether 18-24 h after the last dobutamine injection and were immediately placed in iced saline before attachment to the perfusion apparatus. The perfusate was a modified Krebs-Henseleit buffer at 37°C gassed with a 95% OZ-5% CO2 mixture and contained 15 mM glucose as substrate, 0.01 U/ml regular insulin (Lilly), and 2.0 mM calcium with 0.5 mM EDTA to yield 1.5 mM free calcium. Hearts were suspended by the aorta, and the coronaries were filled retrograde at an aortic pressure of 89 mmHg. A fluid-filled latex balloon at-
INFARCTION
IN
RATS
tached via PE-60 polyethylene tubing to a Statham P23d strain-gauge pressure transducer was inserted into the left ventricular cavity across the mitral valve and inflated so as to establish a spectrum of left ventricular enddiastolic pressures according to the methods established by Apstein et al. (2, 21). The left ventricle was vented to drain Thebesian flow. Pressure measurements were made on the flat portion of the balloon’s compliance curve. An additional catheter was placed in the pulmonary outflow tract, and the effluent was collected for measurement of oxygen tension and myocardial lactate production. Coronary flow in this isolated heart apparatus approximates the right ventricular effluent, which was measured directly. All hearts were paced via the right ventricle at a rate of 350 beats/min. Cardiac function was assessed at two end-diastolic pressures: first at 10 mmHg (moderate) and then at 20 mmHg (high), at a fixed aortic perfusion pressure. Studies were then repeated at the moderate preload to confirm the stability of the preparation. Whenever possible, individual hearts from all experimental and control groups were perfused on the same day. Analog data were digitized on an IBM 9000 laboratory computer, and measurements of dynamic pressure data were determined. At the end of each experiment, the atria and great vessels were dissected free, and the right ventricular free wall was removed. The left ventricle was then divided into infarcted and noninfarcted tissue, weighed, and subsequently saved for contractile protein analysis. Lactate content was determined in the buffer collected from the pulmonary artery outflow, which represents coronary effluent using a, commercially available kit (Behring Diagnostics, La Jolla, CA). Oxygen tension was also measured both in the aortic inflow and the pulmonary effluent using an oxygen electrode (Radiometer). Cardiac contractile proteins. Noninfarcted left ventricular tissue was stored at -80°C in 50% glycerol containing 50 mM KC1 and 10 mM KHPO, before preparation of the myosin extracts. Myosin was extracted and purified by previously described techniques (22, 23) and was shown by sodium dodecyl sulfate gel electrophoresis to be free of actin, troponin, and tropomyosin; no evidence of proteolytic degradation was found. Calcium myosin ATPase activities were assayed at 30°C in 0.3 M KCl, 50 mM tris(hydroxymethyl)aminomethane . HCl (pH 7.6), 10 mM CaC12, 5 mM ATP, and 5 mM sodium azide according to previously described techniques (22, 23). Results are expressed as micromoles of Pi per milligram per minute. Myosin isoenzymes were analyzed by electrophoresis of purified myosin on polyacrylamide gels under nondissociating conditions at 2OC as reported by d’Albis et al. (5). Two to 3 pugof myosin were layered on each gel and run at a constant voltage gradient of 14 V/cm for 20-22 h using a Pharmacia Apparatus (GE 2/4). Gels were stained with Coomassie blue, and densitometric scans were recorded at 605 nm on an E-C densitometer. The semiquantitative estimate of each isoenzyme was calculated from the height of each peak by integration (Hewlett-Packard Integrator, model 3390A, Palo Alto, CA). The relative contribution of V, was equally distributed
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DOBUTAMINE
AFTER
MYOCARDIAL
INFARCTION
IN
H475
RATS
between the V1 and V3 peaks to reflect the myosin heavychain isoform distribution. Statistical analysis. Results were submitted to analysis of variance. The mean square error within groups was then used in a Newman-Keuls multiple comparison test to evaluate differences between hearts from any two groups (32). Significance is reported at the 0.05 level.
chronic drug therapy, myocardial infarction, or CHF. In animals without MI, total heart and left ventricular mass were increased by treatment with dobutamine. MI in the presence of CHF significantly increased the weights of both ventricles. Scar weight was greatest in the animals with CHF, and because noninfarcted ventricular mass was equivalent in all infarcted groups the degree of hypertrophy of the noninfarcted muscle was probably greatest in these animals as well. Dobutamine did not RESULTS cause additive left ventricular hypertrophy in the inThe acute hemodynamic responses to bolus injections farcted animals and also did not appear to influence of dobutamine in sham and infarcted animals are shown infarct size. Of note, animals whose lung weights defined in Table 1. Marked increases in both heart rate and dP/ them as having CHF included those with 11 of the 12 dt were seen in both groups 5 min after drug administralargest infarcts as determined by scar weight. tion, whereas peak left ventricular pressure increased to Figure 1 shows data obtained from the isolated peronly a modest degree. The magnitude of these responses fused heart studies. Data are presented at both moderate in the sham and MI groups was statistically similar. End(10 mmHg) and high (20 mmHg) end-diastolic pressures. diastolic pressure was higher and peak systolic pressure Hearts from dobutamine-treated noninfarcted animals and dP/dt were slightly lower in the infarcted animals. did not show any change in peak left ventricular pressure Those animals with the largest infarcts had higher end- or in positive or negative dP/dt when contrasted with diastolic pressures and less marked increases in both left hearts from placebo-treated animals. Both Db and conventricular pressure and dP/dt in response to dobutatrol hearts from animals subjected to myocardial infarcmine injections, although the chronotropic response to tion not complicated by CHF displayed mechanical functhe drug did not appear to be affected by infarct size. tion similar to controls. The slight reduction in peak left The duration of the drug’s action was equivalent in sham ventricular pressure seen in the Db-MI group did not and MI animals. attain statistical significance. In contrast, those animals Table 2 shows heart and body weight data among the with CHF did have significant impairment in all three various experimental groups. All body weights were sim- measures of mechanical function. Left ventricular volilar and were not affected by the presence of either ume measurements were not systematically obtained and therefore pressure to volume comparisons between the TABLE 1. Acute hemodynamic responses groups cannot be made. to dobutamine administration Table 3 shows data for myocardial oxygen extraction, MVoZ, and lactate production from the isolated perfused Heart Rate, EDP, LVP, dP/dt, Groups hearts at the higher end-diastolic pressure. Control valbeats/min mmHg mmHg mmHg/s ues for 0, extraction, MOON, and lactate production were Sham 0.75 ml/ml 02, 1.37 ml 02*g-1.min-1, and 11.6 ~rnolg-‘. 5,724+514 Initial 220t17 9-+3 114&5 min-l, respectively. Data are presented as a percentage 15,579+808* 5 Min 320&14* 3-+1* 133t7* of control values and, in the case of MVO, and lactate 11,941+743* 60 Min 292t20” 7&2 134t6” MI production, are normalized for viable left ventricular 101t9 4,375+640 Initial 209k9 18+3”r weight. Oxygen extraction decreased in the infarcted 111t14 10,113+2,447* 5 Min 336t25” llt3*? hearts in proportion to the loss of myocardium so that 109+llt 8,503+2,405* 60 Min 301t33” 14tlOt the hearts from animals with CHF, which for the most Data are means t SE. Comparisons were made before, and 5 and 60 part sustained larger infarctions, tended to have lower min after injections of 2 mg/kg SC of dobutamine. EDP, end-diastolic pressure; LVP, left ventricular peak systolic pressure. * P c 0.05 vs. values irrespective of concurrent drug therapy. MVo2 was slightly increased in the animals with MI but was initial; “r P < 0.05 vs. sham. TABLE
2. Body and heart weights MI Weights
Sham
(14)
-CHF Body,
g
Dry heart, mg Dry LV, mg Scar, mg % LV scar, mg/mg Dry viable LV, mg Dry RV, mg Dry/wet (muscle) Dry/wet (scar) Wet lung, g
260&3 149t6 118*5
261&3 168t6* 130t5
31t2 0.16t0.01
38t2* 0.17t0.01
1.12t0.05
Data are means t SE; values in parentheses MI, myocardial infarct; CHF, congestive heart +CHF.
MI-Db
Db (14)
1.14t0.04
264-t4 140tll lllt8 15&2 14t2 96t8 29*3 0.14~0.01 O.lOtO.O1 0.94kO.06
(9)
+CHF
(5)
262-+4 182&17t$ 139+15?$ 36+4$ 28+5$ 103*17 43+4Q 0.15*0.01 0.09t0.01 2.31+0.14$
-CHF
260&2 159t6 122t6 22&4 17*3 102*6 31&4 0.15t0.01 O.lltO.O1 1.02t0.06
(8)
+CHF
(6)
259t4 162klO 116t7 17t2 24+5$ 90t6 46+3-t* 0.14t0.01 0.08t0.01 2.28+0.12$
are no. of animals. LV and RV, left and right ventricle, respectively. Db, dobutamine-treatment; failure; * P < 0.05, sham vs. Db; “f P < 0.05, sham vs. MI or Db vs. MI-Db; $ P < 0.05, -CHF
vs.
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DOBUTAMINE
H476
AFTER
MYOCARDIAL
PEAK LV PRESSURE
INFARCTION
IN
RATS
PEAK 0 dP/dT
PEAK 0 dP/dT
240
180 $ 160 E E 140 120 100
)------I o------(> M FIG. 1. Cardiac mechanical data obtained from isolated isovolumic perfused end-diastolic pressures. Values are means t SE of 5-14 studies. Abbreviations sham vs. MI or Db vs. MI-Db; O P c 0.05, CHF vs. no CHF.
TABLE
m -----a e-----o e-----a
Sham MI-CHF MI+CHF
hearts at moderate (M) or high (H) as indicated in Table 1. ‘P < 0.05,
3. Indexes of myocardial metabolism MI Sham
(14)
O2 extraction MVo, Lactate production
1.00t0.04 1.00~0.11 1.00~0.18
l.Ol-+-0.03 1.01t0.07
0.86t0.08
I~I SE and are expressed as a fraction * P < 0.05 vs. sham; t- P c 0.05, -CHF vs. +CHF. are
means
MI-Db
Db (14) -CHF
Data
Db MIDb -CHF MIDb+CHF
of sham
(9)
1.00t0.05 1.23t0.10
1.55t0.17* values;
not further increased in the animals with CHF, which probably reflected the small increases in coronary blood flow seen in these groups (19% in the MI and 12% in the MI-Db groups with CHF). Parallel changes in lactate production were seen. The metabolic responses of the animals treated with Db did not differ from their matched nontreated cohorts. Figure 2 shows the results of contractile protein analysis. Dobutamine increased calcium-activated myosin ATPase activity in control animals. This increase was not reflected by a measurable shift in isomyosin distribution probably because the %V1 myosin in the control animals was >95% and further increases in this isoform were beyond the limits of detection. Myocardial infarction markedly depressed ATPase activity and shifted the isomyosin distribution towards VS. These biochemical alterations occurred regardless of whether the infarction produced CHF or not. The effect of myocardial infarction on ATPase activity was completely prevented by dobutamine if the infarct was not associated with CHF and was partially prevented even when CHF was present. In the MI-Db group, calcium-activated myosin ATPase activity was moderately depressed in animals with CHF but was near normal in those without failure. Overall, the V, isomyosin content in the Db-MI group was 90% and was not statistically different from either controls
values
+CHF 0.69&0.05-j. 1.15t0.20
1.42t0.21 in parentheses
(5)
-CHF 1.00-c-0.08 1.26rtO.16
1.08t0.10 are no. of animals.
(8)
+CHF
(6)
0.73t0.10 1.38t0.11 1.94+0.28*? Abbreviations
as in Table
1.
or noninfarcted dobutamine-treated animals. A trend toward a depression in the V1 isoform in MI animals with CHF was also observed both in the presence and absence of dobutamine, but inadequate band separation on some of the pyrophosphate gels in these groups preeluded meaningful statistical comparisons. DISCUSSION The present study was conducted to assessthe utility of chronic intermittent dobutamine treatment on the evolution of myocardial infarction in the rat. This agent and mode of administration were studied because it is widely employed in the clinical management of CHF and it has been previously shown in rats to increase both myosin ATPase activity and %V1 myosin (4) and therefore might enhance the force-generating capacity and the velocities of contraction and relaxation in failing hearts with depressed myosin ATPase activities. A paradigm for this is the physiological cardiac hypertrophy seen after exercise conditioning in which myosin ATPase and the velocity of contraction are increased (26). Myocardial infarction was created using standard techniques and a spectrum of left ventricular damage was created, which is consistent with the experience of others (8, 10, 24). Infarct size was estimated by scar
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DOBUTAMINE
Ca++ -activated
AFTER
MYOCARDIAL
INFARCTION
IN
Myosin
H477
RATS
Myosin
Isoenzymes
ATPase
13 . 12 .
80
11 . + 2I 1.0 \F c .9 z =t .8
g
60 .... ... .... ... . . . . .. . ..... .. .... ... . . . . . .. . .. . .. . ..... .. .... ... . . . . .*.*.*.*. . . . . . ..a. . .. . .. . ...a . . .*.*.*.* . . . .*.*.-.*. . . . .. . .. . . ..-.*.-....... . ...... . ...... . . . . . . .. . .. . .. .
. . . . . . . . .
. . . . . . . . . .
. .. . .. . .. . .. .. .. .
*7 , 1
/ /
Sham
Db
+CHF
-CHF
MI FIG.
sham
2. Contractile protein vs. Db or MI vs. MI-Db;
+CHF
t-l
-CHF
anam
Db
MI
MIDb
MIDb
data. Values are means t SE of 4-14 studies. Abbreviations + P < 0.05, sham vs. MI or Db vs. MI-Db; o P < 0.05, CHF
weight, which most likely underestimates the mass of the ventricle that is infarcted (25). Pfeffer et al. (24), for example, reported the development of overt CHF in rats with an infarct size estimated by planimetry of >45%. Approximately one-third of the total animals infarcted in that study were in this group. We also found that CHF developed with a similar frequency, whereas scar weight was -25% of total left ventricular weight. This crude correlate of infarct size was used because of the need to carefully preserve the hearts for con tractile protein analysis. The infarct sizes in th e groups not treated and treated with dobutamine were comparable, and, strikthe mass of the hypertrophied, noninfarcted musWY, cle wa.s similar in all i.nfarcted groups. Because the mass of muscle lost in the animals that developed CHF was probably larger, a greater degree of ventricular hypertrophy in these animals is also likely. This is consistent with the careful morphometric studies of Anversa et al. (1). The apparent exaggerated hypertrophic response of these animals was inadequate in maintaining cardiac compensation, suggesting a limitation to the degree of adaptive hypertrophy available to overloaded muscle. The apparent increase in mass in the MI-CHF group might reflect the relatively small number of animals in this group, although it does not countermand this hypothesis. The increase in ventricular mass after myocardial infarction might have masked a dobutamine-induced hypertrophy in these animals. This has been previously suggested using other hemodynamic stimuli to ventricular hypertrophy (29, 31).
as in Table vs. no CHF.
1. * P < 0.05,
The major finding of the pharmacological portion of the study is that dobutamine treatment results in preservation of normal myosin ATPase activity and myosin heavy-chain isoform distribution in infarcted hearts without any apparent improvement in cardiac performante. In .farction resulted in a 43% decrease in V1 myosin and an -25% depression in calcium-activated myosin ATPase activity, regardless of whether or not the infarct was associated with heart failure. Concurrent treatment with dobutamine prevented the shift in isomyosin distribution and ameliorated the depression in ATPase activity associated with myocardial infarction. The fact that dobutamine totally corrected the ATPase activity in the absence of CHF but only partially corrected it in the presence of CHF suggeststhat the load on the remaining myocardium was greater with CHF and thus ,&myosin heavy-chain gene expression was more active in this setting and could not be overridden by adrenergic stimulation with dobutamine. Lactate production in the isovolumic perfusion apparatus at high end-diastolic pressure was increased in the MI group both with and without CHF, whereas in the MI-Db group normal effluent lactate concentrations were observed except in those animals with extensive infarction, in which lactate production was increased to a degree equivalent to that seen in the MI plus CHF group. This argues against myocardial ischemia during perfusion as an explanation for the lack of any dobutamine-associated improvement in performance. The data also suggest that shifts in the lactate dehydrogenase
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H478
DOBUTAMINE
AFTER
MYOCARDIAL
isoenzyme system might have been induced by the hemodynamic overload (15) and partially ameliorated by dobutamine administration, a situation which would be analogous to that seen with the myosin isoenzymes. The hemodynamic data obtained in the isovolumic perfusion apparatus is consistent with the work of others in that moderately sized infarctions were not associated with altered baseline hemodynamics. It was not until a substantial portion of the ventricle was scarred that depressed left ventricular performance was observed (3, 14). The failure of dobutamine to improve the pressuregenerating capacity of the myocardium is consistent with our previous work (4) and likely reflects a true disassociation between myosin enzymology and contractility, although this might reflect the insensitivity of the perfusion system to detect subtle changes in muscle performance. Because left ventricular end-diastolic volumes were not measured, however, any judgment about force generation must be made with caution. It is conceivable that dobutamine prevented ventricular dilatation postinfarction and thus the Db-MI groups were able to generate similar peak left ventricular pressures from a reduced left ventricular end-diastolic volume when contrasted with MI animals. This would represent a true increase in contractility. The relationship between adrenergic stimulation and isomyosin gene expression is not unique to the present study. Simpson and his colleagues (28), studying neonatal myocytes in culture, have shown that cul-adrenergic stimulation selectively enhances ,&myosin heavy-chain gene expression. More recently, Kawana et al. (16) have shown that ,6-adrenergic stimulation increases the %V1 myosin in denervated rabbit hearts, a result which is more concordant with our own data. A possible mechanism by which catecholamines (and specifically ,&agonists) might mediate these effects is the recent demonstration that cardiac myosin gene expression can be influenced by an adenosine 3’,5’-cyclic monophosphateresponsive element in the a-myosin heavy-chain gene promotor region (13). A potential criticism of this study might be that the infarcted animals with and without failure and the controls did not receive pharmacologically similar doses of the drug. The acute hemodynamic data (Table 1) make this unlikely as the heart rate response to injections and the duration of drug action were similar in sham and MI groups. What is likely, however, was that those animals with large infarcts were unable to generate equivalent peak left ventricular pressures and shortening velocities that might have altered the magnitude of the hemodynamic stimulus associated with drug administration. The lack of an apparent hemodynamic effect of dobutamine in the present study must also be viewed in light of the work of Pfeffer and co-workers (24) and others (9, 11) who have studied the effects of various pharmacological treatments on the evolution of myocardial infarction in the rat model. As described by these workers, the natural history of infarction is one of gradual ventricular dilatation (in proportion to infarct size) associated with a reduction in chamber stiffness and a decrease in maximal ventricular performance. These adaptations can be ameliorated by reducing preload and afterload with drugs
INFARCTION
IN
RATS
such as captopril. Gay et al. (11) have shown a modest improvement in in situ hemodynamics postmyocardial infarction with thyroxine treatment independent of the effects of thyroxine on isomyosin distribution, which may also reflect effects on the peripheral circulation. The present study did not assess ventricular performance in situ but the vasodilatory effect of dobutamine (18) raises the possibility that this agent might exert a beneficial effect on postmyocardial infarction ventricular remodeling independent of the direct cardiac effects of the drug. The authors thank Alwyn Murphy and Kirit Pate1 for technical assistance and Janice Brewton for secretarial help. This project was supported in part by National Heart, Lung, and Blood Institute Grant HL-15498. P. Buttrick is the recipient of a Clinical Investigator Award, HL-01552. Address for reprint requests: P. Buttrick, Div. of Cardiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 E. 210th St., Bronx, NY 10467. Received
10 April
1990; accepted
in final
form
18 September
1990.
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