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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 57:B339-B343 (2002)
© 2002 The Gerontological Society of America

Age-Related Differences in the Effect of Running Training on Cardiac Myosin Isozyme Composition in Rats

Shuichi Machidaa, Hisaya Tsujimotob, Hideki Suzukib, Norikatsu Kasugab, Keizo Kobayashia and Mitsuo Narusawaa

a Department of Exercise Physiology, International Budo University, Chiba, Japan
b Department of Health Science, Aichi University of Education, Japan

Shuichi Machida, Department of Veterinary Biomedical Sciences, University of Missouri—Columbia, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, MO 65211 E-mail: MachidaS{at}missouri.edu.

Decision Editor: James R. Smith, PhD


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
We examined the effect of running training on age-related changes in cardiac myosin isozyme composition in rats. Female Fischer 344 rats (6, 12, 20, and 27 months old) were divided into two groups: sedentary control and trained. The trained group rats were trained by treadmill running for up to 60 minutes per day, 5 days per week for 8 weeks at up to 30 m per minute. In sedentary control rats, the proportion of V1 myosin, that is, {alpha}-myosin heavy chain (MyHC) isoform, decreased progressively from 6 to 27 months of age. In the younger age groups (6 or 12 months old), there was a shift from V1 myosin to V3 myosin (ß-MyHC isoform) in trained hearts. However, the training program did not induce a cardiac myosin isozyme transition in older rats (20 or 27 months old). These results suggest that the mechanisms mediating the responses of cardiac muscle to running training alter during aging.

IT is generally accepted that there is an aging-associated decline in various indices of myocardial contractility (1). One of the components of this performance decline is a change in cardiac myosin isozyme composition, with a corresponding slowing in the velocity of muscle shortening (2)(3)(4)(5). Rat ventricular muscle contains three different myosin isozymes, termed V1, V2, and V3 (6). These isozymes are made up of various combinations of two distinct myosin heavy chain (MyHC) proteins, termed {alpha} and ß. The V1 and V3 isozymes are {alpha}{alpha} and ßß homodimers, respectively, whereas V2 is an {alpha}ß heterodimer (7). The V1 isozyme has a higher adenosine triphosphatase (ATPase) activity than V3, and it is associated with a greater shortening velocity. In addition, V3 exhibits a greater economy of force production than V1 (4)(5)(8). Many investigators have reported that myosin ATPase (or actomyosin ATPase) and the V1 myosin isozyme show a decline during the aging process (2)(3)(9)(10)(11)(12)(13).

It is well known that exercise can attenuate the age-related decline in the intrinsic mechanical performance of the heart (14)(15)(16). Endurance training results in cardiac hypertrophy, characterized by enhanced mechanical performance and increased myosin ATPase activity (17)(18), with a concomitant shift toward the V1 isozyme in rat hearts (18)(19)(20)(21). However, such changes in cardiac myosin ATPase activity or myosin isozyme composition could be demonstrated only after swimming training, not after running training (17). In fact, several studies have failed to find any changes of this type in the heart in running-trained animals (11)(21)(22)(23). In contrast, other studies have reported that running training did have some effect on either myosin ATPase activity or myosin isozyme composition in rat hearts (24)(25)(26). Thus, the published data on the effect of running training on myosin ATPase or isozyme composition are controversial. To our knowledge, only one study has been published on the effect of running training on the age-related change that occurs in cardiac myosin isozyme composition during aging (11). The purpose of the present study, in the rat, was to examine the responses of cardiac muscle to running training at various stages during aging. We demonstrate here that running training had a significant effect on cardiac myosin isozyme composition in young rats (6 or 12 months old). However, the training program did not induce a cardiac myosin isozyme transition in older rats (20 and 27 months old).


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Animal Care and Experimental Groups
Female Fischer 344 rats aged 4 weeks were obtained from Japan SLC, Inc. (Hamamatsu, Japan). They were housed individually in sedentary cages and were not exercised until the physical training period began. At the end of the training period, the rats were 6, 12, 20, or 27 months old. The rats in each age group were divided randomly into sedentary and running-training groups. The numbers of sedentary and running-trained rats were as follows: 6 months old, 7 and 5, respectively; 12 months old, 7 and 9, respectively; 20 months old, 7 and 7, respectively; 27 months old, 6 and 5, respectively. All the animals were kept under controlled conditions throughout the experiment. Temperature was 23 ± 1°C, and the rats were subjected to a 12-hour light–12-hour dark cycle. Chow (CE-2; CLEA, Japan) and water were provided ad libitum. These experiments conformed with the Guiding Principle for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.

Running Training
The rats in the running-training groups were trained on a rodent treadmill (KN-73; Natsume Seisakusyo Co. Ltd., Tokyo, Japan). An electric grid at the rear of the belt was used to supply a running stimulus. All rats ran 5 days per week for 8 weeks. Speed and duration were progressively increased until the rats ran at 30 m per minute for 60 minutes per day on 0% incline.

Muscle Sampling
After the 8-week training program, hearts were removed under pentobarbital sodium anesthesia. The atria and great vessels were dissected away from the rest of the heart, and the ventricular muscle was weighed and then separated into left ventricle and interventricular septum plus right ventricle. The left ventricular (LV) muscles were quickly frozen in liquid nitrogen, and they were stored at -80°C until assayed.

Cardiac Myosin Isozyme Analysis
The cardiac myosin isozymes were analyzed according to the pyrophosphate polyacrylamide gel electrophoresis technique, as described previously (6). The myosin was extracted from each LV sample according to the method of Farrar and colleagues (11), and the extracted myosins were stored at -20°C for electrophoretic analysis. The myosins were loaded onto a 3.88% acrylamide and 0.12% N,N'-methylene-bis-acrylamide gel (T = 4%; C = 3%). The electrophoresis buffer was 20 mM tetrasodium pyrophosphate, 1 mM ethylenediamine tetra-acetic acid (EDTA), 10% glycerol, and 2 mM L-cysteine, pH 8.9. Electrophoresis was performed for 24 hours at 70 V (constant), using a GE-4 apparatus (Pharmacia, Uppsala, Sweden), which allows circulation of the upper and lower chambers with buffer. The temperature was maintained at 2°C. After the run, the gels were stained (0.1% R-250 and G-250 Coomassie blue, 25% methanol, and 10% acetic acid) and destained (30% ethanol and 10% acetic acid). The gels were scanned densitometrically by using a thin layer chromatograph scanner C5-930; Shimazu, Tokyo, Japan) equipped with a laser-source attachment (LSA-9000; Shimazu). The relative amounts of the individual isozymes (V1, V2, and V3) were calculated from the area under each peak height by planimetry. To calculate the percentages of {alpha}- and ß-MyHC proteins in the sample, we used the equations %{alpha} = %V1 + 1/2%V2, and %ß = %V3 + 1/2%V2 (7).

Statistical Analysis
The data are shown as mean ± standard deviation, and they were analyzed by using a two-way analysis of variance, with the statistical significance of differences between group means being assessed by Tukey's method for multiple comparisons. The level of statistical significance was set at a value of .05.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Body Weight and Ventricle Weight
Table 1 shows the effect of aging and running training on body and ventricle weights in rats of various ages. In none of the four age groups was there any difference in body weight between control and trained rats. In both groups of young trained rats (6 and 12 months old), ventricle weights were significantly greater than in the control rats of the same age (by 15% and 9%, respectively). In contrast, there was no difference in absolute ventricle weight between the control and trained groups of older rats (20 and 27 months old). However, the relative heart weight (ventricular-to-body weight ratio) was similarly increased by training in all age groups, although the effect of training was not significant in the oldest rats (27 months old).


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  		Table 1. Aging and Running-Training Effects on Body and Ventricular Weights

 
Cardiac Myosin Isozyme Composition
Fig. 1 shows representative pyrophosphate gel patterns for the various cardiac myosin isozymes, together with densitometric scans of the gels; these were obtained from LV samples from control and trained 6- and 20-month-old rats. Table 2 shows the effects of aging and running training on myosin isozyme composition in the left ventricle of rats of various ages. In sedentary control rats, the proportion of V1 myosin decreased progressively from 6 to 27 months of age. Only in the younger age groups (6 or 12 months old) did the training have any effect on cardiac myosin isozyme composition; in these groups, there was a shift from V1 myosin to V3 myosin in trained hearts. In contrast, training had no effect on cardiac myosin isozyme composition in the older groups (20 or 27 months old). Fig. 2 shows the proportion of the ß-MyHC isoform in the ventricles from control and trained groups at each age. In both of the younger groups, the proportion of the ß-MyHC isoform was significantly greater in trained than in control rats of the same age. However, there was no difference in the proportion of the ß-MyHC isoform between control and trained rats in either of the older groups (20 or 27 months old).



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  		Figure 1. Representative patterns of myosin isozyme composition in the left ventricle of control and trained 6- and 20-month-old rats. Pyrophosphate gels and densitometric scans of the gels are shown. The density of each band (V1, V2, and V3) was quantitated by using a laser densitometer. The composition of each band was calculated on the basis of the area of the resolved Gaussian curves. This classification is based on electrophoretic mobility in the native state, in which the migration rate reflects the combined properties of both of the myosin heavy chains (MyHCs). V1 and V3 are {alpha}- and ß-MyHC homodimers, respectively, whereas V2 is an {alpha}ß-MyHC heterodimer. C = sedentary control; T = trained.

 

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  		Table 2. Aging and Running-Training Effects on LV Myosin Isozyme Composition

 


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  		Figure 2. Effects of aging and running training on the proportion of ß-myosin heavy chain in the left ventricle of control and trained rats of various ages. Values are means ± standard deviation. C = control sedentary; T = trained. **p < .01 (T vs C groups).

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In this study, a profound reduction in the proportion of the V1 myosin isozyme ({alpha}-MyHC isoform) from 82.5% at 6 months of age to 40.5% at 20 months was observed in female Fischer 344 rats. This marked shift from the V1 to the V3 myosin isozyme (ß-MyHC isoform) is similar to that reported by Boluyt and colleagues (27) in female Fischer 344 rats and by both Capasso and colleagues (2) and Farrar and colleagues (11) in male Fischer 344 rats. In our study, we set out to determine whether a higher intensity of running training than previously reported (11) could alter the rate of this age-related change in cardiac myosin isozyme composition. This training program affected cardiac myosin isozyme composition only in young and adult rats (6 or 12 months old). The accelerated shift from the V1 to the V3 isoform with a concomitant cardiac hypertrophy is remarkable because exercise accelerated an effect of maturation. Usually physical activity compensates for decrements occurring with aging; for example, exercise attenuated the age-related decline in the intrinsic mechanical performance of the heart (14)(15)(16). In this case, the maturation-associated shift to V3 myosin isozyme was accelerated by exercise training.

The increase in V3 myosin isoform has been associated with an intermittent increase in hemodynamic load only during running exercise (24). It is generally accepted that chronic pressure or volume overload induces a cardiac hypertrophy, with a decrease in mechanical performance and an increase in the proportion of V3 myosin (2)(28)(29). It is thus possible that a transient pressure or volume overload during exercise would induce a myosin isozyme transition in young rats. Therefore, the increased myocardial contractile activity that was due to intense and prolonged exercise induced a fast (V1 isozyme)-to-slow (V3 isozyme) myosin transformation. This would be analogous to the well-known fast-to-slow fiber type of transformation observed in skeletal muscle after endurance training (30)(31). It is well known that V3 myosin has low ATPase activity and a greater economy of force production than V1 myosin (8). Fitzsimons and colleagues (24) demonstrated that LV contractile performance (i.e., pressure generation) was maintained in trained rats, even though the proportion of V1 myosin was decreased. Perhaps, in the present study, the changes in myosin isozyme composition seen in relatively young trained female rats (6 or 12 months old) allow the heart to economize on cross-bridge cycling without compromising the potential for pressure generation.

The accelerated shift to V3 myosin isozyme in young female rats who exercised confirms a previous report in which only young female rats were exercised (24), but this effect may be gender specific. We recently reported that in young male Sprague-Dawley rats, running training had little effect on cardiac myosin isozyme composition, regardless of the running intensity (32). The reason for the differential responses between the genders remains unclear.

In the present study, the training program did not induce a cardiac myosin isozyme transition in older rats (20 or 27 months old). Farrar and colleagues (11) also reported that cardiac myosin isozyme composition in older rats (24 months old) was not affected by running training at lower intensity (20 m per minute for 60 minutes per day). The question then arises as to why we did not observe any effect of the running training on cardiac myosin isozyme composition in older trained rats. One possible factor is that the proportion of V3 myosin isozyme in the older rats is far higher than it is in the younger rats. We noted an increase in the proportion of V3 myosin isozyme with aging, from 5.2% at 6 months to 31.4% at 27 months. We had hypothesized that a higher intensity of running (30 m per minute for 60 minutes per day) would further decrease V1 myosin isozyme in old rats, but the results did not support the hypothesis. It may be that in the hearts of older rats, which have predominantly V3 myosin, little or no further shift from V1 to V3 myosin isozyme is possible in response to running training. Indeed, the percentage of V3 myosin isozyme in nontrained 20-month-old rats (28.3%) was already greater than in trained 12-month-old rats (19.8%).

In conclusion, running training had a significant effect on cardiac myosin isozyme composition in young female Fischer 344 rats in our study. However, the training program failed to induce a cardiac myosin isozyme transition in older rats. These results suggest that the mechanisms mediating the response of cardiac muscle to training alter during aging.


   Acknowledgments
 
We are grateful to Drs. F. Booth and E. Spangenburg (Department of Veterinary Biomedical Sciences, University of Missouri—Columbia) for their critical reading of the manuscript, and to Mr. F. Kariya (International Budo University, Japan) for his help during the preparation of the manuscript. S. Machida is currently with the University of Missouri—Columbia. H. Tsujimoto is currently with the Institute of Health and Physical Education, Kurume University, Fukuoka, Japan.

Received March 13, 2002

Accepted June 19, 2002


   References
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 Results
 Discussion
 References
 

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