Abstract 12792: Post-oxidative Protein Modification of Mitochondrial Bioenergetic Enzyme Activity in Human Failing Hearts
The aim of this study was to examine whether deficits in the abundance and activity of key mitochondrial enzymes contribute to the diminished bioenergetic capacity purported in human heart failure. Left ventricular free-wall was biopsied from non-failing donor hearts (n=20) and end-stage failing hearts (n=20). Spectrophotometric enzyme activity assays were used to determine that complexes I and IV, the NADH-linked Krebs enzymes isocitrate dehydrogenase and malate dehydrogenase, and aconitase had diminished activity rates in failing hearts (by 27±8%, p=0.007; 37±9%, p=0.01; 40±6%, p=0.001: 20±7%, p=0.0001: 46±6%, p=0.03; respectively), whereas activity of complexes II, III and citrate synthase did not differ between failing and non-failing hearts. Specific protein abundance of each of these, determined by western blotting with their respective antibodies, did not differ between the non-failing and failing heart groups. Post-oxidative protein modification was explored as an underlying cause of enzyme dysfunction by initially measuring total protein carbonylation, which was greater by 31±4.5% (p=0.02) in failing myocardium, compared to non-failing. Subsequently, isolation and partial purification of complex I and IV subunits by immunocapture revealed that the 75, 51, 49 and 24 kDa subunits of complex I, and the 57 and 26 kDa subunits of complex IV, which contain iron-sulphur or heme redox centers were specific targets of post-oxidative modification by carbonylation and protein nitration. Notably the markedly lower mitochondrial activity rates in heart failure, relative to non-failing donor hearts, coincided with significantly higher levels of oxidized glutathione, lower glutathione reductase activity, and lower content of total Coenzyme Q10, cardiolipin, total adenine nucleotides, NADH and NADPH. In conclusion, the energy insufficiency of the failing human heart involves impaired activity of key mitochondrial enzyme subunits, at least in part due to post-oxidative modification.
- © 2010 by American Heart Association, Inc.