Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins.

Rolf Schreckenberg,Rainer Schulz,Klaus‐Dieter Schlüter,Kamilla Gömöri,Hanna Sarah Kutsche,Johann Klein,Éva Sághy,Péter Ferdinandy,Bettina Benczik,Bence Ágg,Péter Bencsik

doi:10.1111/jcmm.15209

Abstract

Ischaemic post‐conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non‐responsiveness to IPoC possibly caused by co‐morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC‐R) and non‐responder (IPoC‐NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre‐conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT‐PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC‐R) or no protection (IPoC‐NR). Twenty‐one miRs were up‐regulated by I/R and modified by IPoC. qRT‐PCR analysis revealed that IPoC‐R differed from other groups by reduced expression of arginase‐2 and bax, whereas the mitochondrial uncoupling protein (UCP)‐2 was induced in IPC and IPoC‐R. IPoC‐R and IPoC‐NR synergistically increased the expression of non‐mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium‐handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC‐NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC‐R. This study underlines the importance of mitochondrial proteins for successful long‐term outcome.

Highlights

Today, early reperfusion is the key strategy to overcome acute ischaemia/reperfusion (I/R) injury
The most important finding of our study is that ischaemic post-conditioning (IPoC) in healthy rats leads to a heterogeneous responsiveness as monitored by an infarct size surrogate parameter
Even in those cases in which IPoC was associated with small infarcts, IPoC differed from ischaemic pre-conditioning (IPC), which was always cardioprotective in the same animal model

Summary

| INTRODUCTION

Early reperfusion is the key strategy to overcome acute ischaemia/reperfusion (I/R) injury. In order to explain the large outcome differences in pre-clinical studies, it may be argued that differences between protocols, source of animals, sex and age of animals, animal handling and other variables are responsible for inconsistent results. To overcome these limitations, we started a study in which experimental variables were minimized. A 5-0 Prolene suture (Ethicon, Johnson & Johnson Kft. Hungary) was placed around LAD artery and a small plastic knob, which was threaded through the ligature and placed in contact with the heart, was used for making occlusion for 30 minutes. Animals were randomly assigned to four experimental groups: (a) sham operated, (b) ischaemic control, (c) ischaemic pre-conditioning (IPC) and (d) ischaemic post-conditioning (IPoC). The study protocol confirm to the recommendation given previously.[20]

| MATERIALS AND METHODS

Findings

| DISCUSSION