Background/Purpose:

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage which is known as ischaemic reperfusion (I/R) injury and can account for up to 50% of cell death. Systemic Lupus Erythematosus (SLE) is a condition associated with a high burden of cardiovascular morbidity and mortality, when compared to the matched healthy population. Patients with SLE have circulating autoantibodies which have been linked to an increased risk of suffering an MI. Studies performed by other groups have shown accelerated I/R injury in other systems such as mesenteric I/R injury in a lupus mouse model, however to date there has been no research focusing on the heart. The purpose of this study is to assess I/R injury in the presence of polyclonal IgG from patients with SLE in an in vitro model of simulated myocardial I/R injury in neonatal rat cardiomyocytes.

Methods:

Polyclonal IgG was isolated by protein G purification from serum of patients with SLE (n=23) and healthy controls (n=11) which were age and gender matched. Endotoxin was removed using Detoxi-Gel columns to a level below 0.225 endotoxin U/mL. It was observed that cells treated with LPS at this level did not have altered caspase-3 cleavage. Fetal calf serum used in the buffers was  also heat inactivated to remove complement mediated mechanisms. We utilised an established in vitro model of anoxia/reoxygenation to I/R injury. Cardiomyocytes were isolated from 1-2 day old rat pups and when beating synchronously were treated with 500 µg/ml polyclonal IgG from each group and the following day exposed to simulated I/R injury for 4hr in a hypoxic chamber (argon, 5% CO₂) followed by 4hr of reoxygenation. Apoptosis was measured by assessment of caspase-3 cleavage using immunoblot.

Results:

In cells exposed to simulated I/R injury caspase-3 cleavage was not significantly increased in the presence of IgG from healthy volunteers (mean increase in caspase-3 cleavage of cells treated with healthy control IgG  above untreated cell baseline – 12.28% ± SD 26.01, n=11). However, in the presence of IgG from patients with SLE, caspase-3 cleavage was increased by 54.79% (±SD 28.9, n=23) above untreated cell baseline and therefore significantly increased in comparison to healthy controls (p=0.0016). The effect observed with IgG from SLE patients was not altered when serum used in the buffers was heat inactivated suggesting a non-complement mediated mechanism.  

Conclusion: In this in vitro simulated I/R injury model IgG purified from patients with SLE enhanced I/R injury as assessed by caspase-3 cleavage. This novel pathogenic role of these antibodies will now be tested in vivo to validate this finding and explore potential mechanisms of action.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/purified-igg-from-patients-with-systemic-lupus-erythematosus-enhances-apoptosis-in-neonatal-rat-cardiomyocytes-exposed-to-simulated-myocardial-ischaemicreperfusion-injury/