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a Division of Kinesiology, The University of Michigan, Ann Arbor.
b Section of Sport and Exercise Sciences, The Ohio State University, Columbus
c Department of Psychiatry, Duke University, Durham, North Carolina.
d College of Health and Human Performance, Oregon State University, Corvallis.
Marvin O. Boluyt, Laboratory of Molecular Kinesiology, The University of Michigan, 401 Washtenaw Avenue/1209 CCRB, Ann Arbor, MI 48109-2214 E-mail: boluytm{at}umich.edu.
Jay Roberts, PhD
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Abstract |
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10%) in the hearts of all three groups of aged rats compared with the adult rats that was unaltered by AC. The percentages of LV myosin heavy chain in the 
-isoform were Cadult, 77%; AC-7adult, 66%; AC-28adult, 66%; Caged, 45%; AC-7aged, 41%; AC-28aged, 32% (age, p < .01; AC, p < .01). Coronary flow per gram of tissue averaged 9% lower in all three of the aged groups compared with the adult rats and was not significantly affected by AC (age, p < .05). The data suggest that a reduction in nonmyofibrillar protein and a reduced coronary flow, rather than changes in calcium sensitivity or myofibrillar protein, are associated with an impairment in the adaptive response of the aged heart. CARDIAC events with pathological implications increase in an exponential manner with advancing age (1). Studies with laboratory rats suggest that an impaired adaptive response of the heart to stress may be one factor contributing to this increased incidence (2). Although some studies indicate an attenuation in the magnitude of myocardial hypertrophy with increased load (3)(4), others have suggested an impairment in function associated with the adaptive response (5)(6). The mechanisms that contribute to the age-associated impairment of the adaptive response are not completely understood. Furthermore, it is not clear whether the age-associated alterations in adaptive response that occur in female rats (6) are simply delayed or whether they persist beyond 7 days after the imposition of a growth challenge.
In response to a pressure-overload challenge, the heart must enlarge to maintain or augment functional capacity. This involves modifying the protein synthesis/degradation balance in favor of protein accumulation. In particular, the concentration of myofibrillar proteins that account for force generation by the myocardium must be maintained in proportion to other proteins if function is to be augmented. In some instances, however, the concentration of myofibrillar proteins is not maintained in the hypertrophic heart. The observation that myofibrillar protein concentration is decreased in patients with heart failure (7) suggested that a reduction in myofibrillar protein concentration might be one factor contributing to the reduction in pressure-generating capacity previously observed in aged compared with adult rats after aortic constriction (AC) (6).
The force-generating capacity of the heart is dependent on calcium signaling to activate the myofibrillar contractile proteins by means of interactions with the regulatory proteins. When the heart is growing and adapting to a pressure-overload stimulus, there is a potential for disruption of the normal signaling by means of excitation–contraction coupling. For example, reduced sensitivity to extracellular calcium was observed in isolated hearts with chronic myocardial infarction (8) and in papillary muscles of aged rats with atrioventricular block (4). Altered excitation–contraction coupling was also observed in the hypertrophic hearts of cardiomyopathic hamsters, as reflected in a reduced augmentation of pressure development to increasing levels of calcium (9). Because a number of membrane proteins that influence calcium fluxes are altered as a function of age and hypertrophy (10)(11), it seemed likely that the sensitivity of the myocardium to extracellular calcium might be reduced in hearts of aged rats with AC, thus contributing to an impaired adaptive response.
When the heart enlarges, angiogenesis must occur or there will be a dilution of capillary density. Dilution of capillary density would result in a reduction in maximal blood flow per unit myocardium. Under circumstances in which maximal coronary flow is necessary for the heart to respond to a hemodynamic challenge, a reduced capillary density or flow per unit tissue would impair function by depriving the working myocardium of optimal levels of metabolic substrates and/or oxygen. There have been numerous reports of reduced coronary flow and/or capillary density associated with cardiac hypertrophy (12)(13)(14) and advancing age (15)(16). It has been suggested that a similar reduction may occur in human patients who exhibit symptoms of myocardial ischemia in the absence of detectable arterial obstructions (14). Flanagan and co-workers (17) report a marked impairment in coronary conductance induced by aortic stenosis of adult sheep, whereas 10-week-old lambs exhibited little or no impairment after the same procedure. Together these observations suggest that reduced coronary blood flow may occur in hearts subjected to pressure overload and that this reduction may be exacerbated by advancing age.
The purpose of this investigation was to examine possible mechanisms of the previously observed age-associated impairment in rat heart function following pressure overload hypertrophy and to determine the age-associated differences in adaptive response that persisted as long as 28 days after the imposition of pressure overload. The following hypotheses were tested: Compared with hearts of adult rats, hearts of aged rats subjected to aortic constriction for 7 and 28 days would (a) have a lower concentration of myofibrillar proteins, (b) exhibit a reduced sensitivity to extracellular calcium, and (c) have a reduced coronary perfusion per gram tissue.
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Methods |
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and 26-month postpartum 
. Rats were maintained on food (Purina Lab Chow #5001) and water ad libitum, housed 2–3 per cage in a barrier facility and kept in a 12-hour:12-hour light:dark cycle. Rats were 9 months to 10 months old (i.e., adult) or 27 months to 28 months old (i.e., aged) at the time of study and were divided into either control or AC groups. Eight aged rats (1 control and 7 experimental) developed small subcutaneous tumors on the abdomen, the chest, or a limb. One rat had obvious neoplastic disease throughout the abdomen and was excluded from the study. Seven of the rats with small tumors appeared otherwise healthy and showed no signs of generalized neoplastic disease at autopsy; thus data from these rats were included in the study. Two rats from the aged cohort died before they were used, and two others died of pathologies during thoracotomy, and the data of one were excluded because its body mass (BM) had dropped by more than 30%. Data of two adult rats were excluded because of technical complications. AC was performed as described by us previously (6) and as described originally by Nair and co-workers (18) and modified by Cutilletta and co-workers (19). A left thoracotomy was performed under ketamine (87 mg/kg) and xylazine (13 mg/kg) anesthesia and the ascending aorta was exposed. A Weck hemoclip was placed around the aorta 4–6 mm superior to the aortic valve, with the gap of the Weck hemoclip applicator set at 1.12 mm. Pleural pressure was reinstated and the wound closed with 5–0 silk suture. Postoperative mortality within 1 day was 8% and 7% for the adult and the aged groups, respectively, with no deaths thereafter.
Functional studies.
Experimental hearts were studied 7 and 28 days following AC as 100% of the adaptive growth occurs by 28 days (3)(18). Hearts were extricated from the animals under ketamine–xylazine anesthesia and studied in the isolated working heart preparation under standard conditions (20)(21). The initial perfusate was a 95% O2–5% CO2-saturated Krebs–Henseleit bicarbonate buffer containing insulin (12 U/ml) and the following: NaCl (118.5 mM), KCl (4.7 mM), MgSO4 (1.2 mM), KH2PO4 (1.2 mM), NaHCO3 (24.7 mM), CaCl2 (3.0 mM), Na2EDTA (0.5 mM), glucose (11 mM), and pyruvate (0.2 mM). The perfusate calcium concentration was subsequently modified when Na2EDTA or CaCl2 was added to achieve the desired level (8). Preload was determined by the height of the atrial filling reservoir and afterload by the height of the aortic outflow tube. Peak afterload was achieved by the complete occlusion of the aortic outflow tube with a clamp.
Antegrade flow was initiated following a 10-minute retrograde washout period. The preparation equilibrated for an additional 10-minute period during which hearts were paced at 5 Hz with right atrial electrodes and the level of the perfusate in the atrial filling reservoir and aortic outflow tube set at 10 and 80 cm above the heart, respectively. Subsequently the workload was varied by altering preload and/or afterload. Each workload was maintained for a minimum of 3 minutes before measurements were recorded. Each heart served as its own control for stability under standard conditions, as at various points the parameters were returned to the initial baseline value. Data were discarded from hearts that exhibited a decline in peak systolic pressure of >10% at this workload.
During the initial equilibration period, a 20-gauge needle attached to a Statham P10EZ pressure transducer was introduced into the left ventricular chamber through the apex of the heart. The transducer was connected in series to a Honeywell Accudata 113 bridge amplifier and a Hewlett-Packard (Model 17402A) chart recorder. A parallel connection joined the amplifier to a Zenith ZW-158-43 personal computer. A program was written with Asyst software to record desired pressure-curve data. The program averaged the data of five pressure curves per measurement. The pressure transducer was calibrated with a column of mercury at the beginning and the end of each experimental session. The effluent that dripped from the heart was collected as coronary flow, and cardiac output was determined by the addition of coronary flow values to those obtained from the aortic outflow tube. Systolic and diastolic pressures and positive and negative dP/dtmax were determined from recordings made at a paper speed of 125 mm/s.
Following functional measurements, hearts were removed from the perfusion apparatus, dissected in iced saline, and weighed. Portions of the right and the left ventricular free walls and septum were used for protein concentration determinations, and other portions of these regions were frozen for subsequent electrophoretic analysis.
Biochemical assays.
With bovine serum albumin used as a standard, the concentration of protein was determined as described by Lowry and co-workers (22) in portions of the left ventricular freewall. Protein determinations were made on crude muscle homogenates and subsequently on a refined preparation of myofibrils from each sample. Myofibrils were prepared with Triton X-100 (23), as described by Baldwin and co-workers (24). Plasma urea nitrogen was determined with Sigma Kit # 535 essentially as described by Crocker (25).
Electrophoresis.
With denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis, a direct determination of the relative proportions of -MHC and ß-MHC (MHC is myosin heavy chain) was performed as described earlier except that 5% nongradient gels were used instead of 4%–9% gradient gels (26). Tissue samples of the right and the left ventricular free walls were homogenized (1:10, w/v) in 62.5-mM tris(hydroxymethyl)aminomethane buffer, pH 6.8. After an aliquot was removed for determination of protein concentration (22), the remaining homogenate was combined with an equal volume of concentrated buffer, yielding a 1:20 tissue homogenate. Standards of known relative quantities of - and ß-MHC were electrophoresed concurrently with experimental samples. Gels were scanned on an LKB Ultroscan XL laser densitometer (Bromma, Sweden; Model #2202) and the - and ß-MHC peaks obtained were quantified by an IBM AT microcomputer and Gelscan software (Version 1.2; LKB, Bromma, Sweden).
Statistics.
Data are expressed as means ± standard error (SE). A two-factor analysis of variance (ANOVA) was used to analyze the data for main effects of age and AC and for interactions between these two variables (27). Where appropriate, a three-factor ANOVA with repeated measures on the third factor was used to examine the interactive effects of age, AC, and multiple measurements on the same rat or heart. The Tukey procedure was used to compare individual means and a p value of 
.05 was considered statistically significant.
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To determine whether aging was associated with a deficit in the adaptive response, pressure-generating capacity was studied in vitro. With preload and afterload maximized, a significant age-associated difference 
in peak systolic pressure was observed after AC (Table 2 ). End-diastolic pressures did not differ because of age (not shown). These findings are similar to a previous study (6) and suggest that the age-associated impairment in left ventricular function is not restored at 28-day post-AC.
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To determine whether the reduced pressure-generating capacity of the aged female rat heart after AC was due to a decrease in contractile units, myofibrillar protein fractions were measured and compared among the groups. Myofibrillar protein concentration was not altered by aortic constriction and did not differ because of age (Table 3 ). There was, however, a lower concentration of nonmyofibrillar protein in the hearts of the aged rats compared with that of the adult rats. There were no differences that were due to AC, nor were there any interactions between age and AC for either total protein or myofibrillar protein fraction. Thus the ratio of myofibrillar protein to total protein was increased in the hearts of the aged rats.
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-MHC and ß-MHC were measured by electrophoretic separation of the dissociated heavy chains. The well-known effect of age to reduce the proportion of -MHC was evident in the control groups. AC decreased the proportion of left ventricular -MHC by the same amount in rats of both ages (Table 3 ), such that the age-associated differences were preserved after AC. To determine whether the age-associated impairment in adaptive response might be attributed to changes in calcium sensitivity, the calcium concentration in the perfusate was varied between 1 and 3 mM, and peak systolic pressure was measured at each of four calcium concentrations. There was no difference associated with age and no effect of AC at either age on sensitivity of left ventricular function to extracellular calcium (Fig. 1). This was true whether the dependent variable tested was aortic flow (data not shown) or peak systolic pressure and was consistently observed at both low (not shown) and high workloads.
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; three-factor ANOVA with repeated measures for workload). There was no significant effect of AC on coronary flow, and there were no interactive effects of age with AC, workload, or both AC and workload on coronary flow.
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Discussion |
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The adaptive response to increased hemodynamic loading conditions is an important feature of the mammalian heart, enabling the organism to survive and thrive in response to a challenge. A reduction in the magnitude of hypertrophy in response to a similar increase in either pressure or volume load has been observed (29). In contrast to those experiments in which the stimulus was held constant, the present study involved conditions that produced an equivalent magnitude of hypertrophic growth in adult and aged rats. In the context of a hypertrophic response of similar magnitude in young and old rats, we evaluated the adaptation in terms of its functional consequences. The data regarding left ventricular function after AC are similar to and extend previous work that demonstrated a significant age-associated impairment in the functional adaptation after 7 days of AC (6). In the present study the data suggest that this impairment is not restored by 28 days after AC in the aged rats (Table 2 ). Although small, this age-associated impairment could have important implications for survival in cases in which the adaptive response of the heart is crucial, as is the case, for example, after myocardial infarction.
It was hypothesized that a reduction in the relative amount of force-generating proteins might underlie the age-associated impairment in pressure-generating capacity. The present results, however, demonstrate that myofibrillar protein concentration was maintained with both age and AC, whereas nonmyofibrillar protein was depressed in the left ventricles of the aged rats. The loss of nonmyofibrillar protein with age may reflect a loss of proteins such as cytochrome c and the Ca2+ ATPase of the sarcoplasmic reticulum that have been reported to decrease in concentration with age or AC (30)(31)(32)(33). It is noteworthy that the Lowry protein assay detects aromatic amino acids only; thus it would not be expected to detect connective tissue proteins that have been shown to increase with age (34)(35). Whether or not the reduction in nonmyofibrillar protein observed is causally related to the functional deficits observed in the hearts of aged rats cannot be inferred from the present data. Future studies should use protein-detection methods that circumvent this limitation as well as methods to evaluate the levels of connective tissue protein.
In previous work by others, total protein concentration and dry:wet weight ratios of cardiac muscle were reported not to change with age (4)(31), whereas volume fractions of mitochondria and myofibrillar proteins were reported to be unchanged with age through the use of an electron micrographic approach (36). Gender differences between the present study and previous studies are unlikely to account for the different results, because we have noted a similar decrease in nonmyofibrillar protein concentration between 12 and 29 months in hearts of male Fischer 344 rats (authors' unpublished results) by using both the Lowry (22) and the biuret protein assays. It may be that the use of multiple groups at each age in the present study provided an increased sensitivity to detect relatively small differences that might have otherwise gone undetected.
The gene coding for the isoforms of MHC are sensitive to influences of hormones (37)(38)(39)(40), physical activity (41)(42), and chronic loading conditions (43)(44). Both age and pressure-overload hypertrophy are associated with a decrease in the proportion of -MHC and a corresponding increase in the proportion of ß-MHC (39), and the alteration is proportional to the magnitude of hypertrophy (43)(44). It is interesting that the alteration in MHC profile concomitant with a 15% hypertrophic response was not altered with age. The decrease in the proportion of -MHC typically observed with pressure-overload hypertrophy was evident in the present study and was similar in magnitude in rats of both ages, indicating that the factors regulating MHC gene expression retain their sensitivity to chronic pressure overload.
Because it had been shown that sensitivity of the rat myocardium to extracellular calcium was reduced after myocardial infarction in younger rats or after atrioventricular blockade in older rats (4)(8), we hypothesized that hearts from rats of advanced age undergoing AC might also be associated with a decreased sensitivity to extracellular calcium. From the data in Fig. 1, this hypothesis was rejected. Inasmuch as both myocardial infarction and atrioventricular block result in a pattern described at least in part as volume overload hypertrophy (4)(45), they differ from hypertrophy associated with aging and AC which are both variations of pressure overload (46). The magnitude of hypertrophy associated with age and superimposed on age by AC in the present study was relatively mild. Thus it is possible that under more strenuous conditions of pressure loading, calcium sensitivity might have been altered.
The small age-associated difference in coronary flow is in agreement with the findings of others (15) and may result from a dilution of capillary concentration that accompanies age-associated hypertrophy (16). In a study that involved a greater magnitude of pressure-overload hypertrophy (45%) than that observed in the present study, hearts of minipigs exhibited a decrease in the numerical density of capillaries in the endocardium (13). In the present study, however, there was no significant effect of AC on coronary flow. Whether or not the age-associated decrease in coronary perfusion capacity contributes causally to the age-associated impairment in adaptive response cannot be determined from the present data.
Overall, the adaptive response of the heart to pressure overload was remarkably preserved with age in the female Fischer 344 rat. Hearts of aged rats demonstrated a capacity for hypertrophy in response to AC with a small, but significant, negative impact of age on the adaptive change in left ventricular function. The data suggest that a reduction in nonmyofibrillar protein and a reduced coronary flow, rather than changes in calcium sensitivity or myofibrillar protein, are associated with the impairment in the adaptive response of the aged heart. The reduction in the MHC profile observed with hypertrophy of the left ventricle was similar in rats of both ages, indicating that factors regulating gene expression retain their sensitivity during aging to alterations in chronic hemodynamic loading conditions.
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Acknowledgments |
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Received January 11, 1999
Accepted December 29, 1999
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; age x aortic constriction, 
; age x perfusate calcium concentration, 
; aortic constriction by perfusate calcium concentration, 
; age by aortic constriction by perfusate calcium concentration, 
.
; age x workload, 
; aortic constriction x workload, 
; age x aortic constric-tion x workload, 
.