Journal of Muscle Research and Cell Motility 25: 69–76, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.

69

Swimming exercise in infancy has beneficial effect on the hearts in cardiomyopathic Syrian hamsters MARIKO TATSUGUCHI1, , ERIKO HIRATSUKA2, , SHUICHI MACHIDA3, TOSHIO NISHIKAWA4, SHIN-ICHIRO IMAMURA2, SATORU SHIMIZU5, MASAHIKO NISHIMURA7, ISSEI KOMURO8, YOSHIYUKI FURUTANI3, MICHIKO FURUTANI3, HIROAKI NAGAO2, KEIKO KOMATSU2, HIROSHI KASANUKI1 and RUMIKO MATSUOKA3,6,* 1 Department of Cardiology; 2Research Division, 3Department of Pediatric Cardiology; The Heart Institute of Japan; 4 Department of Pathology; 5Department of Hygiene and Public Health; 6Division of Genomic Medicine, Institute of Advanced Biochemical Engineering and Science, Graduate School of Medicine, Tokyo Women’s Medical University, Tokyo; 7Institute for Laboratory Animal Research, Nagoya University School of Medicine, Nagoya; 8Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan Received 21 June 2003; accepted in revised form 4 September 2003

Abstract The phenotypic expression of cardiomyopathy is greatly influenced by extrinsic factors other than intrinsic genetic defects, such as environmental stress. Exercise is assumed to be an important extrinsic factor, since sudden death is sometimes seen during exercise in young patients with hypertrophic cardiomyopathy (HCM). However, the longterm effects of mild exercise on phenotypic expression in cardiomyopathy remain unclear. To evaluate the effects of exercise performed during infancy or adolescence in cardiomyopathic patients, cardiomyopathic Syrian hamsters (BIO14.6) were subjected to swimming. BIO14.6 and age-matched congenic normal hamsters (CN) as controls were divided into three groups: sedentary (Sed), and trained during infancy (Inf) and during adolescence (Ado). Histological and biochemical analysis of 41-week-old hamsters revealed that (1) the relative level of b-myosin heavy chain mRNA was significantly lower in the Inf group than in the Sed and Ado groups of BIO14.6. The level in the Inf group of BIO14.6 was compatible with that in the age-matched Sed group of the CN strain; (2) in BIO14.6, degenerative mitochondrial change in the cardiomyocytes was not seen in the Inf group while it was common in the Sed and Ado groups; (3) calcineurin phosphatase activity in the swimming group in 10-week-old CN was significantly higher than that of the age-matched sedentary group, and was as much as that of the swimming and sedentary groups in 10- and 41-week-old BIO14.6.

Introduction Cardiomyopathy is often caused by various mutations and those found so far affect sarcomeric and cytoskeletal proteins (Seidman and Seidman, 1998; Chien, 1999). The natural course of cardiomyopathy is characteristically heterogeneous, ranging from an uneventful clinical course to the most serious complications, i.e., sudden death and severe congestive heart failure (Wigle et al., 1995; Spirito et al., 1997). In hypertrophic cardiomyopathy (HCM), sudden death occurs most commonly in young patients during or after intense physical activity while heart failure and stroke-related deaths occur most frequently in patients beyond mid-life (Hecht et al., 1993). Therefore, various environmental stresses other * To whom correspondence should be addressed: Rumiko Matsuoka, Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: þ81-3-3353-8111, ext. 24067; Fax: þ81-3-3352-3088; E-mail: [email protected]   M. Tatsuguchi and E. Hiratsuka contributed equally to this work.

than genetic defects may be involved in the diverse prognosis of cardiomyopathy. In this regard, exercise may be the culprit as an environmental stress in HCM patients (Geisterfer-Lowrance et al., 1996; Maron et al., 1996; Tabib et al., 1999). Children diagnosed with cardiomyopathy tend to be prohibited from exercising, although there have not been enough basic science studies on the effect of habitual exercise on cardiomyopathy. It is generally accepted that mild exercise can attenuate the age-related decline in cardiac performance (Li et al., 1986; Starnes and Ramsey 1998). In addition, mild exercise may lead to a lower incidence of death from ischemic heart diseases (Siscovick, 1997) and heart failure (Belardinelli et al., 1999). Also, the supposition that mild exercise in cardiomyopathic patients can cause death has not been proven. Several reports have demonstrated that the heart muscle is more responsive to exercise training in younger than in older animals (Chesky et al., 1983) indicating that the effect of exercise training is different according to the age when the exercise was undertaken. We thought it intriguing to

70 investigate whether exercise performed at different ages would affect the prognosis of cardiomyopathic patients. The study of changes in the proportion of cardiac heavy chain (MHC) isozymes has assumed importance because it has been found to be directly related to the level of mechanical performance during hypertrophy and heart failure (Imamura et al., 1991; Bugaisky et al., 1992). Since b-MHC exhibits a greater economy of force production than a-MHC, chronic pressure overload induces cardiac hypertrophy and up-regulates b-MHC, with decreased mechanical performance. In other words, the pathological adaptation has been shown to be associated with an increase in the expression of b-MHC (Holubarsch et al., 1985). On the other hand, physiological cardiac hypertrophy due to exercise training is characterized as enhanced mechanical performance with up-regulation of a-MHC (Rupp, 1981). Swimming exercise is characterized by a high adrenergic drive, and the increased proportion of a-MHC is most likely correlated with the marked catecholamine release during swimming (Chesky et al., 1983; Rupp, 1981). Recently, calcineurin was identified as an important regulator of cardiomyocytes. Previous studies have suggested that calcineurin is one of the signaling molecules implicated in the development of cardiac hypertrophy and failure, and that it also plays a critical role in the activation of the b-MHC promoter (Zhu et al., 2000). Therefore, we evaluated the effect of swimming on the heart in cardiomyopathic Syrian hamsters of the BIO14.6 strain (Strobeck et al., 1979) (BIO) by three independent parameters, histology, myosin heavy chain (MHC) mRNA expression and calcineurin phosphatase activity.

Materials and methods Animals BIO (Bio-Research, Cambridge, MA/USA) was used in this study as an animal model of cardiomyopathy and congenic normal hamsters (CN) were used as normal controls. BIO is a widely studied animal model of cardiomyopathy with a 5¢ deletion located 5¢ upstream of the second exon of the d-sarcoglycan gene (Sakamoto et al., 1997) which has been reported to be found in patients with dilated cardiomyopathy (Tsubata et al., 2000). Two characteristic phases, the hypertrophic phase and the dilated phase, are defined by histological and clinical evidence of BIO. The hypertrophic phase starts from about 20-week-old when cardiomyocytes show pathological hypertrophy. The dilated phase starts from about 40 weeks, when ventricular dilation occurs, up to congestive heart failure (Strobeck et al., 1979). We confirmed deletion of the d-sarcoglycan gene, which was also found in the BIO in our laboratory, whereas this gene was intact in the CN. To generate the CN strain, a normal golden Syrian hamster from a commercial breeder was mated with a cardiomyopathic

inbred hamster (BIO), and their F1 hybrid was again mated with the BIO to produce the N2 generation. N2 animals showing neither hypercreatinemia nor calcification of the tongue were selected and again mated with BIO to produce the N3 generation. After nine generations of backcrossing (N10), the animals were inbred by more than 20 generations of full-sib mating. The BIO and CN were housed at 23 ±2C, with a 12-h light– dark cycle. All hamsters had free access to food and water. Experimental design At first, to evaluate the effect of the swimming training program in this study, the size of cardiomyocytes and calcineurin phosphatase activity in the swimming groups and sedentary groups of the CN and BIO were compared. Both strains of hamsters in the swimming group were subjected to swimming from 3 to 10 weeks, which corresponds to the period of infancy in hamsters, and they were sacrificed 24 h after the last training. Hamsters in the sedentary group were sacrificed at the same time (10 weeks). The duration of swimming was 5 min on the first day which was increased daily in 5-min steps up to 30 min. Swimming was carried out 5 days a week for 7 weeks. The hamsters were placed in a tank (55 · 64 · 38 cm) filled with water kept at 35–37C (Pagani and Solaro 1983). All hamsters were sacrificed under anesthesia with halothane inhalation (5%/animal). There were five hamsters in each group. On the basis of the first study, we scheduled swimming exercise which is summarized in Figure 1. The CN and BIO strains of hamsters were each divided into three groups: (1) sedentary (Sed), (2) trained from 3 to 10 weeks (Inf), and (3) trained from 16 to 23 weeks (Ado), which were abbreviated as CN-Sed, CN-Inf, CNAdo and BIO-Sed, BIO-Inf, BIO-Ado, respectively. After the respective training period, the trained hamsters were kept sedentary and were sacrificed at 41 weeks in the dilated phase (Strobeck et al., 1979). Hamsters sacrificed at 41 weeks were anesthetized with halothane inhalation (5.0%/animal). Table 1 shows the experimental groups. All procedures were approved by the

Fig. 1. The swimming program performed in this study. W, week-old; Sed, sedentary; Inf, swimming in infancy from 3 to 10 weeks; Ado, swimming in adolescence from 16 to 23 weeks.

71 Table 1. Experimental groups Groups

n

CN-Sed CN-Inf CN-Ado BIO-Sed BIO-Inf BIO-Ado

7 5 6 11 9 10

CN – congenic normal hamsters; BIO – cardiomyopathic Syrian hamsters (BIO14.6). Sed: sedentary, Inf: swimming in infancy from 3 to 10 weeks; Ado: swimming in adolescence from 16 to 23 weeks; all hamsters were sacrificed at 41-week-old.

Animal Care and Committee of Tokyo Women’s Medical University and complied with the American Heart Association Guidelines. Tissue removal After body weight was measured, the hearts were removed, washed free of blood with saline and weighed. The thickness of the left ventricular wall was measured. Left ventricular specimens were cut and a few were subjected to light- and electron-microscopic analysis. The remaining specimens were immediately frozen in liquid nitrogen and stored at )80C for molecular analysis.

chain reaction (RT-PCR) was performed. PCR primers were designed on the basis of the reported b-MHC sequence of BIO (Wang et al., 1995): 5¢-CTGAAGCTCCCTGGACATTGACCA-3¢ for a forward primer and 5¢-ctataacattAGGCCCGGATGTTCCACTGG-3¢ for a reverse primer. Ten mer extra sequences (small letters) in an untranslated region were added to the 5¢ end of the reverse primer. The MHC cDNA consisted of a 236 bp fragment, which was fully protected by b-MHC mRNA and partially protected by a-MHC mRNA, as shown by S1-nuclease mapping analysis. (The ratio of the two protected fragments indicates the ratio of a and b-MHC mRNA in a given sample.) Total RNA isolated from ventricular muscle of 250-day BIO hamsters was used as a template for synthesizing double-stranded cDNA. The probe was hybridized with total RNA (18 lg) in 80% formamide for 16 h at 42C. S1 nuclease digestion was carried out for 1 h at 25C, and the digestion products were separated by 8% polyacrylamide 8.3 M urea-sequencing gel. The radioactive density of each band was measured using a computerized system for radiography, BAS 2000 (Fuji Photo Film, Tokyo/Japan). Calcineurin phosphatase activity The activity of calcineurin in the lysates of the left ventricular samples was determined as described previously (Shibasaki and Mckeon, 1995).

Light- and electron-microscopic analysis

Statistical analysis

The specimens for light microscopy were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 3.5lm thick sections, and stained with hematoxylin–eosin and Masson trichrome. Those for electron microscopy were fixed with 3% glutaraldehyde, postfixed with 1% osmium tetroxide, and embedded in epon resin (Epon 812). The diameter of the cardiac myocytes was measured with a micrometer for light microscopy, as previously described (Nishikawa et al., 1990). One hundred myocytes from each sample were measured randomly. Ultrastructural aspects were assessed histopathologically by observing pathologic lesions in the myocytes, including myofibrils and mitochondria, intercalated disks, and interstitial structures, including small vessels. The findings were divided qualitatively into four grades of severity: ) (indicating no pathology) to þ þ þ (indicating severe changes). (Sekiguchi et al., 1978).

The data are presented as mean ± standard error (SE), and intergroup comparisons were made by one-way analysis of variance (ANOVA) followed by the Bonferroni/Dumm method. Statistical significance was defined as P < 0.05.

Analysis of MHC mRNA expression (1) Total RNA extraction: Total RNA from the frozen left ventricular specimens was isolated using RNA zol (TEL-TEST, Houston, TX/USA) as recommended by the manufacturer. (2) Preparation of a probe for S1-nuclease mapping analysis: To obtain MHC cDNA probes for S1-nuclease mapping analysis, the reverse transcription polymerase

Results For the first study, we compared the diameter of cardiac myocytes and the calcineurin phosphatase activity of the CN (n ¼ 10) and BIO (n ¼ 10) strains of the swimming group (n ¼ 5/strain) and the sedentary group (n ¼ 5/ strain) sacrificed at 10 weeks, to evaluate the effect of swimming on the cardiomyocytes (Figure 2A, B). The exercising period of the swimming group was from 3 to 10 weeks which conformed to that of the Inf group in the following experimental design. As shown in Figure 2A, the myocyte size in the swimming groups showed an increase compared to that in the sedentary groups in both CN and BIO, and the difference between the swimming group and the sedentary group in CN was especially significant. Figure 2B shows that in the CN, calcineurin activity in the swimming group was significantly higher than that of the sedentary group, while in the sedentary and swimming groups in the BIO it was at almost the same level,

72

Fig. 2. (A) Myocyte size in the 10-week-CN and -BIO groups. Number of hamsters is indicated in parentheses; (B) [C] calcineurin activity in the 10-week-CN and -BIO groups. CN, congenic normal hamsters; BIO, cardiomyopathic Syrian hamsters (BIO14.6). Values are expressed as mean±SE, statistical significance was assumed at * P < 0.05.

which was as much as that of the swimming group in the CN. From the first study, we confirmed that this swimming program was effective in inducing physiological cardiac hypertrophy. Therefore, we planned a program which would show the effect of the swimming exercise performed at different ages (infancy or adolescence) on the hearts in 41-week-cardiomyopathic hamsters. During this study, three hamsters in the BIO-Inf and two hamsters in BIO-Ado groups died several weeks after exercise, although no death occurred during exercise. The age at death was 19, 20 and 26 weeks in the BIO-Inf group, and 36 and 37 weeks in the BIO-Ado group. No death occurred in any of the CN groups nor in the BIOSed group. Size of cardiac myocytes Figure 3 shows myocyte size in the 41-week-CN and BIO. Although the myocytes in both CN-Inf and BIOInf remained slightly hypertrophic when compared with each strain of the Sed and Ado groups, the size did not differ significantly between the groups. Body weight and heart weight Table 2 shows body weight (BW), heart weight (HW) and the HW-to-BW ratio (HW/BW) in the 41-week-CN and -BIO groups. The HW of swimming hamsters was heavier than that of age-matched sedentary hamsters in CN and BIO. The difference in HW between CN-Sed and CN-Ado was significant. The HW/BW of the Ado groups was significantly increased compared with that of the Sed groups in CN and BIO. The HW/BW of the

Fig. 3. Myocyte size in the 41-week-CN and -BIO groups. CN, congenic normal hamsters; BIO, cardiomyopathic Syrian hamsters (BIO14.6); Sed, Sedentary; Inf, swimming in infancy from 3 to 10 weeks; Ado, swimming in adolescence from 16 to 23 weeks. Number of hamsters is indicated in parentheses.

BIO-Ado was the heaviest. The HW of BIO-Ado was heavier than that of the BIO-Sed. However, the BW of

73 Table 2. Heart and body weight of 41-week-hamsters BW (g) CN-Sed CN-Inf CN-Ado BIO-Sed BIO-Inf BIO-Ado

115 118 117  128
120 101

± ± ± ± ± ±

HW (mg) 2.8 3.5 1.9 4.7 1.2 8.6

 506
560 583 591 637 662

± ± ± ± ± ±

15.6 15.2 23.6 50.1 51.7 49.1

Table 3. Scoring of electron microscopic findings in the cardiac tissue of 41-week-hamsters

HW/BW (mg/g)  4.4
4.8 5.0  4.9
5.3 6.6

± ± ± ± ± ±

0.1 0.1 0.2 0.3 0.4 0.6

CN – congenic normal hamsters; BIO – cardiomyopathic Syrian hamsters (BIO14.6). Sed: sedentary; Inf: swimming in infancy from 3 to 10 weeks; Ado: swimming in adolescence from 16 to 23 weeks; HW – heart weight; BW – body weight. * P < 0.05.

the BIO-Ado was only 79% of that of the BIO-Sed, which led to an increase in the relative heart weight in the BIO-Ado. Morphological findings of the cardiac tissue Among the 41-week-BIO groups, the free wall of the left ventricle and the interventricular septum in the BIO-Inf were hypertrophied, whereas the left ventricular chambers in the BIO-Ado were enlarged. On the other hand, no remarkable morphological changes were seen in the 41-week-CN groups. In the light microscopic study on left ventricular tissue from the 41-week-BIO groups (Figure 4A), no remarkable changes except hypertrophic cardiac myocytes and mild perivascular fibrosis were seen in the BIO-Inf

Mitochondrial Mitochondriosis Scarcity of Dilatation degeneration myofibrils of SR cavity CN-Sed CN-Inf CN-Ado BIO-Sed BIO-Inf BIO-Ado

) ) ) + ) +

) + + ) +++ )

) ) ) ) ) +

) ) ) + ) ++

CN – congenic normal hamsters; BIO – cardiomyopathic Syrian hamsters (BIO14.6). SR – sarcoplasmic reticulum; Sed: sedentary; Inf: swimming in infancy from 3 to 10 weeks; Ado: swimming in adolescence from 16 to 23 weeks. ): no pathology; þ: mild change; þþ: moderate change; þ þ þ: severe change.

(Figure 4A, b). Slight focal thickening of the endocardium and mild fibrosis in the subendocardium were detected in the BIO-Ado (Figure 4A, c), but no remarkable changes, such as degeneration of cardiac myocytes and diffuse fibrosis, were observed. Electron-microscopic findings in left ventricular tissue of the 41-week-BIO groups are shown in Table 3. No significant degenerative changes were seen in the CNgroups. Mild-to-moderate degenerative changes in the mitochondria and myofibrils were seen in the BIO-Sed group (Figure 4B, a). Only slight degenerative change in the myofibrils and a numerical increase in mitochondria without any degeneration was observed in the BIO-Inf

Fig. 4. (A) a: Transverse section of hearts stained with Masson trichrome in the 41-week-BIO-Sed group; b: 41-week-BIO-Inf group and c: 41week-BIO-Ado group. Bar ¼ 2 mm. (B) Electron microscopic findings of cardiomyocytes in the 41-week-BIO groups. a: shows mild-to-moderate degenerative change in the mitochondria (M) and myofibrils in the 41-week-BIO-Sed group. b: shows no apparent degenerative change with a mild increase in mitochondria (M) in the 41-week-BIO-Inf group. c: shows scarcity of myofibrils, degeneration of mitochondria and dilated sarcoplasmic reticular (SR) cavities in the 41-week-BIO-Ado group. L, lipid droplets; N, nucleus; Bar ¼ 2 lm.

74 both groups in 10-week-BIO, and was remarkably higher than that of the sedentary group in the 10week-CN. In 41-week-BIO, although the activity of the Inf group was relatively increased compared with that of the Sed and Ado groups, the differences among the activity of the three groups were not significant (data not shown).

Discussion To our knowledge, this is the first study that shows the effect of exercise performed in cardiomyopathic animals at different ages (infants or adolescents). The present study indicated that swimming exercise performed in infancy produced beneficial effects on most of the hearts of 41-week-cardiomyopathic hamsters. We assessed the left ventricular tissue according to morphology, MHC mRNA expression, and calcineurin phosphatase activity. Effects of exercise on morphology

Fig. 5. (A) Gel electrophoresis showing an example of myosin heavy chain (MHC) mRNA gene expression in the 41-week-CN and -BIO groups. MM, molecular marker; P, probe; (B) the relative level of bMHC mRNA expression in the 41-week-CN and -BIO groups. Values are expressed as mean±SE, statistical significance was assumed at * P < 0.05. MHC, myosin heavy chain; BIO, cardiomyopathic Syrian hamsters (BIO14.6); CN, congenic normal hamsters; Sed, sedentary; Inf, swimming in infancy from 3 to 10 weeks; Ado, swimming in adolescence from 16 to 23 weeks.

(Figure 4B, b). Scarcity of myofibrils, moderately degenerated mitochondria and dilated cavities in the sarcoplasmic reticulum were seen in the BIO-Ado (Figure 4B, c). MHC mRNA expression A representative S1 mapping analysis of the a- and bMHC mRNAs and the relative level of b (b/b + a)MHC mRNA expression of the 41-week-CN and BIO groups are shown in Figure 5A and B. The relative level of b-MHC mRNA in the BIO-Sed and BIO-Ado was greater than that in the CN-Sed. Interestingly, the ratio of b-MHC mRNA in the BIO-Inf (25.7 ± 5.5%) was markedly decreased compared to that of the BIO-Sed and significantly lower than that of the BIO-Ado (P < 0.05). The b-MHC mRNA ratio of the BIO-Inf was comparable with that of the CN-Sed. Calcineurin phosphatase activity The activity of all 41-week-BIO groups was as much as that of the swimming group in the 10-week-CN and

Cardiac hypertrophy of the Inf was more prominent than that of the age-matched Sed and Ado in the 41week-CN and -BIO hamsters. Since younger animals have been reported to adapt more easily to exercise than older ones (Chesky et al., 1983), cardiac morphological adaptation to exercise might be greater in infancy than in adolescence in cardiomyopathic hamsters as well. It has been reported that the enzymatic activity of mitochondrial creatinine kinase, which plays an important role in cytoplasmic energy production was decreased by aging in the BIO (Matsuo, 1991). Long-term exercise has been reported to have effects of increasing the activity of antioxidant enzymes and reducing oxidant production, whereas acute exercise increases oxidant levels and oxidative stress (Atalay and Sen 1999; Leeuwenburgh and Heinecke 2001). In our study, the differences in the cardiac tissue from the BIO-41-week groups studied by light microscopy were not so remarkable; however, those examined by electron microscopy were prominent. The mitochondrial morphology in the 41-week-BIO-Inf remained intact, whereas degenerative mitochondrial change was seen in the cardiac tissue of the 41-week-BIO-Sed and -Ado. The mechanism by which exercise may have altered the biochemistry in the three groups may be that upregulation of antioxidant enzymes and reduction of oxidant production might accompany swimming during infancy, suggesting that cellular energy metabolism (Mallet and Sun 1999) as mitochondrial function was kept intact. On the other hand, swimming during adolescence might have harmful effects, such as increasing oxidant levels that cause oxidative damage. In our study, swimming exercise in infancy worked as a beneficial effect of exercise and it might prevent a decrease in energy production (Mallet and Sun 1999) in the hearts of 41week-cardiomyopathic hamsters.

75 Effects of exercise on MHC mRNA expression To understand the potential functional significance of altered MHC composition in contractility, we evaluated the MHC mRNA expression. Changes in the relative amounts of MHC isoforms are believed to be responsible for altered cardiac performance during hypertrophy and heart failure (Bugaisky et al., 1992). In BIO14.6, it has been reported that the relative level of b-MHC mRNA in cardiac muscles increased with cardiac heart failure and aging (Momomura et al., 1991). In the present study, the relative level of aMHC (a/a + b) mRNA in cardiomyocytes of the 41week-BIO-Inf was high. Since a-MHC has a higher adenosine 5¢-triphosphatase (ATPase) activity than bMHC and is associated with a greater shortening velocity (Schwartz et al., 1981) our finding suggests that cardiac muscle in 41-week-BIO-Inf increases contractility. In addition, degenerative mitochondrial change was not seen in 41-week-BIO-Inf. These results suggest that even in the hearts of hamsters with a genetic deficiency, exercise in infancy may help to maintain cardiac contractility and energy production as in age-matched normal hearts. However, the relative level of b-MHC mRNA in the 41-week-BIO-Ado was as high as that in the 41-week-BIO-Sed and mitochondrial degenerative change in the 41-week-BIO-Ado was severer than in the 41-week-BIO-Sed. According to our results, in contrast with the findings in infancy, exercise performed in adolescence did not apparently show any beneficial effects on cardiac muscle in the 41-week-BIO. This may be because the responses of the cardiac muscle to exercise had been altered by aging. Therefore, exercise in infancy may be beneficial even for cardiomyopathic patients. However, three (25%, 3/12) of the hamsters in the BIO-Inf group subjected to swimming died several weeks after the training. Since no abnormal finding other than cardiomyopathy was seen at autopsy, the training program used in this study might have been too hard for them. These results suggest that the exercise intensity is crucial when using exercise as a therapeutic approach. Effects of exercise on calcineurin activity Calcineurin was initially identified as an important regulator of cardiomyocyte hypertrophy in vivo and in vitro (De Windt et al., 2000). More recent investigation has suggested a role for calcineurin in the regulation of cardiomyocyte apoptosis. Kakita et al. (2001) reported that calcineurin activation promoted an increase in Bcl-2 expression, which suggests downregulation of apoptosis by the enhancement of mitochondrial membrane stability. In contrast, Saito et al. (2000) reported that transgenic mice expressing dominant-negative calcineurin in the heart show increased apoptosis in response to ischemia/reperfusion injury. However, the role that calcineurin plays in regulating cardiomyocyte apoptosis and the potential modulator effects of parallel

regulatory pathways has not been investigated (Molkentin, 2001). The significant increase in calcineurin activity in the swimming 10-week-CN compared with the age-matched sedentary CN is consistent with a study in rats reported by (Eto et al., 2000). They indicated that in normal rats, the calcineurin activity of exercised (voluntary running) animals exhibiting physiological hypertrophy was markedly increased compared with that of sedentary ones. However, in our study, in contrast to the CN, the calcineurin activity in the sedentary and swimming groups was almost the same in the 10-week-BIO. We speculate that the reason for the different response of calcineurin activity between the swimming group of 10-week-CN and 10-week-BIO hamsters is that the cardiac muscle in BIO14.6 is deficient in the gene for d-sarcoglycan, which leads to pathological hypertrophy in the cardiomyocytes. Since d-sarcoglycan helps to stabilize the sarcolemma, cardiac muscle in the BIO may be more susceptible to mechanical stress generated by cardiac contraction (Sakamoto et al., 1997). Furthermore, the level of calcineurin activity in the sedentary 10-week-BIO was already at saturation point, since it was almost the same as that in 41-week-BIO-Sed. These results indicate that pathological hypertrophy may have already started in the 10week-BIO. In humans, the activity level of calcineurin in patients with heart failure is controversial. Lim and Molkentin (1999) reported significant increases in calcineurin activity, whereas Tsao et al. (2000) showed a decrease. Further studies on the role of calcineurin activity in hearts with heart failure are required. In conclusion, the present study demonstrated that exercise performed in infancy might maintain cardiac contractility and energy production until 41 weeks in cardiomyopathic hamsters.

Acknowledgements We thank Dr Minamisawa for critical reading of the manuscript. We also thank Ms Barbara Levene for English correction of the manuscript. This work was supported in part by the Nippon Foundation (1998) and by the Grant for the Promotion of the Advancement of Education and Research in Graduate Schools (2000– 2001) and by a grant for the Promotion of the Advancement of Education and Research in Graduate Schools (1998–2002).

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