Cardiolipin as an oxidative target in cardiac mitochondria in the aged rat

Abstract

The aged heart sustains greater injury during ischemia (ISC) and reperfusion (REP) compared to the adult heart. In the Fischer 344 (F344) rat, aging decreases oxidative phosphorylation and complex III activity increasing the production of reactive oxygen species in interfibrillar mitochondria (IFM) located among the myofibrils. In the isolated, perfused 24 month old elderly F344 rat heart 25 min of stop-flow ISC causes additional damage to complex III, further decreasing the rate of oxidative phosphorylation. We did not observe further progressive mitochondrial damage during REP. We next asked if ISC or REP increased oxidative damage within mitochondria of the aged heart. Cardiolipin (CL) is a phospholipid unique to mitochondria consisting predominantly of four linoleic acid residues (C18:2). Following ISC and REP in the aged heart, there is a new CL species containing three oxygen atoms added to one linoleic residue. ISC alone was sufficient to generate this new oxidized molecular species of CL. Based upon oxidative damage to CL, complex III activity, and oxidative phosphorylation, mitochondrial damage thus occurs in the aged heart mainly during ISC, rather than during REP. Mitochondrial damage during ischemia sets the stage for mitochondrial-driven cardiomyocyte injury during reperfusion in the aged heart.

Figure 1

Ischemia markedly decreases the maximal ADP-stimulated rate of oxidative phosphorylation in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria obtained from the 24 month old Fischer 344 rat heart. Reperfusion does not result in additional decreases in the rate of oxidative phosphorylation. Decreases are observed with glutamate (A), duroquinol (B), and TMPD-ascorbate (C) as substrates. The decrease in the rate of respiration are similar in the mitochondria isolated both following 25′ ischemia and following 25′ ischemia plus 30′ reperfusion. (Mean ± SD; * p<0.05; p=NS Ischemia vs. Ischemia-Reperfusion.)

Figure 1

Ischemia markedly decreases the maximal ADP-stimulated rate of oxidative phosphorylation in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria obtained from the 24 month old Fischer 344 rat heart. Reperfusion does not result in additional decreases in the rate of oxidative phosphorylation. Decreases are observed with glutamate (A), duroquinol (B), and TMPD-ascorbate (C) as substrates. The decrease in the rate of respiration are similar in the mitochondria isolated both following 25′ ischemia and following 25′ ischemia plus 30′ reperfusion. (Mean ± SD; * p<0.05; p=NS Ischemia vs. Ischemia-Reperfusion.)

Figure 1

Ischemia markedly decreases the maximal ADP-stimulated rate of oxidative phosphorylation in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria obtained from the 24 month old Fischer 344 rat heart. Reperfusion does not result in additional decreases in the rate of oxidative phosphorylation. Decreases are observed with glutamate (A), duroquinol (B), and TMPD-ascorbate (C) as substrates. The decrease in the rate of respiration are similar in the mitochondria isolated both following 25′ ischemia and following 25′ ischemia plus 30′ reperfusion. (Mean ± SD; * p<0.05; p=NS Ischemia vs. Ischemia-Reperfusion.)

Figure 2

Ischemia markedly decreases complex III activity (decylubiquinone:cytochrome c reductase) in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria obtained from the 24 month old Fischer 344 rat heart. Reperfusion does not result in additional decreases in complex III activity. The decrease in the rate of respiration are similar in the mitochondria isolated both following 25′ ischemia and following 25′ ischemia plus 30′ reperfusion. (Mean ± SD; * p<0.05; p=NS Ischemia vs. Ischemia-Reperfusion.)

Figure 3

Electrospray ionization mass spectrometry of cardiolipin molecular species separated by reverse phase HPLC (see Methods). (A) Cardiolipin from IFM from an adult rat following ischemia and reperfusion Fragmentation pattern of the 1486 amu (K⁺-adduct of the 1448-intact cardiolipin) major molecular species demonstrating the mass spectral “foot print”: 869-glycerophosphatidic acid fragment; 607-phosphatidic acid; 415-lysophosphatidic acid; 279-linoleic acid. (B) Cardiolipin from IFM from an aged rat following ischemia and reperfusion Fragmentation pattern of the new 1535 amu (K⁺-adduct of the 1496-intact cardiolipin) molecular species demonstrating the mass spectral “foot print”: ; 917-glycerophosphatidic acid fragment; 655-phosphatidic acid; 453-lysophosphatidic acid; 327-oxidized linoleic acid. The collision-induced dissociation demonstrates that the +48 addition tracks thorugh each fragment to the acyl-group.

Figure 3

Electrospray ionization mass spectrometry of cardiolipin molecular species separated by reverse phase HPLC (see Methods). (A) Cardiolipin from IFM from an adult rat following ischemia and reperfusion Fragmentation pattern of the 1486 amu (K⁺-adduct of the 1448-intact cardiolipin) major molecular species demonstrating the mass spectral “foot print”: 869-glycerophosphatidic acid fragment; 607-phosphatidic acid; 415-lysophosphatidic acid; 279-linoleic acid. (B) Cardiolipin from IFM from an aged rat following ischemia and reperfusion Fragmentation pattern of the new 1535 amu (K⁺-adduct of the 1496-intact cardiolipin) molecular species demonstrating the mass spectral “foot print”: ; 917-glycerophosphatidic acid fragment; 655-phosphatidic acid; 453-lysophosphatidic acid; 327-oxidized linoleic acid. The collision-induced dissociation demonstrates that the +48 addition tracks thorugh each fragment to the acyl-group.