Malonate protection against acute infarction in isolated mouse hearts is lost in the presence of insulin
Authors: Wang Q , Xiao Y , Nederlof R , Bakker D , Hollmann M W , Weber N C , Zuurbier C J 
Published: November 2023

Abstract

Background & Objectives
Malonate, an inhibitor of mitochondrial succinate dehydrogenase (SDH), is a promising novel protectant against acute ischaemia-reperfusion injury (IRI) through reduction of ischaemic succinate accumulation and ROS production during early reperfusion. Malonate is still protective when given only at reperfusion, making it an ideal agent for treating AMI patients. However, numerous studies demonstrated that the metabolic milieu could affect the potency of cardioprotective treatments. Therefore, in this study, we explored whether and how the severity of cardiac I/R injury and cardioprotection by malonate is affected by the metabolic milieu. We hypothesized that protection by malonate is affected by metabolic milie.

Methods
Isolated Langendorff-perfused hearts (n=8 to 19 per group) of C57Bl/6N mice were perfused with perfusate to which substrates and hormones were added in a step-wise manner: 1) glucose (G), 2) glucose + glutamine (GG), 3) glucose + glutamine + palmitate (GGP), or 4) glucose + glutamine + palmitate + insulin (GGPI), and subjected to 30 min ischaemia (I) and 90 min reperfusion (R). Heart were treated with malonate (5mM) or vehicle (saline) during the first 5 min of reperfusion only. Cardiac function of the left ventricle was monitored by a balloon in the left ventricle. Coronary effluent was collected at 7 time points of reperfusion to detect the activity of lactate dehydrogenase (LDH) as an indicator of cell death. At the end reperfusion, hearts were used for infarct size determination through TTC staining. Data are presented as mean ± SD.

Results
Going from glucose-only perfusate to a perfusate containing all examined substrates and hormones, the severity of cardiac I/R injury increased: infarct size (%) went from 31.9 ± 10.9% to 37.7 ± 10.6% to 52.3 ± 14.2%, and finally to 68 ± 9.9 %. Similar trends for increased IRI were observed for LDH, End-Diastolic Pressure (EDP) and recovery of the Rate-Pressure Product (RPP). When compared with vehicle control, malonate significantly decreased IS% (control vs malonate: 31.9 ± 10.9% vs. 23.3 ± 4.8%, 37.7 ± 10.6% vs. 20.9 ± 8.5%, 68 ± 9.9% vs. 40.2 ± 9.2 %), and increased the RRP recovery (64.1 ± 16.6 %vs. 83.3 ± 17.9 %, 55.6 ± 11% vs. 72.3 ± 4%, 39.3 ± 15.6% vs. 55.9 ± 8.3%) in the G, GG and GGP group. The EDP was significantly decreased by malonate in the G and GG group (32.8 ± 14.3 mmHg vs. 20.9 ± 9.8 mmHg, 35.1 ± 8.7 mmHg vs. 22.2 ± 8.6 mmHg ). LDH was only significantly decreased by malonate in the GG group (35.2 ± 12.9 vs. 17.1 ± 7.7 U / GWW). Most importantly, adding insulin to the perfusate nullified protection by malonate for all four outcomes.

Conclusions
The severity of cardiac I/R injury is lowest with glucose-only perfusate, and increased with increased complexity of the metabolic milieu. Although malonate is a strong protectant against cardiac IRI for most metabolic milieus, in the isolated heart, protection is lost in the presence of insulin.