Figure 1.
Functional recovery in Intralipid®-treated (Panel A) and acylcarnitine-treated hearts (Panel B) subjected to 15 min of ischemia and 30 min of reperfusion.
LVW, left ventricular work. Basal, average LVW before ischemia. Rep, average LVW during postischemic reperfusion. IR, untreated hearts exposed to 15(IR). IR/IL, hearts exposed to IR and 1% Intralipid® at the onset of reperfusion. IR/C16∶0c, IR/C18∶1c, and IR/C18∶2c, hearts exposed to IR and 1 µM palmitoyl-, oleoyl-, or linoleoylcarnitine at the onset of reperfusion. MPG, N-(2-mercaptopropionyl) glycine (10 µM) added at the onset of reperfusion. *, significantly different from all other groups. N = 6–7 hearts in each group.
Figure 2.
Early activation of Akt and ERK1/2 by ROS in Intralipid®-treated hearts.
Panel A: p-Akt to total Akt immunoblots from tissue samples collected 3 min after reperfusion. Panel B: p-STAT3 to total STAT3 immunoblots from same tissue samples. Panel C: Akt activity measurements. Panel D: p-ERK1/2 to total ERK immunoblots from same tissues. IR, untreated hearts exposed to 15 min of ischemia and 3 min of reperfusion (IR). IR/IL, hearts exposed to IR and 1% Intralipid® at the onset of reperfusion. MPG, N-(2-mercaptopropionyl) glycine (10 µM) added at the onset of reperfusion. Data are mean (SEM). N = 6 hearts in each group.
Figure 3.
Intralipid®-induced formation of ROS in cardiac fibers collected 3 min after reperfusion.
Panel A: Hydrogen peroxide (H2O2) emission capacity from mitochondria as determined by Amplex Red assay. *significantly different from IR. Panel B: Loss of aconitase activity in Intralipid®-treated hearts (PANOVA = 0.002). IR, untreated hearts exposed to 15 min of ischemia and 3 min of reperfusion (IR). IR/IL, hearts exposed to IR and 1% Intralipid® at the onset of reperfusion. Basal, without substrates. +subst, with added substrates. AER, hearts with time-matched aerobic perfusion. Data are mean (SEM). N = 6 hearts in each group.
Figure 4.
Complex IV inhibition by metabolites of Intralipid® fatty acid constituents.
Panel A: Concentration-dependent inhibition of complex IV by palmitoyl(C16∶0c)-, oleoyl(C18∶1c)-, and linoleoylcarnitine (C18∶2c) in permeabilized cardiac fibers. Complex IV inhibition is given as relative decrease in oxygen consumption. Panel B and C: Concentration-dependent hydrogen peroxide (H2O2) emission capacity determined by Amplex Red assay in permeabilized cardiac fibers exposed to increasing concentrations of palmitoyl- and linoleoylcarnitine. *, significantly different from untreated. Data are mean (SEM). N = 6 hearts in each group/concentration.
Table 1.
Assessment of mitochondrial respiratory chain function.
Table 2.
Fitting parameters for the acylcarnitine inhibitor curves.
Figure 5.
Uncoupling by Intralipid® and acylcarnitines.
Panel A: Uncoupling by uncoupling proteins (UCP) and adenine nucleotide translocase (ANT) were measured in cardiac fibers collected at the end of 30 min reperfusion. N-(2-mercaptopropionyl)-glycine (MPG) inhibits ANT- but not UCP-mediated uncoupling. #, significantly different from IR and IR/MPG. *, significantly different from all other groups. Panel B: Cardiac fibers respiring on succinate (leak respiration) were exposed to 150 µM palmitoyl-(C16∶0c) and linoleoylcarnitine (C18∶2c) and UCP- and ANT-mediated uncoupling were determined. IR, untreated hearts exposed to 15 min of ischemia and 30 min of reperfusion (IR). IR+IL, hearts exposed to IR and 1% Intralipid® at the onset of reperfusion. IR/IL+MPG, hearts exposed to IR and 1% Intralipid® +10 µM N-(2-mercaptopropionyl) glycine (MPG) at the onset of reperfusion. *, significantly different from all other groups. @, significantly different from C18∶2c. Data are mean (SEM). N = 6 hearts in each group.
Figure 6.
Accumulation of acylcarnitines and effects on oxidative metabolism in Intralipid®-treated hearts.
Panel A: Accumulation of long-chain acylcarnitines in hearts reperfused for 3 min and 30 min. Panel B: Lack of competition for β-oxidation between exogenous radiolabeled palmitate and fatty acid constituents released from Intralipid® and no alteration in glucose oxidation (Randle cycle). AER, time-matched aerobically perfused hearts. AER/IL, time-matched aerobically perfused hearts treated with 1% Intralipid® for 30 min. IR 3′ or 30′, untreated hearts exposed to 15 min of ischemia and 3 or 30 min of reperfusion, respectively. IR/IL 3′ or 30′, hearts exposed to 15 min of ischemia and 3 or 30 min of reperfusion and 1% Intralipid® at the onset of reperfusion. *, significantly different from the corresponding untreated group. Data are mean (SEM). N = 6 hearts in each group.
Figure 7.
Proposed mechanism of Intralipid®-induced cardioprotection.
Metabolites of fatty acid constituents released from Intralipid® accumulate in the intermembrane space (IMS) in early reperfusion when β-oxidation is still dysfunctional. An initial peak of acyl-CoAs in the mitochondrial matrix may also inhibit carnitine/acylcarnitine translocase (CACT) and carnitine palmitoyl transferase II (CPT II) via product feedback inhibition, which would further boost acylcarnitine accumulation in the intermembrane space. Acylcarnitines, namely palmitoylcarnitine, inhibit complex IV of the respiratory chain (causing superoxide release at complex I (toward matrix) and at complex III (toward IMS). ACS, acyl-CoA synthase cyt c, cytochrome c. CPT I, carnitine palmitoyl transferase I. FFA, free fatty acids. IMM, inner mitochondrial membrane. LC, long-chain. OMM, outer mitochondrial membrane. Q, Q-junction is the point in the respiratory chain where electron flow from complex I and from complex II converge.