Chronic reperfusion mediates restoring the integrity of c‐type cytochrome, limiting superoxide overproduction, and conferring reconditioning mitochondrial function in the postischemic heart
Authors: Chen Yeong-Renn , Chen Chwen-Lih 
Published: April 2020

Background

A serious consequence of acute myocardial ischemia‐reperfusion injury (acute I/R) is oxidative damage which causes mitochondrial dysfunction. Such I/R‐induced mitochondrial dysfunction is observed as impaired state‐3 respiration and overproduction of superoxide. The cascading ROS (reactive oxygen species) propagates heme damage on the array of mitochondrial electron transport chain and cytochrome c (cytc), adding insult to myocardial injury. Chronic reperfusion partially restores mitochondrial function in the postischemic myocardium.

Methods

We employed proteomic analysis with native polyacrylamide gel electrophoresis (native‐PAGE) and LC‐MS/MS to identify heme impairment involved in interrupting the thioether bond formation between c‐type heme and holocytochrome focusing on two key mitochondrial ETC components: complex III (CxIII) and cyt c in the mitochondria of sham control as well as the mitochondria isolated from the infarct region of rat hearts subjected to 30‐min of coronary ligation and 24‐h (acute I/R) and 4‐week (chronic I/R) of reperfusion in vivo. The profile of heme in the mitochondria was assessed by differential UV/VIS spectral analysis. Ubiquinol (Q2H2)‐mediated SOD1‐inhibitable cytochrome c reduction was used to assay superoxide generation by the CxIII in mitochondria.

Results

We detected carbamidomethylated C122 and C125 of the cytochrome c1 subunit of CxIII from alkylation of Cx3 exclusively in the mitochondria of acute I/R heart [detection of the tryptic peptide ion of CxxCH motif: 120QVC122(cam)SSC125(cam)HSMDYVAYR134, m/z 931.88512+, cam denotes carbamidomethylation by iodoacetamide]. The result was consistent with defect of thioether bonds formation of heme c1 and heme damage indicated by UV/VIS spectral analysis of mitochondria following acute I/R. Mitochondria from the infarct region of acute I/R have shown impaired enzymatic activity of CxIII and increased Q2H2‐mediated superoxide generation by CxIII. Acute I/R also mediated down‐regulation of cytochrome c heme lyase or
holo
cytochrome
c
synthetase (HCCS), further aggravating CxIII injury and associated mitochondrial dysfunction caused by thioether defect of c‐type cytochrome. In the mitochondria of risk region after chronic I/R (4‐week postischemic reperfusion), we have observed partial restoration of CxIII activity, protein expression of HCCS, integrity of c‐type heme, and decreasing Q2H2‐mediated superoxide generation. However, we have further observed that chronic reperfusion significantly down regulated the ratio of c‐type heme per c‐type cytochrome and associated c‐type cytochrome biogenesis.

Conclusion

Acute I/R mediates CxIII injury and aggravates mitochondrial dysfunction via impairing the thioether bonds of c‐type heme in the ETC components and increasing ROS production, whereas chronic reperfusion restores the integrity of c‐type cytochrome with downregulation of its biogenesis and limits ROS produciton.

Support or Funding Information

NHLBI/NIH, HL083237