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Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats
1Department of Medicine, New York Medical College, Valhalla, NY, 10595, USA.
Abstract:
Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20+/-0.05; 12 months, 7.21+/-0.07; 24 months, 7.18+/-0.09. In contrast, diastolic Ca2+ determined by the Fura 2-AM method increased progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02). Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.
Insights
Myocyte apoptosis increases with age due to elevated diastolic calcium levels, which activate DNase I. This process occurs independently of p53, Bax, and Bcl-2 protein expression in aging rat hearts.
Area of Science:
- Cardiovascular Biology
- Cellular Aging
- Molecular Cardiology
Background:
- Myocyte apoptosis (programmed cell death) increases with age.
- Molecular mechanisms driving age-related myocyte apoptosis are not fully understood.
- Potential factors include altered calcium (Ca2+) homeostasis, pH, and expression of apoptosis-related proteins like p53 and Bcl-2 family members.
Purpose of the Study:
- To investigate age-related changes in intracellular pH, cytosolic Ca2+, DNase I and II activity, and the expression of p53, Bcl-2, and Bax in rat myocytes.
- To identify molecular events contributing to increased myocyte apoptosis with aging.
Main Methods:
- Isolation of ventricular myocytes from Fischer 344 rats at 3, 12, and 24 months of age.
- Measurement of intracellular pH using BCECF fluorescence.
- Quantification of diastolic Ca2+ using Fura 2-AM.
- Assay of DNase I and DNase II activity.
- Determination of p53, Bcl-2, and Bax protein expression via Western blotting and immunocytochemistry.
Main Results:
- Intracellular pH remained constant across all age groups.
- Diastolic Ca2+ levels progressively increased with age (from 3 to 24 months).
- DNase I activity significantly increased with age (3.2-fold), while DNase II activity remained stable.
- Expression levels of p53, Bax, and Bcl-2 proteins did not change significantly with age.
Conclusions:
- Age-related myocyte apoptosis is associated with increased diastolic Ca2+.
- Elevated diastolic Ca2+ may activate DNase I, leading to apoptosis.
- This apoptotic pathway appears independent of age-related changes in p53, Bax, and Bcl-2 expression.