Intermedin protects against ischaemia and reperfusion injury in human ventricular cardiomyocytes: receptor subtype involvement

Abstract

Purpose: Early reperfusion following myocardial infarction is essential but results in ischaemia/reperfusion (I/R) injury, whereby myocardial cells die largely due to oxidative stress. Intermedin (IMD; adrenomedullin-2), a novel peptide related to adrenomedullin (AM), is up-regulated in cardiovascular disease and is protective against I/R injury in rodent hearts and human coronary microvascular endothelial cells and cardiac non-myocytes. Protection is dependent on calcitonin receptor-like receptor (CRLR) signalling in association with one of three receptor activity-modifying proteins (RAMPs). IMD has yet to be investigated in human cardiomyocytes for a cardioprotective role. Aims: To identify if IMD and its receptor components are expressed in human ventricular cardiomyocytes; to investigate whether IMD protects these cells against simulated I/R injury and if so, determine receptor subtype involvement. Methods: Left ventricular adult human cardiomyocytes (HCM) were subjected to normoxia or simulated ischaemia for 4 h or I/R (3 h normoxia followed by 1 h reperfusion) all in the absence or presence of IMD. Cell viability was assessed using Trypan blue dye exclusion and oxidative damage by commercial Oxyblot assay. mRNA expression and protein distribution of IMD, AM, CRLR and RAMPs1-3 were assessed by Quantitative Real-Time PCR and indirect immunofluorescence microscopy, respectively. The silencing of the peptides or their receptor components using siRNA knock-down was employed to determine endogenous peptide and receptor subtype involvement in protection against I/R. Results: IMD, AM and their receptor component mRNAs were expressed in HCM; RAMP2 was the most abundant RAMP, while IMD was less abundantly expressed than AM. Ischaemia or I/R reduced the HCM cell count by 40% and 60% respectively (p<0.05 for both). IMD (10-9 mol.L-1) protected against the deleterious effects of ischaemia and I/R injury (∼20% salvaged for both conditions, p<0.05), with preservation of cell structure when examined by phase contrast microscopy and decreased oxidative stress noted on Oxyblot (p<0.001 vs. non-IMD counterpart for ischaemia or I/R). Protection was greatest when IMD was given during reperfusion (p<0.001) and was abolished by silencing CRLR (p<0.05) and RAMP2 (p<0.001). However, silencing endogenous IMD and/or AM expression did not augment extent of I/R injury in HCM. Conclusion: Exogenous IMD is protective against I/R in HCM, attributed to AM1 receptor subtype involvement. IMD administration during cardiac reperfusion could improve levels of morbidity and mortality in patients following myocardial infarction.