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Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart
M Nakamura1, N P Wang, Z Q Zhao
1Department of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA 30365-2225, USA.
Objective:
Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins.
Methods:
Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n = 10) or without IPC (n = 8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay.
Results:
IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6 +/- 2.8 to 3.4 +/- 0.9 (P < 0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm2 myocardium) in the ischemic zone from 243 +/- 19 to 118 +/- 19 (P < 0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r = 0.8, P < 0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204 +/- 57% in the control group to 76 +/- 7% (P < 0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group.
Conclusion:
These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax.
Insights
Ischemic preconditioning (IPC) significantly reduces heart cell death after blood flow is restored. IPC achieves this by decreasing neutrophil accumulation and lowering Bax protein levels, protecting the heart from injury.
Area of Science:
- Cardiovascular Science
- Cellular Biology
- Pathophysiology
Background:
- Ischemic preconditioning (IPC) is a phenomenon where brief periods of ischemia followed by reperfusion protect the myocardium from subsequent longer ischemic insults.
- Myocardial apoptosis (programmed cell death) is a significant contributor to heart damage following ischemia and reperfusion.
- Neutrophil (PMN) accumulation and the expression of apoptosis-related proteins like Bcl-2 and Bax are implicated in myocardial injury.
Purpose of the Study:
- To investigate the protective mechanisms of IPC against ischemia/reperfusion-induced myocardial apoptosis.
- To determine if IPC inhibits neutrophil accumulation in the ischemic myocardium.
- To analyze the effect of IPC on the expression of Bcl-2 and Bax proteins in the context of myocardial apoptosis.
Main Methods:
- Rats underwent 30 minutes of coronary artery occlusion followed by 180 minutes of reperfusion, with or without IPC.
- Myocardial apoptosis was assessed using the TUNEL assay and DNA laddering.
- Neutrophil infiltration was quantified via immunohistochemistry (CD18 antibody).
- Bcl-2 and Bax protein expression levels were determined using Western blot analysis.
Main Results:
- IPC significantly reduced the percentage of TUNEL-positive cells in the ischemic myocardium (28.6% to 3.4%).
- IPC markedly attenuated neutrophil accumulation in the ischemic zone (243 cells/mm² to 118 cells/mm²).
- A strong positive correlation was observed between TUNEL-positive cells and neutrophil accumulation, which was reduced by IPC.
- IPC significantly downregulated the expression of Bax protein, while Bcl-2 expression remained unchanged.
Conclusions:
- Ischemic preconditioning effectively reduces myocardial apoptosis following ischemia and reperfusion.
- The cardioprotective effects of IPC are associated with the inhibition of neutrophil accumulation.
- Down-regulation of Bax protein expression by IPC contributes to the reduction in myocardial apoptosis.