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Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue
Alfredo Grilli1, Maria Anna De Lutiis, Antonia Patruno
1Department of Biomorphology, University G. D'Annunzio, 66013 Chieti, Italy.
Abstract:
The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (iNOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of iNOS. Expression of iNOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). iNOS expression was evaluated through immunohistochemical techniques (Ab anti-iNOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of iNOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for iNOS in the same experimental conditions. In addition, the iNOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 +/- 0.01 vs 0.02 +/- 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating iNOS.
Insights
Chronic low oxygen tension in rat hearts induces apoptosis and activates inducible nitric oxide synthase (iNOS). This study reveals how hypoxia impacts cardiac cell death and iNOS expression, crucial for understanding heart conditions.
Area of Science:
- Cardiovascular Biology
- Cellular Physiology
- Molecular Medicine
Background:
- Cellular oxygen levels critically influence cardiac function.
- Hypoxia can trigger cellular stress responses, including apoptosis and altered gene expression.
- Inducible nitric oxide synthase (iNOS) plays a role in various physiological and pathological processes in the heart.
Purpose of the Study:
- To investigate the effects of chronic low oxygen tension on apoptosis in rat heart tissue.
- To determine the impact of hypoxia on the transcription and translation of inducible nitric oxide synthase (iNOS).
Main Methods:
- Rats were exposed to normoxic and hypoxic conditions for varying durations.
- Apoptosis was assessed using DNA fragmentation assays (electrophoresis, end labeling).
- iNOS expression and activity were evaluated via immunohistochemistry, Western blot, and RT-PCR.
Main Results:
- Hypoxic exposure induced oligonucleosomal DNA fragmentation, indicative of apoptosis.
- iNOS protein and mRNA expression were significantly elevated in hypoxic rat hearts.
- iNOS activity was markedly increased under low oxygen tension conditions.
Conclusions:
- Chronic exposure to low oxygen tension is a potent inducer of programmed cell death (apoptosis) in the heart.
- Hypoxia activates both the transcription and translation of inducible nitric oxide synthase (iNOS) in cardiac tissue.
- These findings highlight the role of hypoxia-induced iNOS activation and apoptosis in cardiac pathophysiology.