The effects of phosphatidic acid (PA), a known inotropic agent, on Ca2+ transients and contractile activity of cardiomyocytes in congestive heart failure (CHF) due to myocardial infarction were examined. In control cells, PA induced a significant increase (25%) in active cell shortening and Ca2+ transients. The phospholipase C (PLC) inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbonate, blocked the positive inotropic action induced by PA, indicating that PA induces an increase in contractile activity and Ca2+ transients through stimulation of PLC. Conversely, in failing cardiomyocytes there was a loss of PA-induced increase in active cell shortening and Ca2+ transients. PA did not alter resting cell length. Both diastolic and systolic [Ca2+] were significantly elevated in the failing cardiomyocytes. In vitro assessment of the cardiac sarcolemmal (SL) PLC activity revealed that the impaired failing cardiomyocyte response to PA was associated with a diminished stimulation of SL PLC activity by PA. Our results identify an important defect in the PA–PLC signaling pathway in failing cardiomyocytes, which may have significant implications for the depressed contractile function during CHF.

Objective: Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P-2) is not only a precursor to inositol 1,4,5-trisphosphate (Ins 1,4,5-P-3) and sn-1,2 diacylglycerol, but also essential for the function of several membrane proteins. The aim of this study was to evaluate the changes in the level of this phospholipid in the cell plasma membrane (sarcolemma, SL) of cardiomyopathic hamster (CMPH) heart. Methods: We examined the cardiac SL PtdIns 4,5-P-2 mass and the activities of the enzymes responsible for its synthesis and hydrolysis in 250-day-old UM-X7.1 CMPH at a severe stage of congestive heart failure (CHF) and in age-matched controls (Syrian Golden hamsters). Results: The SL PtdIns 4,5-P-2 mass in CMPH was reduced by 72% of the control value. The activities of PtdIns 4 kinase and PtdIns 4-P 5 kinase were depressed by 69 and 50% of control values, respectively. Although, the total phospholipase C (PLC) activity was moderately, although significantly, decreased (by 18% of control), PLC delta (1) isoenzyme activity in the SL membrane was elevated, with a concomitant increase in its protein content, whereas PLC beta (1) and gamma (1) isoenzyme activities were depressed despite the increase in their protein revels. A 2-fold increase in the Ins 1,4,5-P-3 concentration in the cytosol of the failing heart of CMPH was also observed. Conclusions: Reduced SL level of PtdIns 4,5-P-2 may severely jeopardize cardiac cell function in this hamster model of CHF. In addition, the profound changes in the profile of heart SL PLC isoenzyme could alter the complex second messenger responses of these isoenzymes, and elevated Ins 1,4,5-P-3 levels may contribute to intracellular Ca2+ overload in the failing cardiomyocyte. (C) 2001 Elsevier Science BN; All rights reserved.

Phosphatidylinositol 4,5-bisphosphate (Ptdlns 4,5-P2) is an important signaling factor as it is a membrane attachment site for proteins containing PH domains and/or an essential requirement for several proteins, which are associated with the cardiac cell plasma membrane (sarcolemma, SL) for normal cardiac function (,). Ptdlns 4,5-P2 is synthesized in the SL membrane by the coordinated and successive action of Ptdlns 4-kinase (which catalyses the phosphorylation of Ptdlns to Ptdlns 4-phosphate) and Ptdlns 4-phosphate 5-kinase (which catalyses the phosphorylation of Ptdlns 4-phosphate to Ptdlns 4,5-P2) (). Further, Ptdlns 4,5-P2 can be phosphorylated by phosphatidylinositol 3-kinase to another membrane-delimited messenger, phosphatidylinositol 3,4,5, trisphosphate (Ptdlns 3,4,5-P3) (). Changes in the membrane phosphoinositide levels due to the activation of the phosphoinositide signaling pathway () may be taken into consideration as being part of the receptor-mediated signaling events via this pathway in the heart ().

The cardiac sarcolemmal membrane cis -unsaturated fatty acid-sensitive phospholipase D hydrolyzes phosphatidylcholine to form phosphatidic acid. The functional significance of phosphatidic acid is indicated by its ability to increase [Ca2+]iand augment cardiac contractile performance via the activation of phospholipase C. Accordingly, we tested the hypothesis that a defect occurs in the membrane level of phosphatidic acid and/or the responsiveness of cardiomyocytes to phosphatidic acid in congestive heart failure due to myocardial infarction. Myocardial infarction was produced in rats by ligation of the left coronary artery while sham-operated animals served as control. At 8 weeks after surgery, the experimental animals were at a stage of moderate congestive heart failure. Compared to sham controls, phosphatidic acid-mediated increase in [Ca2+]i, as determined by the fura 2-AM technique, was significantly reduced in failing cardiomyocytes. Immunoprecipitation of sarcolemmal phospholipase C isoenzymes using specific monoclonal antibodies revealed that the stimulation of phospholipase C γ1andδ1 phosphatidylinositol 4,5-bisphosphate hydrolyzing activities by phosphatidic acid was decreased in the failing heart. Although the activity of phospholipase C β1in the failing heart was higher than the control, phosphatidic acid did not stimulate this isoform in control sarcolemma, and produced an inhibitory action in the failing heart preparation. Furthermore, the specific binding of phosphatidic acid to phospholipase C γ1andδ1 isoenzymes was decreased, whereas binding to phospholipase β1was absent in the failing heart. A reduction in the intramembranal level of phosphatidic acid derived via cis -unsaturated fatty acid-sensitive phospholipase D was also seen in the failing heart. These findings suggest that a defect in phosphatidic acid-mediated signal pathway in sarcolemma may represent a novel mechanism of heart dysfunction in congestive heart failure.

Although an excessive amount of circulating catecholamines is known to induce cardiomyopathy, the mechanisms are poorly understood. This study was undertaken to investigate the role of oxidative stress in catecholamine-induced heart dysfunction. Treatment of rats for 24 h with a high dose (40 mg/kg) of a synthetic catecholamine, isoproterenol, resulted in increased left ventricular end diastolic pressure, depressed rates of pressure development, and pressure decay as well as increased myocardial Ca2+ content. The increased malondialdehyde content, as well as increased formation of conjugated dienes and low glutathione redox ratio were also observed in hearts from animals injected with isoproterenol. Furthermore, depressed cardiac sarcolemmal (SL) ATP-dependent Ca2+ uptake, Ca2+-stimulated ATPase activity, and Na+-dependent Ca2+ accumulation were detected in experimental hearts. All these catecholamine-induced changes in the heart were attenuated by pretreatment of animals with vitamin E, a well-known antioxidant (25 mg/kg/day for 2 days). Depressed cardiac performance, increased myocardial Ca2+ content, and decreased SL ATP-dependent, and Na+-dependent Ca2+ uptake activities were also seen in the isolated rat hearts perfused with adrenochrome, a catecholamine oxidation product (10 to 25 microg/ml). Incubation of SL membrane with different concentrations of adrenochrome also decreased the ATP-dependent and Na+-dependent Ca2+ uptake activities. These findings suggest the occurrence of oxidative stress, which may depress the SL Ca2+ transport and result in the development intracellular Ca2+ overload and heart dysfunction in catecholamine-induced cardiomyopathy.

Cardiac sarcolemmal (SL) cis-unsaturated fatty acid sensitive phospholipase D (cis-UFA PLD) is modulated by SL Ca(2+)-independent phospholipase A(2) (iPLA(2)) activity via intramembrane release of cis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca(2+) concentration and contractile performance of the cardiomyocyte, changes in iPLA(2) activity may contribute to abnormal function of the failing heart. We examined PLA(2) immunoprotein expression and activity in the SL and cytosol from noninfarcted left ventricular (LV) tissue of rats in an overt stage of congestive heart failure (CHF). Hemodynamic assessment of CHF animals showed an increase of the LV end-diastolic pressure with loss of contractile function. In normal hearts, immunoblot analysis revealed the presence of cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)) in the cytosol, with cPLA(2) and iPLA(2) in the SL. Intracellular PLA(2) activity was predominantly Ca(2+) independent, with minimal sPLA(2) activity. CHF increased cPLA(2) immunoprotein and PLA(2) activity in the cytosol and decreased SL iPLA(2) and cPLA(2) immunoprotein and SL PLA(2) activity. sPLA(2) activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA(2) isoenzymes, suggesting that PLA(2)-mediated bioprocesses are altered in CHF.

A. Vecchini, F. Del Rosso, L. Binaglia, N. S. Dhalla and V. Panagia. Molecular Defects in Sarcolemmal Glycerophospholipid Subclasses in Diabetic Cardiomyopathy. Journal of Molecular and Cellular Cardiology (2000)32, 1061–1074. Although still scarcely studied, the phospholipid component of the cell membrane is of absolute importance for cell function. Experimental evidence indicates that individual molecular species of a given phospholipid can influence specific membrane functions. We have examined the changes in molecular species of diacyl and alkenylacyl choline/ethanolamine glycerophospholipid subclasses and those of phosphatidylserine in purified cardiac sarcolemma of healthy and streptozotocin-induced insulin dependent diabetic rats without or with insulin treatment. The relative content of plasmalogens increased in all the phospholipid classes of diabetic sarcolemma under study. Phosphatidylcholine and phosphatidylethanolamine were mostly enriched with molecular species containing linoleic acid in sn -2 position and deprived of the molecular species containing arachidonic acid. The molecular species of phosphatidylserine containing either arachidonic or docosahexaenoic acid were less abundant in membranes from diabetic rats than in membranes from controls. Insulin treatment of diabetic rats restored the species profile of phosphatidylethanolamine and overcorrected the changes in molecular species of phosphatidylcholine. The results suggest that the high sarcolemmal level of plasmalogens and the abnormal molecular species of glycerophospholipids may be critical for the membrane dysfunction and defective contractility of the diabetic heart.

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide-phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine-xanthine oxidase-ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine-xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an *OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of *OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 microM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.

Although fibroblast growth factor-2 (FGF-2) plays an important role in cardioprotection and growth, little is known about the signals triggered by it in the adult heart. We therefore examined FGF-2-induced effects on phosphoinositide-specific phospholipase C (PI-PLC) isozymes, which produce second messengers linked to the inotropic and hypertrophic response of the myocardium. FGF-2, administered by retrograde perfusion to the isolated heart, induced an increase in inositol-1,4,5-trisphosphate levels in the cytosol, as well as an increase in total PI-PLC activity associated with sarcolemmal and cytosolic fractions. Furthermore FGF-2 induced a time-dependent elevation in cardiomyocyte membrane-associated PLC gamma1 and PLC beta1 activities, assayed in immunoprecipitated fractions, and moreover, increased the membrane levels of PLC beta1 and PLC beta3. Activation of PLC beta is suggestive of FGF-2-induced cross-talk between FGF-receptor tyrosine kinase and G-protein-coupled signaling in adult cardiomyocytes and underscores the importance of FGF-2 in cardiac physiology.

We have examined the changes in quantity and activity of cardiac sarcolemmal (SL) phosphoinositide-phospholipase C (PLC)-beta(1), -gamma(1), and -delta(1) in a model of congestive heart failure (CHF) secondary to large transmural myocardial infarction (MI). We also instituted a late in vivo monotherapy with imidapril, an ANG-converting enzyme (ACE) inhibitor, to test the hypothesis that its therapeutic action is associated with the functional correction of PLC isoenzymes. SL membranes were purified from the surviving left; ventricle of rats in a moderate stage of CHF at 8 wk after occlusion of the left anterior descending coronary artery. SL PLC isoenzymes were examined in terms of protein mass and hydrolytic activity. CHF resulted in a striking reduction (to 6-17% of controls) of the mass and activity of gamma(1)- and delta(1)-isoforms in combination with a significant increase of both PLC beta(1) parameters. In vivo treatment with imidapril (1 mg/kg body wt, daily, initiated 4 wk after coronary occlusion) improved the contractile function and induced a partial correction of PLCs. The mass of SL phosphatidylinositol 4,5-bisphosphate and the activities of the enzymes responsible for its synthesis were significantly reduced in post-MI CHF and partially corrected by imidapril. The results indicate that profound changes in the profile of heart SL PLC-beta(1), -gamma(1), and -delta(1) occur in CHF, which could alter the complex second messenger responses of these isoforms, whereas their partial correction by imidapril may be related to the mechanism of action of this ACE inhibitor.

LipidsVolume 34, Issue S1Part1 p. S73-S74 Phospholipase Abnormalities in sarcolemmal phospholipase D and phospholipase C isoenzymes and in their interactions in post-infarcted failing hearts V. Panagia, Corresponding Author V. Panagia cvso@sbrc.umanitoba.ca Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaTo whom correspondence should be addressed. E-mail: cvso@sbrc.umanitoba.caSearch for more papers by this authorP. S. Tappia, P. S. Tappia Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorC. Yu, C. Yu Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorN. Takeda, N. Takeda Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorN. S. Dhalla, N. S. Dhalla Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this author V. Panagia, Corresponding Author V. Panagia cvso@sbrc.umanitoba.ca Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaTo whom correspondence should be addressed. E-mail: cvso@sbrc.umanitoba.caSearch for more papers by this authorP. S. Tappia, P. S. Tappia Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorC. Yu, C. Yu Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorN. Takeda, N. Takeda Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this authorN. S. Dhalla, N. S. Dhalla Institute of Cardiovascular Sciences, St. Boniface G.H. Research Centre, Canada Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, CanadaSearch for more papers by this author First published: 01 January 1999 https://doi.org/10.1007/BF02562234Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume34, IssueS1Part1January 1999Pages S73-S74 RelatedInformation

To understand cardiac dysfunction in diabetes, the activity of protein kinase C (PKC) and protein contents of its isozymes (PKC-α, -β, -ε, and -ζ) were examined in diabetic rats upon injection of streptozotocin (65 mg/kg iv). The hearts were removed at 1, 2, 4, and 8 wk, and some of the 6-wk diabetic animals had been injected with insulin (3 U/day) for 2 wk. The Ca2+-dependent PKC activity was increased by 43 and 51% in the homogenate fraction and 31 and 70% in the cytosolic fraction from the 4- and 8-wk diabetic hearts, respectively, in comparison with control values. The Ca2+-independent PKC activity was increased by 24 and 32% in the homogenate fraction and 52 and 89% in the cytosolic fraction from the 4- and 8-wk diabetic hearts, respectively, in comparison with control values. The relative protein contents of PKC-α, -β, -ε, and -ζ isozymes were increased by 43, 31, 48, and 38%, respectively, in the homogenate fraction and by 126, 119, 148, and 129%, respectively, in the cytosolic fraction of the 8-wk diabetic heart. The observed changes in heart homogenate and cytosolic fractions were partially reversible upon treatment of the diabetic rats with insulin. The results suggest that the increased myocardial PKC activity and increased protein contents of the cytosolic PKC isozymes are associated with subcellular alterations and cardiac dysfunction in the diabetic heart.

Earlier studies have demonstrated that palmitoyl carnitine (PC), a long chain acyl carnitine, accumulates in the ischemic myocardium. Although perfusion of hearts with PC is known to induce contractile dysfunction which resembles ischemic contracture, the mechanisms underlying this derangement are not clear. In this study, we examined the effect of exogenous PC on the intracellular concentration of calcium ([Ca(2+)](i)) in freshly isolated cardiomyocytes from adult rat hearts. The results showed that PC elevated [Ca(2+)](i)in a dose-dependent (5-20 microm) manner; 15 microm PC evoked a marked and reversible increase in [Ca(2+)](i)without having any significant action on cell viability. The PC (15 microm)-induced increase in [Ca(2+)](i)was slightly depressed but delayed in the absence of extracellular Ca(2+). Pre-incubation of cardiomyocytes with sarcolemmal (SL) l -type Ca(2+)-channel blockers, verapamil or diltiazem, and inhibitors of SL Na(+)-Ca(2+)exchanger such as Ni(2+)or amiloride, depressed the PC-evoked increase in [Ca(2+)](i)significantly. Ouabain, a Na(+)-K(+)ATPase inhibitor, and low concentrations of extracellular Na(+)enhanced the PC-induced increase in [Ca(2+)](i). Depletion of the sarcoplasmic reticulum (SR) Ca(2+)stores by low micromolar concentrations of ryanodine (a SR Ca(2+)-release channel activator) or by thapsigargin (a SR Ca(2+)-pump ATPase inhibitor) depressed the PC-mediated increase in [Ca(2+)](i). Combined blockade of the l -type Ca(2+)channel, Na(+)-Ca(2+)exchanger and the SR Ca(2+)-pump had an additive inhibitory effect on the PC response. These observations suggest that the PC-induced increase in [Ca(2+)](i)is dependent on both Ca(2+)-influx from the extracellular space and Ca(2+)-release from the SR stores. Thus, the accumulation of PC in the myocardium may be partly responsible for the occurrence of intracellular Ca(2+)overload in ischemic heart.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

The effect of phospholipase A2(PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenouscis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100μm, respectively; their effect was maximal at 250μmand declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r=0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1μmE-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca2+-independentvCa2+-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca2+-independent, high molecular weight (110 kDa) PLA2in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca2+-independent PLA2and subsequent endogenous mobilization ofsn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2as well as in heart diseases characterized by PLA2pathological dysfunction.

Phosphatidic acid (PA) is mainly formed by the hydrolysis of phosphatidylcholine due to the activation of phospholipase D (PLD). PA is also generated by phosphorylation of diacylglycerol (DAG) due to the action of DAG kinase and is converted to DAG under the action of PA phosphohydrolase. Most of the positive inotropic agents which are known to stimulate cardiac hypertrophy, have been shown to increase the level of PA in cardiac sarcolemma. Although the growth factor-like effect of PA has been recognized in a wide variety of tissues, there is a lack of similar information in adult cardiomyocytes. By using single cardiomyocytes, we have now shown that PA increased the basal [Ca2+]i level without significant effect on the amplitude of Ca2+ transients. PA (10-50 mu M) also increased the [Ca2+]i in cardiac cell suspension. PA has also been shown to stimulate protein synthesis in cardiomyocytes, which is inhibited by a PKC inhibitor as well as a Ca2+ chelator. PA at the concentration of 1-50 mu M was observed to stimulate the activity of PLC in cardiac sarcolemma; this effect was attenuated by a PLC inhibitor. Since DAG, formed due to the activation of PLC, is considered to play a crucial role in regulating the activity of protein kinase C (PKC), the positive feedback effect of PA on this pathway may be essential for maintaining the sustained elevation in the activity of PKC during the development of cardiac hypertrophy. In view of these observations and other facts available in the literature, it is suggested that PA may be a potential signal transducer for the development of cardiac hypertrophy.

In order to examine the mechanisms of ischemia-reperfusion induced changes in beta-adrenoceptor-linked signal transduction pathway, isolated rat hearts perfused in the absence or presence of superoxide dismutase (SOD) plus catalase (CAT) were made ischemic for 30 min and then reperfused for 60 min. The left ventricular developed pressure as well as the rare of contraction and rate of relaxation were markedly decreased, whereas the left ventricular end-diastolic pressure increased in the ischemic hearts. A significant increase in the density and affinity of beta 1-adrenoceptors without any changes in the characteristics of beta 2-adrenoceptors was evident in cardiac membranes obtained from the ischemic hearts. The recovery of contractile abnormalities in the ischemic heart was depressed upon reperfusion; the ischemic-reperfused hearts also showed attenuated inotropic responses to isoproterenol. The affinities and densities of beta- and beta-adrenoceptors were decreased in the ischemic-reperfused hearts; the magnitude of changes in beta 1-adrenoceptors was greater than that in beta 2-adrenoceptors. The isoproterenol-stimulated adenylyl cyclase activity was depressed in both ischemic hearts and ischemic-reperfused hearts. The basal and forskolin-stimulated adenylyl cyclase activities were unaltered due to ischemia but were increased upon reperfusion. The NaF- and 5'-Guanylyl-imidodiphosphate[Gpp(NH)p]-stimulated adenylyl cyclase activities were depressed in the ischemic hearts and increased in the ischemic reperfused hearts. Cholera toxin (CT)-stimulated adenylyl cyclase as well as the CT-catalysed ADP-ribosylation activity and stimulatory G protein (Gs protein) immunoreactivity were decreased in the ischemic hearts and increased in the reperfused hearts. Pertussis toxin (PT)-stimulated adenylyl cyclase activity was unaltered in both ischemic and ischemic-reperfused hearts, whereas the PT-catalysed ribosylation and inhibitory G protein (Gi protein) immunoactivity were slightly increased in the reperfused myocardium. Thus the inability of isoproterenol to stimulate adenylyl cyclase in the ischemic-reperfused hearts may be due to alterations mainly in the characteristics of beta 1-adrenoceptors including density, affinity and coupling with the adenylyl cyclase. Scavenging of oxyradicals by the addition of SOD plus CAT in the perfusion medium prevented the reperfusion-induced changes in contractile function, inotropic responses of the heart to isoproterenol, activation of adenylyl cyclase by isoproterenol, as well as densities and affinities of beta-adrenoceptors in cardiac membranes. These results suggest that the depressed contractile activity and the attenuated inotropic responses of ischemic-reperfused hearts to isoproterenol as well as the defects in beta-adrenoceptor-linked signal transduction may be due to the formation of oxyradicals in the myocardium.

Although phosphoinositide-specific phospholipase C (PLC) is involved in signal transduction mechanisms of the myocardial cell, very little is known about its status in congestive heart failure (CHF). We have examined the PLC activity in sarcolemmal and cytosolic fractions isolated from the viable left ventricle of rats at 8 weeks (moderate stage of CHF) and 16 weeks (severe stage of CHF) after occlusion of the left anterior descending coronary artery; the hypertrophied right ventricle was used for comparison. At 8 weeks, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by sarcolemmal PLC was reduced by 37% of sham control values only in the left ventricle, whereas at 16 weeks, PLC-mediated hydrolysis was depressed in both left and right ventricles by 25% and 30%, respectively. The hydrolysis of phosphatidylinositol 4-monophosphate (PIP) was reduced by 25% of control value only in the severely failing left ventricle, while the phosphatidylinositol (PI) hydrolysis remained unaltered. Kinetic studies of PLC activity in the left ventricle showed a depression of Vmaxat moderate and severe failure stages, whereas the affinity for the substrate was increased in the left ventricle at 8 weeks and decreased in the right ventricle at 16 weeks. The only difference observed between experimental and control groups at the cytosolic level, was a significant enhancement of PLC activity in the severely failing left ventricle when PIP was given as a substrate, and in the corresponding right ventricle when PI was the substrate. The results of this study identify time-related defects in sarcolemmal PLC in right and left ventricles during the development of CHF due to myocardial infarction.