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Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart
E Szabados1, G M Fischer, F Gallyas
1Department of Biochemistry, University Medical School Pecs, Hungary.
Abstract:
Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.
Insights
Lipoamide, an antioxidant, protects the heart from ischemia-reperfusion injury by reducing reactive oxygen species (ROS) and halting poly-ADP-ribose polymerase (PARP) activation, thereby preserving cellular energy and function.
Area of Science:
- Cardiovascular Research
- Biochemistry
- Oxidative Stress Biology
Background:
- Poly-ADP-ribose polymerase (PARP) is implicated in oxidative cell damage.
- Ischemia-reperfusion injury increases reactive oxygen species (ROS), potentially activating PARP.
Purpose of the Study:
- To investigate if lipoamide, an antioxidant, can reverse ischemia-reperfusion-induced ADP-ribosylation and cell damage.
- To evaluate lipoamide's effect on ROS levels and PARP activation.
Main Methods:
- Isolated Langendorff hearts subjected to ischemia-reperfusion.
- Assays for ROS, malondialdehyde, DNA breaks, NAD+ content, and protein ADP-ribosylation.
- Western blot analysis for protein ADP-ribosylation.
Main Results:
- Ischemia-reperfusion increased ROS, malondialdehyde, DNA damage, and NAD+ catabolism, activating PARP.
- Lipoamide treatment reduced ROS, malondialdehyde, and DNA damage, abolished NAD+ catabolism, and reverted PARP activation.
- Lipoamide significantly decreased cell membrane damage and improved myocardial recovery.
Conclusions:
- Ischemia-reperfusion activates PARP via ROS and DNA breaks, leading to NAD+ depletion and cell damage.
- Lipoamide mitigates ischemia-reperfusion injury by reducing ROS, inhibiting PARP activation, and preventing membrane damage, thus improving postischemic heart recovery.