Figure 5.

Ca²⁺-dependent activation of phospholipase(s) in non-failing human heart mitochondria releases arachidonic acid, which is dramatically attenuated in failing human heart mitochondria. Isolated mitochondria from human non-failing control (n = 7) and failing (n = 9) hearts were placed in 10 mm HEPES buffer (pH 7.4) containing 10% glycerol and 1 mm DTT, and homogenized by sonication on ice. Activation of phospholipase(s) was measured following the addition of either 2 mm EGTA or 0.6 mm free Ca²⁺ at 35 °C. After 10 min of incubation, the reaction was terminated by addition of 2 ml of chloroform/methanol (1:1) in the presence of internal standards (d₄-16:0-fatty acid, 17:0-LPC) followed by phase separation. Extracted fatty acid (A) and LPC (B) molecular species were identified and quantified in the negative and positive ion modes, respectively, utilizing a mass spectrometer. §, p < 0.001; **, p < 0.005, and ¶, p < 10⁻⁵ when compared with EGTA treatment. C, mitochondrial homogenates obtained from non-failing (control, n = 5) and failing (n = 5) hearts were incubated with 0 (2 mm EGTA), 5, 15, 75, 180, and 600 μm free Ca²⁺ for 10 min at 35 °C. AA was quantified by mass spectrometry. *, p < 0.05, and **, p < 0.005 when compared with non-failing controls.