Panasiuk O. The effect of phospholipid components on the functional state of the endothelium and heart mitochondria

The dissertation is devoted to investigation of the influence of omega-3 polyunsaturated fatty acids (omega-3 PUFA) and lysophospholipids (LPL) on the function and signalling of myocardial mitochondria and vascular endothelium. The differences in the effects of omega-3 PUFA therapy on the sensitivity of mitochondrial permeability transition pore (MPTP) opening elicited by Ca2+ overload between the two functionally and structurally heterogeneous fractions of myocardial mitochondria were revealed in the work. The results of the study indicate that the interfibrillar (IF) and subsarcolemmal (SS) fractions of mitochondria have the same sensitivity to Ca2+-induced swelling. Four weeks treatment of rats with Epadol containing 45% omega-3 PUFA, was found to reduce the sensitivity of MPTP to calcium-induced opening of both myocardium mitochondrial fractions. It was found that the protective effect of omega-3 PUFA therapy is much more pronounced for the IF than for the SS fraction of mitochondria, indicating that the functional role of mitochondrial heterogeneity increases under pathological conditions accompanied by Ca2+ overload. The effect of omega-3 PUFA-enriched diet on the functioning of IF and SS fractions of rat myocardial mitochondria under isoproterenol-induced myocardial damage was also studied. It is shown that under such conditions, omega-3 PUFA-enriched diet prevents Ca2+-induced mitochondria swelling, pointing for protective effect of omega-3 PUFA on isoproterenol-induced heart damage. This effect was found to be more pronounced for the IF mitochondrial fraction, When studying mitochondrial respiration, it was found that in mitochondria isolated from rats with isoproterenol-evoked myocardial damage, the respiration rate in state 3, the respiratory control and phosphorylation efficiency were significantly restored by omega-3 PUFA-enriched diet. Isoproterenol injections have been shown to result in altered mitochondrial respiration during succinate oxidation: respiration rate in state 4 under such conditions did not change significantly. The ability of mitochondria to respond to the addition of Ca2+ in the group with omega-3 PUFA was also restored. To elucidate the mechanisms providing the protective effect of omega-3 PUFA on myocardial and vascular function, their effects on the electrical properties of isolated mitochondria and endothelial cells (EC) were investigated. In patch-clamp experiments on isolated mitoplasts (mitochondria without an outer membrane), single channel activity of Ca2+-dependent potassium channels of big conductance (BKCa) was demonstrated. The channel activity was increased by the administration of 10-5 M docosahexaenoic acid (DHA), a member of omega-3 PUFA, to the external solution. Addition of paxilin, a selective blocker of BKCa channels, led to a significant inhibition of the channel activity, indicating the involvement of BKCa channels of the inner mitochondrial membrane in the effect of DHA. Addition of DHA elicited hyperpolarizion of endothelial cells. In patches excised from endothelial cells, the addition of DHA to the inner membrane surface enhanced the BKCa single channel activity in a concentration-dependent manner. After removal of membrane cholesterol with methylcyclodextrin, the addition of DHA did not stimulate the activity of BKCa channels. In inside-out patches, lysophosphatidylcholine (LPH) and lysophosphatidylinositol (LPI) were shown to stimulate the BKCa channel activity. Endothelial cells were hyperpolarized in response to these compounds, however, the hyperpolarization to histamine and acetylcholine (Ac) was inhibited by LPH and LPI. It was shown that this effect is mediated by inhibition of the sodium-calcium exchanger.