Cardiac Protection by Preconditioning Is Generated via an Iron-Signal Created by Proteasomal Degradation of Iron Proteins

<div><p>Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after <em>ex vivo</em> ischemia-and-reperfusion (I/R) were found in hearts from <em>in vivo</em> hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing <em>de-novo</em> apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.</p> </div

Hemodynamic parameters measured throughout the <i>ex vivo</i> procedure following <i>in vivo</i> hypoxic-preconditioning.

<p>a. Heart rate (beats per minutes). b. Developed pressure (mmHg). c. Working index (A.U. = heart rate x developed presure). Results are means±SE of 5–6 experiments. Hypoxia- white square, Normoxia- black square. Stab = stabilization period.</p

Ferritin and ferritin-bound iron levels in hearts subjected to I/R.

<p>Ferritin and ferritin-bound iron levels with or without prior IPC and a ‘delay’ period. a. Relative ferritin protein levels (% of the post stabilization period). b. Ferritin-bound iron (number of iron atoms per molecule of ferritin). c. Relative ferritin protein levels (% of the post stabilization period) in hearts infused with a cocktail of protease inhibitors (MG132-3 µmol/L, leupeptin-11.7 µmol/L, pepstatin-A-4.4 µmol/L): perfusion -black diamond, IPC+‘delay’ +I/R - black circle, ‘delay’+I/R- white square. Results are means±SE of 6 experiments. SE for perfusion and ‘delay’+I/R groups are too small to be seen.</p

Schematic illustration of the protocols for the experimental groups.

<p>Ischemia- dark gray background; 3 min during IPC or 35 min in the prolonged ischemia. Protease inhibitor cocktails- black oblique lines; 3 min before or after IPC. Heme-oxygenase inhibitor, SnMP - light gray background, 20 min before IPC. In vivo hypoxia (9% O2)- black background 8 h per day for 3 days.</p

Parameters for recovery of hearts subjected to 3 days of <i>in vivo</i> hypoxia followed by <i>ex vivo</i> I/R.

<p>Hemodynamic parameters; the heart’s hemodynamic parameters at the end of a 10 min stabilization period are considered 100%.</p><p>Ferritin levels given as µg/mg protein.</p><p>mRNA levels given as arbitrary units per unit β-actin.</p><p>Results are mean±SE. Numbers in parentheses represents the number of repetitious experiments.</p>*<p> = Significantly different from Normoxia (p<0.05).</p

Representative EMSA using extracts of hearts subjected to I/R with and without prior IPC.

<p>a. Original phosphorimaging blots. Upper panel shows active IRPs. Lower panel displays total IRPs (+βME). b. Graphic presentation of active IRP; three scanned blots each (±SE). Perfusion only- black, I/R- gray, IPC+ I/R- light gray. No efforts were made to distinguish between IRP1 and IRP2.</p

Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver.

<p>(A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in SDS-PAGE with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H₂O₂ (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.</p

HepG2 clones overexpressing heparanase showed a reduction of hepcidin expression and indices of iron loading.

<p>Two stable clones of HepG2 cells transfected with pcDNA3.1-HPA (HPA3 and HPA6) were analyzed for hepcidin expression, BMP/SMAD signaling and indices of iron status. (A) qPCR was assessed to analyze the level of HPA mRNA. (B) Western blot for HPA shows the level of the latent (65 kDa) and active (50 kDa) forms. Densitometry quantification of the two forms was performed in relation to actin as calibrator. (C) qPCR was performed to analyze the level of hepcidin mRNA, (D) and the level of Id1 mRNA in relation to Hprt1. (E) WB of phosphorylated SMAD5 and their densitometry quantification referred to actin and WB of total SMAD5, (F) WB of transferrin Receptor 1, Tfr1, and its densitometry, (G) WB of ferritin light chain, FTL and its densitometry; (H) WB of Ferroportin (FPN) and its densitometry. The values are expressed as–dCt (for HPA mRNA) or as fold change over the control (MOCK) (for hepcidin and Id1 mRNA). The images are representative from three different analyses</p

Transgenic mice overexpressing heparanase showed reduced liver hepcidin mRNA and serum protein.

<p>(A) Liver hepcidin mRNA levels of wild type (WT) and transgenic HPA mice (TG-HPA) normalized to Hprt1. The values are expressed as fold change of wild type mice. (B) Quantification of serum hepcidin by SELDI-TOF in the same mice.</p

Transgenic mice overexpressing Heparanase showed increased BMP6/SMAD signaling.

<p>(A) evaluation of BMP6 mRNA in the liver of WT and TG-HPA mice (B) evaluation of Id1 mRNA, in relation to Hprt1. (C) Western blot of pSMAD5, of total SMAD5 and of GAPDH as housekeeping for normalization in densitometry quantification. The values are expressed as fold change of wild type mice.</p