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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 3, 2006 as doi:10.1096/fj.05-5116fje. |
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* Redox Biology and Cell Signaling Laboratory, Texas A&M University, College Station, Texas, USA;
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
1Correspondence: Redox Biology and Cell Signaling Laboratory, Department of Health and Kinesiology, Texas A&M University, College Station, TX 77843-4243, USA. E-mail: jml2621{at}neo.tamu.edu
SPECIFIC AIMS
Aging causes progressive remodeling of the heart that includes loss of cardiac myocytes through apoptosis and necrosis, reactive hypertrophy of the remaining myocytes, and increased connective tissue. Endurance exercise may be protective of heart function in the elderly, although the mechanisms are not well understood. We sought to determine whether 12 wk of exercise training would attenuate remodeling, apoptosis, and signaling through the Bcl-2 apoptotic pathway in left ventricle of the aging rat heart. The current study provides the first evidence that regular exercise ameliorates age-associated loss of myocytes, increased apoptosis, elevated proapoptotic signaling through the Bcl-2 pathway, and increased connective tissue.
PRINCIPAL FINDINGS
1. Twelve wk of exercise training attenuates loss of myocytes/area and reactive hypertrophy in the aging rat left ventricle
Three- and 24-month-old Fischer-344 rats (n=20 in each age group) were divided into sedentary and exercise training groups. Rats in the exercise training groups ran for 12 wk (5 days/wk) on a motorized treadmill for 60 min/day at an intensity of 75% of VO2max for the old group. After the training period at 6 and 27 months of age, the rats were anesthetized with 50 mg/kg Na pentobarbital and the hearts removed. The left ventricles were dissected, frozen in liquid nitrogen, and used for protein and histological analyses.
Cross sections of left ventricles were stained with hematoxylin for morphological analysis in 4 groups: young (6 month) sedentary (YS), young exercise trained (YE), old (27 month) sedentary (OS), and old exercise trained (OT). Heart mass was not significantly different for either age or exercise. However, exercise resulted in a substantial attenuation of ventricular remodeling in the aging heart (Fig. 1
). Mean (±SE) myocyte number per unit area of the left ventricle was 89.3 ± 3.5 cells/100,000 µm2 in YS. Myocyte number did not differ in the left ventricle between young sedentary and exercise-trained (89.0±3.1 cells/100,000 µm2). Mean myocyte number per cross-sectional area was 55% lower in the old, sedentary group (40.4±6.2 cells/100,000 µm2) than the young sedentary group (Fig. 2
). Mean left ventricle myocyte number per cross-sectional area was 54% greater in the old rats that exercised (62.5±2.4 cells/100,000 µm2) than old rats that were sedentary. We also calculated the number of myonuclei/cell area. Given no significant change in heart mass and thus volume in our animals, these data indicate that exercise training provided significant, partial protection against cell loss. Young sedentary and young exercise left ventricles averaged 91 myonuclei/100,000 µm2 and 93 myonuclei/100,000 µm2, respectively. However, left ventricles from old exhibited far fewer myonuclei per unit area (55 myonuclei/100,000 µm2) than in young rats. Conversely, regular exercise resulted in partial protection against the reduction of myonuclei (69 myonuclei/100,000 µm2).
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Myocyte cross-sectional area increased 492% from 320.5 ± 10.6 µm2 in YS to 2216.5 ± 270.2 µm2 in OS, indicating reactive hypertrophy associated with cell loss and remodeling. Conversely, exercise significantly reduced myocyte cross-sectional area in the older age group by 69% to 690.0 ± 34.2 µm2 in the old age group. These data indicate that 12 wk of treadmill exercise retarded myocyte hypertrophy in the aging heart.
2. Exercise training reduces age-associated accumulation of connective tissue and remodeling of the left ventricle
Accumulation of connective tissue is typical of age-induced remodeling of the heart. Indeed, left ventricles from old, sedentary rats possessed more connective tissue area (20.7±3.6%) than left ventricles from young, sedentary animals (7.4±1.8%). Exercise training did not alter amount of connective tissue per unit area in left ventricles from young rats. However, exercise did retard elevation of connective tissue content in the left ventricle of old rats, with 9.2 ± 2.5% of left ventricle area being comprised of connective tissue, down from over 20.7% in OS. In addition, the number of fibroblast nuclei in the left ventricle increased with age, but was decreased from 185 nuclei/100,000 µm2 to 111 nuclei/100,000 µm2 by exercise training in the old group.
The geometric pattern of connective tissue was far more linear and regular in the old exercise group than the old sedentary heart. For example, the geometry of connective tissue in the aging sedentary heart was more web-like (Fig. 1)
than discrete sheaths, with a long axis bounded by connective tissue, typical in heart of young rats. However, exercise resulted in a geometric pattern left ventricles from the old group that was more similar to young exercised hearts than old sedentary animals.
3. Exercise training attenuates age-induced increases in TUNEL+ staining and DNA fragmentation in left ventricle
Histone-associated DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive fluorescence were used as direct markers of apoptosis. Aging significantly increased histone-associated DNA fragmentation in the left ventricle by 62% in OS compared with YS (Fig. 2)
. Conversely, exercise training attenuated DNA fragmentation (–44.0%) in the left ventricle of the old age group, but not in the young age group.
Apoptotic nuclei were visualized using the TUNEL method and fluorescence microscopy. DAPI (4,6-diamidino-2-phenylindole) was used to mark all nuclei. TUNEL+ nuclei in the left ventricles of young sedentary rats accounted for 0.24% of all nuclei, while 0.47% of DAPI+ nuclei were TUNEL+ in the young exercise group. Left ventricles from the old sedentary group displayed a far higher incidence (3.32%) of TUNEL+ nuclei. However, exercise training greatly reduced the rate of myocytes nuclei undergoing, with 1.62% found as TUNEL+ in OE. These indicate that exercise training ameliorates the apoptotic process of myocytes in the left ventricle of aging hearts.
4. Exercise training ameliorates caspase-3 cleavage in the aging left ventricle
Caspases are cysteine-dependent aspartate-specific proteases functioning as endonucleases integral in the final execution of nuclei and cell death. A critical step in the final execution of the apoptotic program eliciting DNA fragmentation is the cleavage of caspase-3. Caspase-3 cleavage is downstream of the Bcl-2 family apoptotic cascade and may integrate apoptotic signaling from the cytokine/fas and Ca2+/ER pathways as well. Low levels of cleaved caspase-3 were detected in the left ventricles of young sedentary animals. In addition, the 17 kDa subunit was undetectable in both young sedentary and young exercise trained groups. In contrast, cleaved caspase-3 levels were dramatically increased (+123%) by aging in the left ventricle, primarily affecting the 17 kDa fragment. Exercise training resulted in significant diminishment (–33%) of cleaved caspase-3 protein expression, with the 17 kDa fragment again most affected.
5. Exercise training reduces Bcl-2 pathway signaling in the aging left ventricle
We then tested the hypothesis that endurance exercise would attenuate the effect of aging on Bcl-2 family (e.g., caspase-9, Bax/Bcl-2 ratio) upstream to caspase-3. Aging increased protein expression (+89%) of caspase-9 in the rat left ventricle. Conversely, exercise training resulted in a down-regulation of caspase-9 levels in left ventricle samples from both the young (–52.6%) and old (–74%) age groups.
In addition, protein expression of proapoptotic Bax in the left ventricle was much higher (+176%) in the old sedentary group than in the young sedentary group. In contrast, exercise training resulted in a significant decrease (–53%) in Bax protein expression of the left ventricle in the old trained group when compared with old controls. Bax protein expression was not different between young sedentary controls and young trained rats. Levels of antiapoptotic Bcl-2 protein in the left ventricle tended to decrease by 26.3% with aging. However, exercise training in the old age group resulted in a significant up-regulation of Bcl-2 protein expression (+118%) in the left ventricle. There was no significant exercise effect on Bcl-2 protein expression in the young group. Thus the calculated Bax/Bcl-2 ratio for the left ventricle was 272% higher in the old sedentary group vs. the young adult sedentary group. Exercise training attenuated Bax/Bcl-2 ratio markedly (–79%) in the aging left ventricle. In contrast, there was no significant effect of exercise on Bax/Bcl-2 ratio in the young age group. Our findings are consistent with the hypothesis that alterations in Bax/Bcl-2 ratio with exercise regulate downstream caspase-driven apoptosis in the aging heart.
CONCLUSIONS AND SIGNIFICANCE
In conclusion, 12 wk of exercise training provided protection against age-induced remodeling of the left ventricle that includes myocyte loss, reactive hypertrophy of the remaining cells and increased connective tissue in the aging heart. Regular exercise also down-regulated markers of apoptosis (TUNEL+ nuclei, DNA fragmentation, cleaved caspase-3) in concert with a reduction in caspase-9 protein expression and Bax/Bcl-2 ratio. Our findings are consistent with the hypothesis that exercise training modulation of the mitochondrial Bcl-2 pathway can ameliorate age-induced apoptosis and ventricular remodeling.
Impairment of mitochondrial and contractile function is a hallmark of aging. A major pathway leading to progressive apoptosis in the aging heart is associated with the mitochondria and the Bcl-2 family pathway. Aging also increases the rate of apoptosis through the Bcl-2 pathway caused by the anticancer drug doxorubicin. Although apoptosis is important in regulating development, cell proliferation, and removal of precancerous cells in adult mitotic tissues (e.g., liver, kidney), dysregulation of apoptosis is now recognized as a mechanism fundamental to numerous pathologies. Excessive cell loss in predominantly postmitotic tissues such as the heart is dire, since ability of the heart to replace removed lost cells with adult stem cells appears to be very poor with advancing age. Our findings indicate that exercise training can retard the processes of apoptosis, cell loss, and remodeling in the aging heart. These data should lead toward investigation of potential mechanisms by which exercise training can ameliorate cell loss in the aging heart, including reduction of oxidative stress and up-regulation of signaling through cell protective stress proteins (Fig. 3
). Once the specific cellular signaling mechanisms by which exercise confers protection in the aging heart are better understood, then more effective combination therapeutics can be developed that will reduce impairment of cardiac function.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5116fje;
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