Journal of Applied Physiology
Vol. 81, No. 4, pp. 1488-1494, October 1996
EXERCISE AND MUSCLE

Exercise training improves lusitropy by isoproterenol in papillary muscles from aged rats

George E. Taffet, Lloyd A. Michael, and Charlotte A. Tate

Sections of Cardiovascular Sciences and Geriatrics, Baylor College of Medicine, Houston 77030; and Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Texas 77204

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Taffet, George E., Lloyd A. Michael, and Charlotte A. Tate. Exercise training improves lusitropy by isoproterenol in papillary muscles from aged rats. J. Appl. Physiol. 81(4): 1488-1494, 1996.Aging is associated with a decreased cardiac responsiveness to -adrenergic stimulation. We examined the effect of endurance exercise training of old Fischer 344 male rats on -adrenergic stimulation of the function of isolated left ventricular papillary muscle. Three groups were examined: sedentary mature (SM; 12-mo old), sedentary old (SO; 23-24 mo old), and exercised old (EO; 23-24 mo old) that were treadmill trained for 4-8 wk. The isometric contractile properties were studied at 0.2 Hz and 0.75 mM calcium. Without -adrenergic stimulation, there were no group differences for peak tension, maximum rate of tension development (+dP/dt), or maximum rate of tension dissipation (dP/dt). The time to peak tension was longer (P < 0.05) for both EO and SO than for SM rats. Half relaxation time (RT_1/2) was prolonged (P < 0.05) for SO compared with SM and EO (which did not differ). The three groups did not differ in the -adrenergic stimulation by isoproterenol of peak tension, dP/dt, time to peak tension, or contraction duration. The inotropic response (+dP/dt) of SM was greater (P < 0.05) than that in SO or EO rats (which did not differ); however, the lusitropic response (RT_1/2) was lesser (P < 0.05) in SO than in SM or EO rats (which did not differ). Thus exercise training of old rats improved the lusitropic response to isoproterenol without altering the age-associated impairment in inotropic response.

beta-adrenergic; heart; old; tension; treadmill

INTRODUCTION

EXERCISE IS WIDELY RECOMMENDED for the elderly (32). It can improve their functional status and quality of life. Exercise training of older people and older animals clearly results in improved exercise tolerance and increased maximum oxygen consumption (6, 11). However, the mechanisms of this improvement in performance, which in part depends on cardiac function, are uncertain (5, 6).

Alterations in resting cardiac function after training do not explain the increased performance. With training of old men and rats, there is a small decrease in resting heart rate (5, 43). Systolic function determined by resting left ventricular ejection fraction in situ (6, 31, 35) or isometric tension development in vitro (15, 34, 39) is unchanged with training. There are, however, significant improvements in cardiac diastolic function as determined noninvasively by echocardiogram or radionuclide studies in older humans (16). Indeed, these indirect measurements of diastolic function are the strongest correlates of maximum oxygen consumption in the old person (16, 42). This is, in part, because the Starling mechanism is extensively utilized by the older person in response to exercise to increase cardiac output (7-9, 25). Young persons do not appear to rely on increasing left ventricular end-diastolic volumes or pressures to the same extent to increase their cardiac output (25).

Much of the cardiovascular response to stress, such as exercise, is mediated by the sympathetic nervous system (15). The importance of -adrenergic responsiveness, chronotropic, inotropic, and lusitropic (the facilitation of relaxation), is evident in increasing cardiac output for exercise. Reduced -adrenergic responses contribute to the decreased cardiac function at maximal exercise seen with aging (7). The chronotropic and inotropic responsiveness of the senescent heart is decreased significantly in both rat and human (4, 10, 14, 35). In humans, the maximum heart rate decreases ~1 beat/yr, and this decrement is not altered by exercise training (5). Exercise training may improve the decreased inotropic response somewhat in older persons (6); however, this is not a uniform finding (5, 31, 35). The response of left ventricular filling to isoproterenol (Iso) infusion has recently been shown to be unaltered by training in older men (36). However, noninvasive measurements reflect many other influences beside cardiac lusitropy (36).

For the rat, the age-related changes in lusitropic responsiveness to -adrenergic stimulation has not been examined as completely as the inotropic response, although there is evidence that it is impaired in unloaded myocytes and perfused ventricles isolated from senescent rats (12, 45). We observed that the lusitropic response is decreased with age in left ventricular papillary muscles from very old (30 mo) rats (unpublished preliminary data). Overall, the aging heart is less able to respond to the increased sympathetic nervous stimulation associated with exercise, and an improvement in responsiveness could result in an important improvement in exercise performance. The purpose of this study was to determine whether treadmill training of older rats would improve cardiac inotropic and lusitropic response to the -adrenergic agonist Iso. By using isometrically contracting left ventricular papillary muscles, these separate effects could be studied in isolation.

METHODS

Animals. Male, retired breeder, Fischer 344 (F-344) rats were obtained from the pathogen-free colony maintained for the National Institute on Aging (Harlan, Indianapolis, IN). Animals were either 10 (Mature) or 22 mo of age (Old) before the start of the experimental protocol. The rats were individually caged in a positive-airflow room maintained at 21-22°C. The animals were fed NIH-31 rat chow and water ad libitum. Access to the room was limited to personnel working directly on this project who wore surgical gowns, masks, caps, and shoe covers that were fresh daily. The cages, bedding, water bottles with acidified water, and chow were sterilized before use. Additionally, all equipment was disinfected before being brought into the room with twice-weekly cleaning thereafter. Between shipments of animals, the room was cleaned thoroughly with household bleach. Blood was drawn via the abdominal aorta, and randomly chosen rats had analysis for pathogens on serum samples. All rats were negative on the pathogen screens. Body weights of all rats were recorded weekly.

Training program. Animals were grouped as sedentary mature (SM), sedentary old (SO), and exercise-trained old (EO) for 4 or 8 wk. The training protocol, carried out 5 days/wk, has been described elsewhere (39). The exercise-trained rats were able to run for 60 min/day at 16 m/min at a 5% grade.

Isometric contractile function of left ventricular papillary muscle. The isometric contractile studies were performed as described previously (39). Animals were anesthetized with an intramuscular injection of a combination anesthetic (43 mg ketamine-HCl, 8.6 mg xylazine-HCl, and 1.4 mg acepromazine/ml) at 0.7 ml/kg. The hearts were removed and placed in modified Krebs-Ringer solution preequilibrated with 95% O₂-5% CO₂ at 30°C. Two animals from different groups were studied each day. The two left ventricular papillary muscles were removed for study. The muscles were placed in separate water-jacketed baths. The ends of the muscles were secured by Lucite clips with the upper clip attached to a Statham UC-2 force transducer positioned above the bath. The muscles were suspended in modified Krebs-Ringer solution of the following composition (in mM): 117.4 NaCl; 25 NaHCO₃; 1.2 NaH₂PO₄; 11.1 glucose; 1.2 MgSO₄; 3.6 KCl; and 2.5 CaCl₂. The muscle bath solution was kept at 30°C and continuously bubbled with a mixture of 95% O₂-5% CO₂, maintaining a pH of 7.4. Two platinum-plated electrodes were arranged vertically on opposite sides of the muscle. The muscles were field stimulated with a square-wave pulse 5 ms in duration, 20% above threshold voltage, and at a rate of 0.2 Hz. Outputs were recorded on a Grass 7 polygraph with an electronic differentiator. Outputs were also collected by a data-acquisition board into a personal computer. Analysis was made by using a spreadsheet to average three consecutive twitches. Verification of the computer analysis was made by measuring the Grass polygraph hard copy by hand. Animals that had both muscles function adequately were represented by the average of the two muscles.

Study protocol. The muscles were allowed to equilibrate for 1 h, after which they were stretched to attain the resting length at which developed tension was maximal (L_max). It required 40-60 min to reach L_max, and stress relaxation was allowed to dissipate at each length change. After L_max was attained, the muscles again were allowed to equilibrate for an additional hour, at which time baseline values were determined. The bath solution was then changed to a bath with no added calcium, nominal calcium concentration >5 µM, until twitches were no longer visible (at most 20 min). The bath solution was then switched to 0.75 mM calcium for the studies. The 0.75-mM studies were started after 45 min of equilibration in the low-calcium bath solution. Iso was made fresh daily in the Krebs-Ringer and added in stepwise and additive fashion to the baths, and data collection was started after 2 min. This procedure was followed for each Iso concentration (10¹⁰, 10⁹, 10⁸, 10⁷, and 10⁶ M). If at any point a muscle became automatic (having an intrinsic contraction rate greater than the stimulation rate), then the muscle was no longer considered in the dose curve, the Iso was washed out, and the muscle was bathed in the 0.75-mM calcium solution without Iso until the end of the protocol. Because in preliminary studies automaticity was found frequently at Iso concentrations >10⁶ M, we did not utilize concentrations above this.

After completion of the Iso concentration curve, the baths were again changed to "no added calcium" to wash out Iso followed by a return to 2.5 mM calcium. The muscles all underwent minor, but significant, decreases in peak tension. After the 3- to 4-h protocol, the EO muscles decreased 16 ± 2%, the SM 17 ± 2%, and the SO 15 ± 2%. There were no significant differences in this decrement among the groups. No increases in resting tension were observed.

After the experimental protocol was completed, muscles were trimmed and weighed. Cross-sectional area (CSA) was calculated by assuming that the muscles were cylindrical and that the density of muscle was 1.065 (39). Muscles that generated <1 g/mm² under baseline conditions were not included in the analysis. Muscles that were >1.2 mm² in CSA were also excluded because of the likelihood of central ischemia (33) and the potential problem with diffusion of Iso.

Statistical analysis. The results are presented as means ± SE for the listed number of animals per group. The initial analysis employed analysis of variance for measurements determined at 10⁶ M Iso. When group differences were significant at 10⁶ M Iso, the Iso dose-response data were analyzed by using a rank transformation of the data (3) and repeated-measures analysis of variance techniques from the SAS program (Cary, NC). This corrected for the relatively small size of the data set and the possible presence of interaction effects. A probability of 0.05 was established for significance.

RESULTS

Papillary muscle descriptors. There were no group differences in length, mass, or CSA of the muscles (Table 1).

Table 1. Muscle characteristics SM SO EO n 9 6 13 Lmax, mm 8.37 ± 0.18  8.07 ± 0.34  7.86 ± 0.42  Mass, mg 6.46 ± 0.32  7.04 ± 0.53  6.15 ± 0.52  CSA, mm2 0.72 ± 0.03  0.82 ± 0.05  0.75 ± 0.06 Values are means ± SE; n, no. of rats. SM, sedentary mature; SO, sedentary old; EO, old rats after exercise training. Lmax, length at which maximum tension was developed; CSA, cross-sectional area.

Isometric function at 0.75 mM calcium. At 0.75 mM calcium, the same pattern of age-related alterations seen previously at 2.5 mM calcium was present (39). Because in a preliminary analysis there were no differences between animals undergoing 4 and 8 wk of training, the results were pooled for the analysis of Iso response (Table 2). There were no age-related or exercise-related differences in peak tension, maximum rate of tension development (+dP/dt), and maximum rate of tension dissipation (dP/dt). Exercise training resulted in an abbreviation of half relaxation time (RT_1/2), such that the EO group was significantly less than SO (P < 0.05) and did not differ from SM. Both time to peak tension (TPT) and contraction duration (CD) were significantly faster (P < 0.05) in SM than in SO and EO rats; the two older groups did not differ, indicating a lack of training effect on these two variables.

Table 2. Isometric contraction characteristics at baseline PT, g/mm2 +dP/dt, g · mm2 · s1  dP/dt, g · mm2 · s1 TPT, ms RT1/2, ms CD, ms SM (n = 9) 1.80 ± 0.34  17.7 ± 2.7  12.5 ± 2.1  122 ± 4.5  93 ± 3.0  367 ± 11  SO (n = 6) 1.98 ± 0.28  16.3 ± 2.3  10.6 ± 1.2  154 ± 6.0* 114 ± 4.3* 420 ± 14* EO (n = 13) 1.90 ± 0.27  16.0 ± 2.1  10.6 ± 1.3  140 ± 5.4* 102 ± 3.0  401 ± 14* Values are means ± SE; n, no. of rats. Baseline data were obtained at Lmax. Stimulation rate was 0.2 Hz, with bath calcium 0.75 mM. PT, peak developed tension; +dP/dt, maximum rate of tension development; dP/dt, maximum rate of tension dissipation; TPT, time to peak tension; RT1/2, half relaxation time; CD, contraction duration. * P < 0.05 vs. SM.

Inotropic effect of Iso. The inotropic responses of the muscles from the SO animals to Iso were decreased compared with SM F-344 rats. As shown in Fig. 1A, the increase of +dP/dt was significantly less in the muscles from the SO left ventricles than in those from the SM rats (P < 0.05). The response of +dP/dt for EO group was not different from SO group and was also significantly less than in SM animals (P < 0.05). Approximately 50% of the inotropic response seen at 10⁶ M Iso was present at 10⁷ M Iso for all three groups. For both peak tension (Fig. 1B) and dP/dt (Fig. 1C), the age-related and exercise training-related differences did not attain statistical significance.

Lusitropic effect of Iso. Figure 2A demonstrates the effects of Iso on RT_1/2. There was an age-related decrease in lusitropic responsiveness to Iso (SO vs. SM, P < 0.05). Exercise training resulted in an increased lusitropic response compared with the aged sedentary counterparts (SO vs. EO, P < 0.05). SM and EO groups were not different. There were no statistical differences in responsiveness of TPT to Iso among the groups (Fig. 2B). CD is a composite measurement of TPT and 100% relaxation time (as opposed to RT_1/2). There was a significant difference in responsiveness of CD between SM and SO groups (Fig. 2C, P < 0.05) but not between SM and EO groups.

DISCUSSION

This study examined the effects of exercise training of old rats on inotropic and lusitropic cardiac responses to the -adrenergic agonist Iso. The age-related blunting in adrenergic responsiveness is one of the major determinants of the deterioration in exercise performance with aging and would be a likely site of improvement after exercise training (5). This is especially true with the lusitropic response, because diastolic function at rest (and perhaps during exercise) has been shown to be one of the most important correlates of maximum oxygen consumption in older men (16, 42). Furthermore, the delineation of the modification of adrenergic responsiveness is important in understanding the mechanisms of endurance exercise training.

We found that treadmill endurance exercise resulted in an increased lusitropic responsiveness (RT_1/2) to Iso but in unchanged inotropic (+dP/dt) responsiveness in left ventricular papillary muscles from senescent F-344 rats. Thus exercise training of the senescent rat may improve cardiac function at times of increased output by altering both unstimulated and -adrenergic stimulated relaxation.

The mechanism for the differential effects of training on inotropic and lusitropic responses to -adrenergic stimulation is unclear. Impaired adenosine 3,5-cyclic monophosphate (cAMP) generation with aging has been implicated in the decreased cardiac responses to -adrenergic stimulation (14, 29). However, the limiting component of the lusitropic response may be different from that limiting the inotropic response, to explain the disparate effects of exercise training on RT_1/2 and +dP/dt responsiveness.

-Adrenergic lusitropic response in the senescent sedentary and trained rat heart. The lusitropic responsiveness to -adrenergic stimulation is decreased with age but it has been examined less completely than the inotropic response. When dP/dt was used as the primary measure of relaxation, early studies found minor age-related changes in response to Iso or norepinephrine (10). However, Mattiazzi et al. (18) have produced convincing evidence that under isometric conditions dP/dt is not a good measure of relaxation and that RT_1/2 better reflects the lusitropic state. Lusitropy is decreased in the perfused isolated rat left ventricle from senescent rats (12). Xiao et al. (45) reported an age-related impairment in responsiveness to norepinephrine measuring the sum of TPT and RT_1/2 of isolated myocytes.

The mechanisms for the age-related impairment in lusitropic responsiveness and subsequent improvement by exercise training are unknown. Numerous changes in the receptor-signal transduction cascade that are present both proximal to and involving cAMP production occur with aging and result in decreased second-messenger formation after stimulation (24, 29). -Adrenergic-receptor density is unaltered with age and is not modified by training (1, 29). However, quantification of the total number of receptors may be inadequate. Xiao and Lakatta (44) reported that, although stimulation of the ₁-receptor has both inotropic and lusitropic effects, the ₂-receptor has only inotropic effects. Although the ratio of left ventricular ₁- to ₂-receptors does not change with aging (41), modification by training of their relative contents could result in improved lusitropy and unaltered inotropy.

Total cAMP production does appear to be the site of the training effect. Its production in response to Iso was decreased in both sedentary and exercise-trained older groups compared with 20-wk-old animals (2). Scarpace et al. (30) also found decreased cAMP production in sedentary and exercised old rat hearts. Although 14 wk of forced treadmill running did not alter forskolin-stimulated cAMP production (implying that total cyclase activity was preserved with age and unaltered by training), exercise training decreased inhibitory G protein content (2). Because inhibitory G protein is increased with aging, the antagonistic effects of adenosine on -adrenergic stimulation may be enhanced (2, 4). Relative decreases in stimulatory G proteins with aging have been reported by others (22). Alterations with aging and modification by training of specific G proteins or other regulators affecting distinct pools of cAMP are also plausible and would not be assessed using total cAMP values.

The interaction of the regulatory protein phospholamban with the sarcoplasmic reticulum (SR) calcium pump [SR-Ca-adenosinetriphosphatase (SR-Ca-ATPase)] is a final step of the lusitropic -adrenergic response. With stimulation, phospholamban is phosphorylated, and the affinity of the SR-Ca-ATPase for calcium is increased (37). In the study by Jiang et al. (13), the age-related decrease in response to a submaximal dose of Iso (10⁷ M) was 20% for cAMP production; the phospholamban phosphorylation was reduced by 40%. The maximal response to phosphorylation is maintained with age (12, 13); however, the physiological relevance of the maximum response is uncertain.

The rate of calcium sequestration by the SR is reduced with aging, and the reduction is due to a decreased content of the SR-Ca-ATPase (38). With exercise training of old rats, increases SR calcium sequestration occur in parallel with improved isometric relaxation (19, 34, 39). We recently showed that training of old rats increases their content of the SR-Ca-ATPase (40). If the limit on lusitropic responsiveness was the content of Ca-ATPase (38) available for phosphorylation-augmented function, then with training lusitropy might be expected to improve. Alternatively, if phospholamban is in excess in the sedentary aged animal, as it is in the failing heart (20), then the increase in Ca-ATPase with training might bring the two into a more appropriate ratio, leading to improved lusitropy. These hypotheses are all plausible, merit direct testing, and are not exclusive.

Phosphorylation of troponin I is another end effect of -adrenergic stimulation. Troponin I phosphorylation results in decreased affinity of troponin C for calcium, enhancing relaxation. With aging, troponin I phosphorylation is decreased (27). It is uncertain whether modification of the age-related effects occurs with training.

-Adrenergic inotropic response in the senescent sedentary and trained rat. Exercise training has been reported to increase (19) or not alter (14) the inotropic response to -adrenergic stimulation in middle-aged rats. Our data reveal no training-induced alteration in inotropic responsiveness and confirm that the inotropic responsiveness of the senescent heart is decreased significantly compared with that seen in the mature muscle. The age-related impaired inotropic responsiveness is intrinsic to the myocyte (26, 27), but the mechanism of the decreased responsiveness is uncertain, in part because the mechanisms of inotropy distal to cAMP production are complex.

Transgenic mice having no phospholamban have elevated ventricular contractile function at baseline, but no inotropic response to Iso, implicating phospholamban phosphorylation as a potential key to the inotropic response (17). However, wild-type mouse atria have no phospholamban but do have significant inotropic responses to -adrenergic stimulation (21). Therefore, modification of other proteins after -adrenergic stimulation must be hypothesized.

Two sarcolemmal proteins are also phosphorylated after exposure to Iso. Modification of the sarcolemmal calcium channel and a 15-kDa sarcolemmal protein may contribute to the increased calcium transient and inotropic response with -adrenergic stimulation (12). Relative changes with aging or exercise of these proteins are unknown.

Study limitations. We recognize the numerous limitations of this work. The stimulation rate was kept low, the rate routinely used in papillary muscle studies (14, 33, 34, 39). This is done to obviate the possible development of ischemia; however, age may modify the force-frequency relationship in the rat (12). Jiang et al. (12) showed that at higher stimulation rates (4 Hz) the aged ventricle generated lower force, +dP/dt, and dP/dt; however, we did not find this (Table 2 and Ref. 39). Therefore, extrapolation from slow rates we used in vitro to the much faster rates observed in vivo must be made cautiously.

During our protocol, the muscles were exposed transiently to low-calcium baths so that the calcium concentration in the 0.75 mM bath would not be altered by admixing with residual 2.5 mM calcium. The muscles did not appear to be injured by this exposure, although the possibility of "calcium paradox"-induced damage in a very small percentage of cells exists. Specifically, there was no evidence of altered resting tension, and the decrement in developed tension was modest over the protocol.

The stepwise addition of Iso may result in desensitization of -adrenergic receptors during the protocol (23), and the appearance of automaticity in preliminary studies limited the exposure to 10⁶ M Iso. Therefore, the dose response-curves may not be saturating. However, these technical factors are not likely to explain the observed results.

Finally, extrapolation of these in vitro findings to the human in vivo situation may be unwise. In older men, the response of left ventricular filling to Iso infusion is unaltered by training (36). However, peak flow rates measured noninvasively reflect many other influences beside cardiac lusitropy (36). Corroboration of this observation with the use of independent techniques is warranted.

Summary. As shown by others, endurance exercise training, even of moderate intensity, increases the maximum oxygen consumption and cardiac function of sedentary older animals (15, 29, 34, 39). This paper provides evidence that one of the mechanisms by which this improved function occurs may be via increased lusitropic response to -adrenergic stimulation. The relative contribution of this mechanism is uncertain, but improved lusitropy may be vital to the improved cardiac function that occurs in the elderly with endurance exercise training.

ACKNOWLEDGEMENTS

We extend our appreciation to Ed Hudson and Thorunn Helgason for animal husbandry and training of the animals.

FOOTNOTES

Address for reprint requests: G. E. Taffet, Huffington Center on Aging, M-320, Baylor College of Medicine, Houston, TX 77030-3498.

Received 15 February 1995; accepted in final form 17 April 1996.

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