INTRODUCTION

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AGING IN HUMANS is associated with changes in the cardiac muscle characterized by increased myocyte size and decreased sensitivity to -adrenergic stimulation (reviewed in Refs. 15 and 16). In addition there is a prolongation of contraction produced, at least in part, by an increase in the time course of the diastolic relaxation. A decrease in the myocardial sarco(endo)plasmic reticulum content has been reported in humans (8), which contributes to the prolongation of contraction by slowing the removal of Ca²⁺ following contraction. To be a useful model of human aging, an animal model must exhibit age-related changes similar to those seen in humans. Therefore, an animal model of human cardiac aging must also display a prolongation of cardiac contraction with age. Rats appear to fulfill this requirement and are widely used to study aging. In aged Wistar and Fischer 344 rats, the time to peak tension and peak shortening is increased and the duration of contraction is prolonged (4, 12, 22). This is likely due to a decrease in the rate of Ca²⁺ uptake by the sarcoplasmic reticulum produced by a decrease in either the Ca ATPase content (26) or a decrease in the Ca ATPase activity (13). There is also a shift in the myosin heavy chain (MHC) isoform in rats from -MHC to the slower -MHC, which is also expected to contribute to a prolongation of contraction. The tension-pCa relation remains unchanged (7).

The difficulty with using inbred rat strains to study aging is the high incidence of pathology in these strains that occurs with advanced age. For example, Fischer 344 rats frequently exhibit chronic nephropathy (19, 18). These pathologies become important when studying aging in cardiac muscle because they can cause changes, such as increased vascular resistance or changes in blood composition, which secondarily lead to altered cardiac function. Thus the presence of pathology makes it difficult to separate the effects of aging from the effects of age-related disease. Because the varying genetic background of different rat strains can produce widely varying responses to experimental interventions, it is important that the physiological and/or pathophysiological performance of any given rat model be established. The Fischer 344 x Brown Norway F1 hybrid rat (F344xBN) has recently become a popular model for aging due to the decreased incidence of pathologies in this strain, leading to a significantly increased longevity in comparison with other rat strains (18). Although no differences in terms of cardiac function have yet to be reported between the F344xBN and other rat strains, there have been no published accounts of the effects of aging on intact single cardiac myocytes in F344xBN rats. This is a significant gap in the aging literature because of the central role the heart plays in determining the overall health of the animal. We therefore evaluated the contractile properties of cardiac myocytes isolated from these F344xBN rats to test the hypothesis that decreased cardiac function is a primary result of the aging myocardium and not a secondary consequence of noncardiac pathologies. Female rats were used because they show a somewhat lower amount of cardiomyopathies than male F344xBN rats (18), and female rats also do not undergo the large increase in body weight observed in aging males. Because the contractile properties of the heart are determined at the cellular level, the isolated cardiac myocyte preparation was used. This is appropriate because isolated myocytes have been shown to retain the contractile properties of the bulk cardiac muscle (e.g., Ref. 10) but lack the confounding effects of the intercellular attachments and uncertain myocyte orientations that make mechanical measurements in the intact heart difficult. We examined the effect of aging on the Ca²⁺ transient and subsequent sarcomere shortening in isolated intact myocytes and the tension-pCa relation in skinned myocytes from the F344xBN rat. To our knowledge, this is the first report of the effect of aging on sarcomere shortening and the Ca²⁺ transient in cardiac myocytes from F344xBN rats. Furthermore, it is well established that ischemic conditions result in a significant decrease in the intracellular pH of cardiac myocytes and that older rats are more susceptible to the effects of ischemia (1, 2, 5). Therefore, the tension-pCa relation was also examined in cardiac myocytes from young and old F344xBN rats under both normal and acidic (pH 6.2) conditions to determine whether the age-related susceptibility to cardiac ischemia is partially determined by a differential response to acidosis in the tension-pCa relationship.

	METHODS

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Animals. Female virgin F344xBN rats were obtained from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN). Rats were kept in cages on a 12:12-h light-dark cycle and were allowed to acclimate for a minimum of 2 mo before use. Normal rat chow and water were provided ad libitum.

Myocyte isolation. Intact myocytes were isolated from rat hearts at 6 (young) and 32-33 (old) mo of age by enzymatic digestion as previously described (29). Briefly, the heart was removed from an anesthetized rat and perfused with Krebs- Henseleit buffer (KHB) + 1 mM Ca²⁺ for 5 min on a modified Langendorff perfusion apparatus. The heart was then perfused with Ca²⁺-free KHB for 5 min followed by the addition of collagenase (0.5 mg/ml) and hyaluronidase (0.2 mg/ml) for 15 min. The solution was then titrated to 1 mM Ca²⁺ and perfusion continued for 10-15 min. The ventricles were then minced and digested 2x for 10 min and 2x for 15 min in the enzyme solution. The last two enzyme digestions were collected by brief centrifugation and resuspended in KHB + 1 mM Ca²⁺ + 2% BSA, and the solution was titrated to 1.8 mM Ca²⁺ over 15 min.

SDS-PAGE. Cardiac myocytes (5-10) were placed in SDS sample buffer and stored at 80°C for analysis of protein content by SDS-PAGE as described previously (20). Gels for SDS-PAGE were prepared with 3.5% acrylamide in the stacking gel and 12% acrylamide in the running gel. Samples were loaded on the gel and the proteins were separated by electrophoresis at constant current (20 mA). Gels were fixed in 10% glutaraldehyde overnight, washed, silver stained, and dried between Mylar and cellophane sheets.

Ca²⁺ measurements. Ca²⁺ measurements were made using confocal microscopy with the fluorescent indicator fluo-3. Fluo-3 acetoxymethyl ester (AM) (50 µg) was dissolved in 10 µl of DMSO and diluted to 5 µM in KHB + 5 mM reduced glutathione (GSH) + 1.8 mM CaCl₂. Freshly isolated myocytes were plated on laminin-coated coverslips in DMEM + 5% serum at 1 x 10⁵ cells/ml. After 2 h the medium was replaced with serum-free DMEM, and the coverslips were kept in an incubator at 37°C until use later that same day. A coverslip containing the plated myocytes was then mounted in a custom-made chamber on a heated microscope stage. The coverslip formed the bottom of the chamber, and field stimulation was provided through platinum electrodes. After mounting was completed, the coverslip was washed briefly with KHB + 5 mM GSH + 1.8 mM CaCl₂. This solution was subsequently replaced with the fluo-3 AM solution for loading. Myocytes were loaded at 37°C for 10 min, and an additional 20 min in KHB were allowed for deesterification. Fluorescence was stimulated by an argon laser at 488 Hz and collected through a 500/25 barrier-pass filter. Images were collected at 480 Hz for later analysis as described previously (28).

Sarcomere length detection. The sarcomere length of freshly isolated myocytes in medium 199 + 5 mM GSH was followed using laser diffraction. Myocytes were placed in a temperature-controlled chamber with a glass bottom through which the output of a 10-mW HeNe laser was focused with an achromatic lens. Platinum electrodes provided a 5-ms square-pulse electrical field stimulation. A myocyte was placed into the beam, and the first-order diffraction pattern was focused onto a linear position detector (LSC 30D, UDT Sensors, Hawthorne, CA) using a cylindrical lens. The output of the detector was amplified and stored on a digital oscilloscope for later analysis. The response from 10 twitches was signal averaged for each myocyte. After the above protocol, the detector was removed and a screen was placed in the beam path. The sarcomere length was then calculated from the distance from the zero to first-order diffraction pattern. The sarcomere length was 1.81 ± 0.09 µm (n = 46) in myocytes from young rats before shortening and was not significantly different in aged rats. Although this initial sarcomere length is still within the physiological range of sarcomere lengths (14), the unloaded initial sarcomere length is likely to be somewhat shorter than the initial sarcomere length of myocytes in the intact heart, where myocytes are strained by the preload. However, the initial sarcomere length is the same in myocytes from both young and old rats, and therefore any sarcomere length dependence that may exist is eliminated.

Tension-pCa curves. Activating and relaxing solutions were prepared using the program of Fabiato (10a) and contained 7 mM EGTA, 14.5 mM creatine phosphate, 20 mM imidazole, and 4 mM MgATP, 1 mM Mg²⁺, and sufficient KCl to bring the ionic strength to 180 mM. Final pH was adjusted with KOH. Tension-pCa curves were constructed at 15°C as previously described (20). Briefly, single myocytes were permeabilized by brief exposure (~20 s) to 0.1% Triton X-100 and attached with silicone adhesive (Dow Corning) between a moving coil galvanometer and a force transducer (Cambridge Technology). At each pCa, tension was allowed to develop to a steady state, and the myocyte was then rapidly slackened to obtain the tension baseline. The myocyte was then relaxed at pCa 9.0. Total tension was determined as the difference between the developed steady-state and baseline tension. Active tension was then calculated by subtracting the resting tension, obtained by slackening the relaxed myocyte at pCa 9.0, from the total tension. Tension-pCa data were fit to the Hill equation in the form P = [Ca²⁺]ⁿ/(Kⁿ + [Ca²⁺]ⁿ), where P is the tension relative to maximum (pCa 4.0), K is the pCa at 50% tension (pCa₅₀), n is the Hill coefficient (n_H) and brackets denote concentration.

Data analysis and statistics. A Student's two-tailed t-test was used to determine significance between two groups. A probability level of P < 0.05 was used to indicate significance. Values are given as means ± SE.

	RESULTS

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Protein isoform expression patterns. SDS-PAGE followed by silver staining was used to evaluate the myosin isoform expression pattern and stoichiometry in myocytes from young (6 mo) and old (32-33 mo) female F344xBN rats. Densitometry of the protein bands was used to evaluate the amount of protein relative to the total MHC content. A well-established effect of aging in rats is a shift in the MHC isoform expression from -MHC to the more slowly cycling -MHC. As can be seen in Fig. 1, there was a substantial shift in MHC isoform with -MHC making up 27.6 ± 1.8% (n = 2) of the total MHC in young animals and increasing to 46.3 ± 3.0% (n = 5) in old animals. In addition, densitometry of the bands for actin and myosin light chains 1 and 2 revealed that there were no changes in the stoichiometry of these proteins, and no expression pattern changes in other contractile proteins were apparent from the gel.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Myofilament protein expression in myocytes from young (lanes 1 and 2) and old (lanes 3 and 4) rats. Silver-stained gel with 5-10 myocytes is represented per lane. Representative lanes from silver-stained SDS-polyacrilamide gel showing myosin heavy chain (MHC) (A) and lower molecular weight protein isoforms (B). Protein bands are identified for MHC (- and -MHC), actin, and myosin light chains (LC1 and LC2).

Tension-pCa relationship. We recently found that a shift in MHC isoform from -MHC to -MHC induced in hypothyroid Sprague-Dawley rats is accompanied by a rightward shift in the tension-pCa relationship (20). A similar, although less complete, shift in the MHC complement of cardiac myocytes was observed here with aging in F344xBN rats. Therefore, tension-pCa curves were constructed in permeabilized myocytes from young and old rats to determine whether a rightward shift similar to that seen in hypothyroid rats would occur (Fig. 2). At pH 7.0, adult myocytes isolated from young F344xBN rats showed a pCa₅₀ of 5.78 ± 0.02 (n = 7) versus the significantly lower pCa₅₀ of 5.66 ± 0.03 (n = 13) observed in myocytes from old rats (Fig. 3A). Thus the tension-pCa curve was shifted to the right by 0.12 pCa units with age. This is a physiologically significant shift because the Ca²⁺ transient typically peaks in the central portion of the tension-pCa curve during a cardiac twitch. As can be seen in Fig. 2, a 0.12 pCa unit change in this central region of the tension-pCa curve can reduce the developed tension from 50% to 35% of the maximum tension. No significant change with aging was observed in either the Hill coefficient (n_H) [young, 2.71 ± 0.28 (n = 7); old, 2.15 ± 0.16 (n = 13)] or maximum isometric tension production [young, 22.6 ± 7.0 kN/m² (n = 8); old, 20.6 ± 2.4 kN/m² (n = 13)].

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Tension-pCa curves for skinned single adult cardiac myocytes from young (open symbols, dashed line) and old (filled symbols, solid line) rats. Curves were constructed at 15°C at pH 7.0 (triangles) and 6.2 (squares). Curves are normalized to the maximum Ca2+-activated isometric tension produced at each pH. Points represent means ± SE for 4-13 myocytes from 4 to 6 animals.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Change in Ca2+ sensitivity of tension with age. Parameters derived from tension-pCa curves shown in Fig. 2. Open bars, young myocytes; solid bars, old myocytes. A: pCa at 50% tension (pCa50) at pH 7.0 and 6.2. B: pCa50 defined as pCa50 (pH 7.0)  pCa50 (pH 6.2). *Significantly different from young, P < 0.05.

Ca²⁺ transients in intact myocytes. Fluorescence changes in intact cardiac myocytes loaded with fluo-3 at 37°C were used to determine the effect of aging on the Ca²⁺ transient. Figure 4 shows the mean normalized fluorescence change in fluo-3-loaded myocytes from young and old animals, and Table 1 gives the time to peak fluorescence, time from peak to one-half fluorescence decay, and width at half maximum of the fluorescence trace. As is plainly evident, no significant difference in the fluo-3 fluorescence transient between myocytes obtained from young and old rats was observed. Although changes in Ca²⁺ handling have been proposed to underlie age-related decreases in human heart function (8, 15), these fluo-3 results imply that there has been no age-related change in the Ca²⁺ handling ability in myocytes isolated from female F344xBN rats.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mean fluorescence intensity vs. time for myocytes from young and old rats. Ca2+ transients were followed using fluo-3 fluorescence. Myocytes loaded with fluo-3 were stimulated, and the subsequent fluorescence change was recorded and signal averaged for three stimulations per myocyte. The average fluorescence change was normalized to the maximum fluorescence change and the normalized traces averaged for 8 young (dashed line) and 57 old (solid line) myocytes. No change in the time course of the fluorescence was detected.

Sarcomere shortening in intact myocytes. Intact myocyte function was assessed by using laser diffraction to follow the unloaded shortening and relengthening of sarcomeres. Sarcomere length detection is preferable to using edge detection to follow whole myocyte shortening because attachment of the myocytes to the surface limits the free shortening of the myocyte, leading to distortion of the myocyte shortening measurement. In contrast, sarcomere shortening is relatively unaffected by cell surface attachments. A prolonged contraction time was observed using sarcomere shortening as a measure of contractility (Fig. 5). This prolongation of contraction was quantified as the full width of the shortening trace at half the maximum shortening (t_FWHM). With aging, t_FWHM increased from 55.7 ± 1.6 ms (n = 48) in myocytes from young rats to 64.2 ± 1.6 ms (n = 72) in myocytes from old rats. As can be seen in Fig. 5 and Table 2, this prolongation of contraction is due to a significantly decreased rate of shortening (+dL/dt) leading to an increased in the time to peak shortening (t_peak) from 50.5 ± 1.3 ms in single adult myocytes isolated from young rats to 58.9 ± 1.0 ms in myocytes isolated from old rats. These changes are as expected following a MHC shift to a slower isoform. Interestingly, the relengthening parameters [time from peak to one-half maximum fluorescence decay (t_1/2R) and maximum rate of relengthening (dL/dt)] were not significantly altered in these isolated myocytes.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Examples of sarcomere shortening following electrical stimulation. Average first-order laser diffraction pattern movement from 10 stimulations of single cardiac myocytes from a young (solid line) and old (dashed line) rat normalized to the maximum length change.

	DISCUSSION

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One of the difficulties in studies on aging is separating the effects of age from the effects of age-related pathology. Recently, the F344xBN rat has been shown to have a significantly lower rate of age-related pathology compared with other commonly used rat strains (18). This study represents a significant improvement over previous studies of cardiac myocyte function with advanced age through the use of intact myocytes isolated from female F344xBN rats, thereby allowing investigation of the effects of aging without the complication of age-related pathology. In intact single myocytes isolated from F344xBN rats, we observed a decrease in the rate of sarcomere shortening with aging, as seen in the decreased +dL/dt and increased t_peak of aged hearts, leading to an increase in the contraction time as measured by t_FWHM. These results are consistent with an age-dependent shift from the -MHC to the slower -MHC observed with SDS-PAGE as can be seen in Fig. 1. This shift in MHC with age has also been observed in other laboratories (e.g., Ref. 11). As discussed below, the change in MHC isoform appears to be sufficient to explain the results obtained here. We observed no change in either the Ca²⁺ transient (Fig. 4, Table 1) or the relengthening parameters of dL/dt and t_1/2R (Fig. 5, Table 2), indicating that there is no age-associated change in the mechanism of relaxation in single intact myocytes isolated from F344xBN rats. This is in contrast to most of the results observed in other rat strains (4, 6, 7, 9) and also differs from the reported decreased velocity of relengthening with aging in humans (8). Given that the decreases in relaxation capacity seen in other studies is likely to be due to a prolonged Ca²⁺ transient produced by compromised sarcoplasmic reticulum function (13, 26, 30), we conclude that the sarcoplasmic reticulum function is maintained during aging in female F344xBN rats. The absence of an effect on the Ca²⁺ transient is surprising, because it has been clearly shown that there is a decrease in the Ca²⁺ ATPase activity in sarcoplasmic reticulum vesicles obtained from Fischer 344 rats (13, 26, 30). Unlike most previous studies on aging rats, which have used either other strains (e.g., Refs. 4, 6, 7, 9, 12, 13, 22, 26, 30) or male F344xBN rats (11), in the present study we used female F344xBN rats, which have been shown to suffer from fewer pathologies, and with a later onset of pathology, than in other rat strains or in male F344xBN rats (18). It is therefore reasonable to conclude that the lack of an effect of aging on either the Ca²⁺ transient or the relengthening of sarcomeres is due to the absence of compensatory changes induced by pathologies present in other strains.

The present experiments were performed under unloaded conditions. It could therefore be argued that the effect of aging on the Ca²⁺ transient is only observable under loaded conditions. However, this is unlikely because several of the previous reports indicating an aging effect on relaxation in other rat strains were also under unloaded conditions (6, 9). Also, sarcoplasmic reticulum vesicles prepared from aged Fischer 344 rat hearts show a decrease in Ca²⁺ ATPase activity (13, 26, 30), and it is unlikely that sarcoplasmic reticulum vesicle experiments are conducted under conditions that mimic the environment inside loaded myocytes. Another possibility is that there is a difference in the phosphorylation state of troponin I (TnI) between the present and previous studies that compensates for an aging-related decrease in sarcoplasmic reticulum function. This also seems unlikely, especially because phosphorylation would be expected to involve the -adrenergic pathway, and the -adrenergic receptor density and function are reported to be decreased in cardiac tissue from F344xBN rats (17).

An interesting result of this study was the shift in the tension-pCa curve to the right with aging at physiological pH (7.0) in myocytes isolated from F344xBN rats (Figs. 2 and 3A). A rightward shift in pCa₅₀ is also observed in hypothyroid rats, which undergo an almost complete remodeling of the myosin heavy chain isoform from -MHC to -MHC (20). A similar, although less complete, MHC isoform shift was observed here and was the only detected change in sarcomeric protein content (Fig. 1). Therefore, it is concluded that this shift from -MHC to -MHC is likely to underlie the change in Ca²⁺ sensitivity. The exact mechanism of this decrease in Ca²⁺ sensitivity is uncertain, but we have previously demonstrated that changes in myosin kinetics are capable of inducing changes in the Ca²⁺ sensitivity of contraction by altering the distribution of cross-bridge states involved in thin filament activation (27). The decreased Ca²⁺ sensitivity observed here can be understood in terms of our previous model (27) as a decrease in the proportion of cross bridges in a strongly bound, pretension-bearing state. Because a decrease in the strongly bound, pretension-bearing state would also tend to decrease the maximum tension, a result that was not observed with the aging-associated shift from -MHC to -MHC, the rate of cross-bridge detachment of -MHC must also be reduced relative to -MHC to maintain the number of tension-bearing cross bridges. Such a decrease in cross-bridge detachment would then produce the decreased rate of cross-bridge cycling previously reported for -MHC relative to -MHC (25). Surprisingly, the difference in Ca²⁺ sensitivity between young and old myocytes was abolished at lower pH (Fig. 3A, pH 6.2). This result is puzzling in light of the persistence of a difference in Ca²⁺ sensitivity between myocytes from control and hypothyroid rats at low pH (unpublished observations). However, in the hypothyroid animals the myosin is entirely -MHC, whereas in the aged rats studied here -MHC makes up only ~50% of the total myosin content. This difference in MHC content may underlie the difference in the response to acidic conditions between cardiac myocytes from aged and hypothyroid rats. Alternatively, the rightward shift in the tension-pCa curve could be the result of an increase in TnI phosphorylation (23). However, the decreased response to -adrenergic stimulation observed with age in rats (13, 17) would be expected to result in a decreased amount of TnI phosphorylation with age. In addition, acidosis in myocytes from adult rats does not abolish the rightward shift produced by TnI phosphorylation but instead further increases pCa₅₀ (M. V. Westfall and J. M. Metzger, unpublished observations), indicating that TnI phosphorylation and acidosis are separate effects. In contrast, the age-related shift in the tension-pCa curve was not increased at low pH but was abolished (Figs. 2 and 3A). This result indicates that the rightward shift in pCa₅₀ with age was not due to an increased amount of TnI phosphorylation, which would have been expected to increase pCa₅₀, but occurs through some other mechanism.

-MHC cross bridges cycle more slowly than -MHC cross bridges (25). Because the rate of cell shortening is strongly dependent on the rate of myosin cross-bridge cycling, the shift in the MHC is also likely to be responsible for the increase in t_peak. In agreement with this, studies using a caged Ca²⁺ to rapidly activate myocytes from F344xBN rats, thereby eliminating possible effects of a compromised sarcoplasmic reticulum, also show a decrease in the rate of tension development with advancing age (11). The decreased cross-bridge cycling rate produced by a change in myosin isoform might also be expected to decrease the rate of cell relengthening. This was not observed here (Fig. 5, Table 2). However, the mechanisms of relaxation, particularly from a twitch, have not been thoroughly explored and are not well understood. The lack of an effect on relengthening in this report indicates that the cross-bridge cycle may not be rate limiting from the unloaded twitches used in this report.

In summary, the goal of this study was to determine the effects of aging on cardiac myocytes in the absence of underlying pathology. Unlike previous investigators, we utilized the female F344xBN rat, which is subject to fewer pathologies than other rat strains or even the male F344xBN (18). The results presented here from cardiac myocytes isolated from female F344xBN rats indicate that the primary effect of aging in cardiac myocytes appears to be a shift in the myosin isoform from -MHC to -MHC. This shift in MHC appears to be responsible for the slowing of the rate of sarcomere shortening and the rightward shift of the tension-pCa relation observed here. No effect of aging on the Ca²⁺ transient or sarcomere relengthening rate was observed in myocytes isolated from F344xBN rats (Figs. 4 and 5; Tables 1 and 2). From this information we conclude that the decrease in diastolic parameters frequently reported in aging studies using other rat strains are in fact the compensatory effects of some underlying pathology not present in the F344xBN strain. For example, hypertension also produces many of the structural changes in the heart commonly associated with aging (15). Alternatively, because the results presented here were obtained under unloaded conditions, it could be argued that the slowing of relaxation with age is a load-dependent phenomenon. Indeed, there has been a recent echocardiographic report of decreased diastolic function in intact F344xBN rats (24). However, it is unclear what would be the mechanism by which such a load-dependent phenomenon could occur, particularly because a slowing of relaxation has also been observed in unloaded single myocytes from Fischer 344 rats (9). It is possible that altered kinetics in intact hearts are due to structural changes in the heart that are not evident at the single myocyte level. For instance, an increase in extracellular fibrosis or myocyte hypertrophy, perhaps in reaction to a loss in myocyte number with age (4, 12), could potentially affect the compliance of the intact heart, and hence the contractile parameters, without a change in the kinetics of the isolated myocytes. In any event, the increase in the time course of relaxation observed in humans is not apparent in the intact single myocytes isolated from F344xBN rats used here. On the surface, this lack of a decrease in cardiac relaxation parameters calls into question the suitability of the F344xBN rat as a model of human aging, at least in terms of cardiac function. However, humans are subject to a wide variety of age-related pathologies that may develop over prolonged time periods. It is therefore possible that the observed decrease in relaxation in humans is a secondary result of some undetected disease process, even in presumably healthy patients. This possibility warrants further study.