Abnormalities in Sarcolemmal Phospholipase D and Phospholipase C Isoenzymes and in Their Interactions in Post-Infarcted Failing Hearts V. Panagia*, P.S. Tappia, C. Yu, N. Takeda, and N.S. Dhalla Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre and Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

Phospholipase D (PLD) activity associated with the myocardial cell membrane (sarcolemma, SL) specifically hydrolyzes phosphatidylcholine to form phosphatidic acid (PtdOH). As reported for other cells, SL PLD is coupled to a PtdOH phosphohydrolase for the coordinate production of a sn-1,2-diacylglycerol (DAG) pool originating from phosphatidylcholine (t). PLD is part of the SL signaling system (2). Its importance for the heart function is shown by PtdOH-induced changes in cellular Ca 2+ movements and contractile performance, as well as by its potential involvement in the development of cardiac hypertrophy (2). We reported biochemical evidence for the presence, at the SL level, of a cis-unsaturated fatty acid (UFA)-sensitive PLD activity (cis UFA-PLD); arachidonic, oleic and docosahexaenoic acids being the most efficient stimulants among the fatty acids tested) (3,4) which is affected by oxidants and thiol modifiers (5). SL phosphoinositide-phospholipase C (PLC), which participates in many different physiological processes of the cardiac cell (6), is also inhibited by the oxidative stress (7). Oxidants generation, which alters functional protein thiots, is an important feature of congestive heart failure (CHF) following myocardial infarction (8). Therefore, cis UFA-PLD and PLC [5l, Y1 and 81, which are the most relevant and well-characterized variants of PLC in mammalian cells, were studied in SL membranes purified from the surviving left ventricle of rats at 8 wk after occlusion of the left-anterior descending coronary artery, when the animals were in a moderate stage of CHF (9). A significant reduction of the SL level of signaling PtdOH was observed in CHF, due to the imbalance between increased PtdOH synthesis (124% of controls) by cis UFA-PLD and comparatively higher PtdOH dephosphorylation ( 135% of controls) by PtdOH phosphohydrolase. As expected, the specific DAG pool derived from cis UFA-PLD/PtdOH phosphohydrolase combined activities was significantly augmented in CHE Lipids 34, $73-$74 (1999).

*To whomcorrespondenceshouldbe addressed. E-mail: [email protected] Abbreviations:CHF, congestiveheart failure; DAG, diacylglycerol;PLC, phospholipaseC; PLD,phospholipaseD; I~dOH,phosphatidicacid; SL, sarcotemma;UFA, unsaturatedfattyacid.

Copyright © 1999 by AOCS Press

The rank order of PLC isoenzymes hydrolytic activity toward phosphatidylinositol 4,5-bisphosphate in control SL was PLC 81 > PLC Y1 > PLC ~1, while that of the protein mass was PLC Yl > PLC 81 > PLC ~l" CHF resulted in a drastic reduction ofPLC Yi and 81 activity (11 and 14% of controls, respectively) and protein mass (7 and 18% of controls, respectively), in direct contrast with a significant increase of both PLC [~t parameters. In vivo treatment with the angiotensinconverting enzyme inhibitor imidapril, instituted 4 wk postinfarct, significantly improved the contractile function of the 8-wk postinfarcted failing hearts with partial correction of the PLC isoenzymes parameters and normalization of cis UFA-PLD and PtdOH phosphohydrolase activities. PtdOH stimulates the total SL PLC activity in normal hearts (2). As the level of cis UFA-PLD-derived PtdOH was diminished in CHF, we investigated whether or not the response of PLC isoenzymes to PtdOH was also altered in this disease state. In controls, in vitro stimulation by exogenous PtdOH (30 ~tM) was limited to the Yt and ~l variants. In CHF, these PtdOH-stimulated PLC activities were significantly lower than the correspondent control values. We also showed that PLC is one of the mechanisms for PtdOH-induced increase in intracellular Ca 2÷ ([Ca2+] i) and contractile performance of the normal heart (10). In CHE PtdOH-mediated rise of cardiomyocytal [Ca2+] i was significantly reduced; this might have been caused, at least partly, by the attenuated response of PLC Y1 and 81 to PtdOH. Our results indicate that, in post-MI CHF: (i) the bioprocesses mediated by the SL PtdOH and DAG pool originating from the cis UFA-PLD/PtdOH phosphohydrolase pathway may be impaired; (ii) SL PLC I]l-dependent function is amplified with almost complete loss of the PLC Y1 and 61-related signaling; this event would impact negatively on the complex second-messenger response of PLC-linked receptors; and (iii) the possibility of valid interactions between SL cis UFA-PLD and PLC signaling pathways may be hampered by the diminished level of SL PtdOH as well as by the striking decrease of PLC Y1 and 51 and their insufficient response to PtdOH, with possible consequences for [Ca2+]i homeostasis and contractile performance of the post-infarcted failing

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V. PANAGIA ETAL.

heart. Moreover, the reversibility of PLC, cis UFA-PLD, and PtdOH phosphohydrolase pathological changes by the imidapril therapy may confer pathophysiological significance to these enzymes and may be related to the mechanism of the beneficial action of this angiotensin-converting e n z y m e inhibitor.

ACKNOWLEDGMENT

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Supported by a grant (V.P.) from the Medical Research Council Group in Experimental Cardiology, Canada.

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REFERENCES

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1. Panagia, V., Ou, C., Taira, Y., Dai., J., and Dhalla, N.S. (1991) Phospholipase D Activity in Subcellular Membranes of Ral Ventficular Myocardium, Biochim. Biophys. Acta 1064, 242-250. 2. Dhalla, N.S., Xu, Y.-J., Sheu, S.-S., Tappia, P.S., and Panagia, V. (1997) Phosphatidic Acid: A Potential Signal Transducer for Cardiac Hypertrophy, J. Mol. Cell Cardiol. 29, 2865-2871. 3. Dai, J., Williams, S.A., Ziegelh6ffer, A., and Panagia, V. (1995) Structure-Activity Relationship of the Effect of cis-Unsaturated

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Fatty Acids on Heart Sarcolemmal Phosphotipase D Activity, Prostaglandins Leukotrienes Essent. Fatty Acids 52, 167-171. Liu, S.-Y., Tappia, P.S., Dai. J, Williams, S.A., and Panagia, V. (1998) Phospholipase A2-Mediated Activation of Phospholipasae D in Rat Heart Sarcolemma, J. Mol. Celt Cardiol. 30 1203-1214. Dai, J., Meij, J.T.A., Padua, R., and Panagia, V. (1992) Depression of Cardiac Sarcolemmal Phospholipase D Activity by Oxidant-Induced Thiol Modification, Circ'. Res. 71,970-977. De Jonge, H.W., van Heugten, H.A.A., and Lamers, J.M.J. (1995) Signal Transduction by the Phosphatidylinositol Cycle in Myocardium, J. Mol. Cell Cardiol. 27, 93-106. Meij, J.T.A., Suzuki, A., Panagia, V., and Dhalla, N.S. (1994) Oxidative Stress Modifies the Activity of Cardiac Sarcolemmal Phospholipase C, Biochim. Biophys. Acta 1199, 6-12. Hill, M.F., and Singal, P.K. (1997) Right and Left Myocardial Antioxidant Responses During Heart Failure Subsequent to Myocardial Infarction, Circulation 96, 24 t4-2420. Ju, H., Zhao, S., Tappia, P.S., Panagia, V., and Dixon, I.M.C. (1997) Expression of Gq and PLC- in Scar and Border Tissue in Heart Failure Due to Myocardial Infarction, Circulation 97, 892-899. Xu, Y.-J., Panagia, V., Shao, Q., Wang, X., and Dhalla, N.S. (1996) Phosphatidic Acid Increases Intracellular Free Ca 2÷ and Cardiac Contractile Force, Am. J. Physiol. 271, H651-H659.