Cardiac Sarcolemmal Membranes Research Articles

Emerging Roles of Phospholipase C Beta Isozymes as Potential Biomarkers in Cardiac Disorders.

Phospholipase C (PLC) enzymes represent crucial participants in the plasma membrane of mammalian cells, including the cardiac sarcolemmal (SL) membrane of cardiomyocytes. They are responsible for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into 1,2-diacylglycerol (DAG) and inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), both essential lipid mediators. These second messengers regulate the intracellular calcium (Ca2+) concentration, which activates signal transduction cascades involved in the regulation of cardiomyocyte activity. Of note, emerging evidence suggests that changes in cardiomyocytes' phospholipid profiles are associated with an increased occurrence of cardiovascular diseases, but the underlying mechanisms are still poorly understood. This review aims to provide a comprehensive overview of the significant impact of PLC on the cardiovascular system, encompassing both physiological and pathological conditions. Specifically, it focuses on the relevance of PLCβ isoforms as potential cardiac biomarkers, due to their implications for pathological disorders, such as cardiac hypertrophy, diabetic cardiomyopathy, and myocardial ischemia/reperfusion injury. Gaining a deeper understanding of the mechanisms underlying PLCβ activation and regulation is crucial for unraveling the complex signaling networks involved in healthy and diseased myocardium. Ultimately, this knowledge holds significant promise for advancing the development of potential therapeutic strategies that can effectively target and address cardiac disorders by focusing on the PLCβ subfamily.

Pathogenic Mechanisms Underlying Cirrhotic Cardiomyopathy.

Cardiac dysfunction associated with cirrhosis in the absence of preexisting heart disease is a condition known as cirrhotic cardiomyopathy (CCM). Cardiac abnormalities consist of enlargement of cardiac chambers, attenuated systolic and diastolic contractile responses to stress stimuli, and repolarization changes. CCM may contribute to cardiovascular morbidity and mortality after liver transplantation and other major surgeries, and also to the pathogenesis of hepatorenal syndrome. The underlying mechanisms of CCM are poorly understood and as such medical therapy is an area of unmet medical need. The present review focuses on the pathogenic mechanisms responsible for development of CCM. The two major concurrent mechanistic pathways are the inflammatory phenotype due to portal hypertension, and protein/lipid synthetic/metabolic defects due to cirrhosis and liver insufficiency. The inflammatory phenotype arises from intestinal congestion due to portal hypertension, resulting in bacteria/endotoxin translocation into the systemic circulation. The cytokine storm associated with inflammation, particularly TNFα acting via NFκB depresses cardiac function. They also stimulate two evanescent gases, nitric oxide and carbon monoxide which produce cardiodepression by cGMP. Inflammation also stimulates the endocannabinoid CB-1 pathway. These systems inhibit the stimulatory beta-adrenergic contractile pathway. The liver insufficiency of cirrhosis is associated with defective synthesis or metabolism of several substances including proteins and lipids/lipoproteins. The protein defects including titin and collagen contribute to diastolic dysfunction. Other protein abnormalities such as a switch of myosin heavy chain isoforms result in systolic dysfunction. Lipid biochemical changes at the cardiac sarcolemmal plasma membrane result in increased cholesterol:phospholipid ratio and decreased membrane fluidity. Final common pathway changes involve abnormal cardiomyocyte intracellular ion kinetics, particularly calcium. In conclusion, cirrhotic cardiomyopathy is caused by two pathways of cellular and molecular dysfunction/damage due to hepatic insufficiency and portal hypertension.

Increase of ATP-sensitive potassium (KATP) channels in the heart of type-1 diabetic rats

BackgroundAn impairment of cardiovascular function in streptozotocin (STZ)-diabetic rats has been mentioned within 5 days-to-3 months of induction. ATP-sensitive potassium (KATP) channels are expressed on cardiac sarcolemmal membranes. It is highly responsive to metabolic fluctuations and can have effects on cardiac contractility. The present study attempted to clarify the changes of cardiac KATP channels in diabetic disorders.MethodsStreptozotocin-induced diabetic rats and neonatal rat cardiomyocytes treated with a high concentration of glucose (a D-glucose concentration of 30 mM was used and cells were cultured for 24 hr) were used to examine the effect of hyperglycemia on cardiac function and the expression of KATP channels. KATP channels expression was found to be linked to cardiac tonic dysfunction, and we evaluated the expression levels of KATP channels by Western blot and Northern blot analysis.ResultsThe result shows diazoxide produced a marked reduction of heart rate in control group. Furthermore, the methods of Northern blotting and Western blotting were employed to identify the gene expression of KATP channel. Two subunits of cardiac KATP channel (SUR2A and kir 6.2) were purchased as indicators and showed significantly decreased in both diabetic rats and high glucose treated rat cardiac myocytes. Correction of hyperglycemia by insulin or phlorizin restored the gene expression of cardiac KATP in these diabetic rats.ConclusionsBoth mRNA and protein expression of cardiac KATP channels are decreased in diabetic rats induced by STZ for 8 weeks. This phenomenon leads to result in desensitization of some KATP channel drugs.

Abstract 17095: Caveolin-1 Mediates Cellular Protective Effects of a High-Molecular Weight Polyethylene Glycol-Based Copolymer, PEG 15-20 During Hypoxia-Reoxygenation in Cardiac Myocytes

Ischemic heart disease is the primary etiology for the development of heart failure. We recently demonstrated that PEG 15-20 protects cardiac myocytes from hypoxia-reoxygenatiion (H-R)-induced cell death and preserves ventricular function. In the present study we investigated our hypothesis that PEG 15-20 exerts its beneficial effect by stabilizing sarcolemmal lipid rafts particularly by its effect on Caveolin-1 (Cav-1). Cav-1 alongwith Cav-3, the principal structural components of caveolar domains are abundantly expressed in the myocardium and implicated in the pathogenesis of ischemic injury. Cardiac myocytes were exposed to 30 minutes of hypoxia (2% O 2 , 5% CO 2 , 93% N 2 ) followed by 3 hours of reoxygenation (5% CO 2 , 95% air) with cells treated with 5% PEG 15-20 during reoxygenation. Adenoviral-mediated knockdown of Cav-1 in cardiac myocytes by RNA interference reversed the anti-apoptotic effect of PEG 15-20 both in terms of caspase 3 activity (6.7±1.2 vs. 3.8±0.76 fluorometric units/mg protein) and TUNEL staining (38±7 vs. 17±3 apoptotic cells). Cav-1 knockdown also abrogated the decrease in oxidative stress mediated by PEG 15-20 during H-R as measured by superoxide radical generation (4.3±0.65 vs. 1.4±0.32 nmoles/min/mg protein with PEG 15-20 alone). Further Cav-1 knockdown mitigated the increased pro-survival phosphorylation of Akt, GSK-3β and ERK1/2 observed with PEG15-20 during H-R. Importantly maintenance of cardiac myocyte β-adrenergic signaling that is critical for myocardial function that was observed with PEG 15-20 treatment during H-R in cardiac myocytes was also reversed with knockdown of Cav-1(16±1.8 vs. 28±2.6 pmol cAMP/mL with PEG 15-20 alone). Finally, confocal microscopy revealed that PEG 15-20 mediated preservation of sarcolemmal lipid-raft architecture during H-R was abolished in cells deficient in Cav-1as seen by increased lipid raft coalescence. These studies demonstrate that PEG 15-20 primarily exerts its cardiac-specific pro-survival and anti-apoptotic effects during H-R by binding and stabilizing Cav-1 in the caveolar domains. Therapeutic strategies to augment maintenance of lipid raft micro architecture in the cardiac sarcolemmal membrane may improve survival after ischemic injuries.

Anti-La (SS-B) but not anti-Ro52 (SS-A) antibodies cross-react with laminin--a role in the pathogenesis of congenital heart block?

Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sjögren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8.7 to 14.9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9.4 to 14.9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development.

Abstract 1011: SUR2 Short Form-Based Channels Confer A Glibenclamide Insensitive, ATP-Sensitive Potassium Current In Cardiac Myocytes

K ATP channels are widely distributed in various tissues and play important physiological roles in regulating insulin secretion in pancreatic beta-cells, providing ischemic protections to the heart and modulating vascular tone in smooth muscles. It is generally agreed that the sarcolemmal K ATP is a hetero-octamer consisting of a Kir6.0 pore and a sulfonylurea receptor (SUR). A SUR2 mutant mouse was generated by disrupting the first nucleotide-binding domain (NBD1), where a previously reported N-end component for the glibenclamide action site was located. In ventricular myocytes isolated from relatively young mutant mice, a novel ATP-sensitive current ( I KATPn ) was detected in 33% single-channel recordings with an average amplitude of 12.3 ± 5.4 pA per patch (n = 15), which was 1/4 of the conventional I KATP (55.4 ± 10.0 pA, n = 19). The IC 50 (ATP) for K ATPn was 10 μM compared to 39.3 μM for the conventional K ATP suggesting that K ATPn could respond faster to low-ATP conditions. Mean burst duration for K ATPn (20.6 ± 1.8 ms, n=4) was half of that for the conventional K ATP (44.2 ± 9.5 ms, n=4). K ATPn was insensitive to 10 μM glibenclamide and could not be re-activated by 0.1– 0.5 mM pinacidil in the presence of ATP. Newly designed SUR2-isoform or variant-specific antibodies identified several novel SUR2 short forms in the sizes of 28 – 68 kDa in addition to a 150-kDa long form in a purified WT cardiac membrane fraction. The 150-kDa long form was absent in the mutant as expected but the SUR2 short forms remained intact. We hypothesized that channels constituted by these short forms, presumably lacking NBD1, confer I KATPn . The SUR2 short forms co-immunoprecipitated with Kir6.0 suggesting that the short forms may function like hemi-transporters reported in other ATP-binding cassette transporter families. Our results show that different K ATP compositions based on the novel SUR2 short forms may exist in cardiac sarcolemmal membrane.

Cardiac sulfonylurea receptor short form-based channels confer a glibenclamide-insensitive K ATP activity

The cardiac sarcolemmal ATP-sensitive potassium channel (K ATP) consists of a Kir6.2 pore and an SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. K ATP channels have been proposed to play protective roles during ischemic preconditioning. An SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current ( I KATPn) was detected in 33% of single-channel recordings with an average amplitude of 12.3 ± 5.4 pA per patch, an IC 50 to ATP inhibition at 10 μM and a mean burst duration at 20.6 ± 1.8 ms. Newly designed SUR2 isoform- or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1 confer I KATPn. The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive K ATP currents ( I KATP) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different K ATP compositions may co-exist in cardiac sarcolemmal membrane.

Exercise training and β-blocker treatment ameliorate age-dependent impairment of β-adrenergic receptor signaling and enhance cardiac responsiveness to adrenergic stimulation

Cardiac β-adrenergic receptor (β-AR) signaling and left ventricular (LV) responses to β-AR stimulation are impaired with aging. It is shown that exercise and β-AR blockade have a favorable effect on cardiac and vascular β-AR signaling in several cardiovascular diseases. In the present study, we examined the effects of these two different strategies on β-AR dysregulation and LV inotropic reserve in the aging heart. Forty male Wistar-Kyoto aged rats were randomized to sedentary, exercise (12 wk treadmill training), metoprolol (250 mg·kg−1·day−1 for 4 wk), and exercise plus metoprolol treatment protocols. Ten male Wistar-Kyoto sedentary young rats were also used as a control group. Old trained, old metoprolol-treated, and old trained plus metoprolol-treated rats showed significantly improved LV maximal and minimal first derivative of the pressure rise responses to β-AR stimulation (isoproterenol) compared with old untrained animals. We found a significant reduction in cardiac sarcolemmal membrane β-AR density and adenylyl cyclase activity in old untrained animals compared with young controls. Exercise training and metoprolol, alone or combined, restored cardiac β-AR density and G-protein-dependent adenylyl cyclase activation in old rats. Although cardiac membrane G-protein-receptor kinase 2 levels were not upregulated in untrained old compared with young control rats, both exercise and metoprolol treatment resulted in a dramatic reduction of G-protein-receptor kinase 2 protein levels, which is a further indication of β-AR signaling amelioration in the aged heart induced by these treatment modalities. In conclusion, we demonstrate for the first time that exercise and β-AR blockade can similarly ameliorate β-AR signaling in the aged heart, leading to improved β-AR responsiveness and corresponding LV inotropic reserve.

Phospholipid-mediated signaling systems as novel targets for treatment of heart diseaseThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

The phospholipases associated with the cardiac sarcolemmal (SL) membrane hydrolyze specific membrane phospholipids to generate important lipid signaling molecules, which are known to influence normal cardiac function. However, impairment of the phospholipases and their related signaling events may be contributory factors in altering cardiac function of the diseased myocardium. The identification of the changes in such signaling systems as well as understanding the contribution of phospholipid-signaling pathways to the pathophysiology of heart disease are rapidly emerging areas of research in this field. In this paper, I provide an overview of the role of phospholipid-mediated signal transduction processes in cardiac hypertrophy and congestive heart failure, diabetic cardiomyopathy, as well as in ischemia-reperfusion. From the cumulative evidence presented, it is suggested that phospholipid-mediated signal transduction processes could serve as novel targets for the treatment of the different types of heart disease.

Measurements of intracellular volumes by59Co and2H/1H NMR and their physiological applications

Determination of the intracellular water volumes using NMR spectroscopy was performed using the NMR-visible nuclei: 59Co and 2H or 1H. Accurate measurement of intracellular water in cell suspensions and perfused organs is an important physiological parameter in the context of electrolyte homeostasis and energy metabolism, in particular when these parameters are monitored by non-invasive NMR spectroscopy. Furthermore, repeated or continuous monitoring of intracellular water provided significant insights into the physiology of cardiac muscle and sarcolemmal membrane permeability and integrity.

Expression of phospholipase D isozymes in scar and viable tissue in congestive heart failure due to myocardial infarction

The phospholipase D (PLD) associated with the cardiac sarcolemmal (SL) membrane hydrolyses phosphatidylcholine to produce phosphatidic acid, an important phospholipid signaling molecule known to influence cardiac function. The present study was undertaken to examine PLD isozyme mRNA expression, protein contents and activities in congestive heart failure (CHF) subsequent to myocardial infarction (MI). MI was induced in rats by occlusion of the left anterior descending coronary artery. At 8 weeks after the surgical procedure, hemodynamic assessment revealed that these experimental rats were at a moderate stage of CHF. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that PLD1 and PLD2 mRNA amounts were unchanged in viable left ventricular (LV) tissue of the failing heart. Furthermore, this technique demonstrated the presence of PLD1 and PLD2 mRNA in the scar tissue. While SL PLD1 and PLD2 protein contents were elevated in the viable LV tissue of the failing heart, SL PLD1 activity was significantly decreased, whereas SL PLD2 activity was significantly increased. On the other hand, although PLD1 protein was undetectable, PLD2 protein and activity were detected in the scar tissue. Our findings suggest that differential changes in PLD isozymes may contribute to the pathophysiology of CHF and may also be involved in the processes of scar remodeling.

Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K(+)-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the alpha-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K(+)-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K(+)-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.

Nucleoside Diphosphate Kinase–Mediated Activation of Heterotrimeric G Proteins

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to receptor independent G protein activation. Apparently, the NDPK B isoform forms complexes with Gbetagamma dimers and thereby phosphorylates His266 in Gbeta1 subunits. Phosphorylated His266 mediates G protein activation by a transfer of the high energetic phosphate onto GDP, thus leading to de novo synthesis of GTP. Moreover, it has been demonstrated that the sarcolemmal content of NDPK isoforms is increased in hearts with terminal congestive heart failure leading to enhanced G protein activation. Similar data were reported in a rat model for beta-adrenoceptor-induced cardiac hypertrophy. We therefore describe in this chapter several methods which can be used for analysis of NDPK mediated G protein activation: (1) The quantification of NDPK isoforms in highly purified cardiac sarcolemmal membranes, (2) the enrichment of the NDPK B/Gbetagamma-complex from preparations of the retinal G protein transducin, (3) the analysis of the enhanced NDPK activated and high energy phosphate transfer in a neonatal rat cardiac myocyte derived cell line stably overexpressing NDPK (H10 cells), and (4) the increased activation of adenylyl cyclase by the enhanced receptor-independent activation of the stimulatory G protein alpha subunit in these cells.

Nitric Oxide Protects Cardiac Sarcolemmal Membrane Enzyme Function and Ion Active Transport against Ischemia-induced Inactivation

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.

Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca 2+ movements and the association of abnormal Ca 2+ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.

Spontaneous release of GDP from Gi proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes.

Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'- O-(2-thiodiphosphate; GDPbetaS). Here, we studied the mechanism underlying this unconventional activation. Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg2+ concentration, decreased rapidly during incubation at 37 degrees C. This decrease in enzyme activity was paralleled by a rapid release of GDP from Gi proteins, amounting to 75% release of total Gi-bound GDP within 10 min at 37 degrees C. In contrast, no GDP release was observed at 4 degrees C, and adenylyl cyclase activity remained stable for up to 20 min at 4 degrees C. GDPbetaS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37 degrees C or 4 degrees C, but almost fully prevented the decrease in enzyme activity occurring at 37 degrees C. Hence, after 10 min of incubation at 37 degrees C, adenylyl cyclase activity in the presence of GDPbetaS was increased by 150%, while no difference in activity was observed at 4 degrees C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory Gi proteins, i.e., at high concentrations of Mg2+ or Mn2+, adenylyl cyclase activity remained stable even at 37 degrees C and GDPbetaS did not stimulate activity. In conclusion, Gi proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active Gi proteins.

Depresssed Responsiveness of Phospholipase C Isoenzymes to Phosphatidic Acid in Congestive Heart Failure

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.

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles.

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.

G(s) and adenylyl cyclase in transverse tubules of heart: implications for cAMP-dependent signaling.

The transverse tubules are highly specialized invaginations of the cardiac sarcolemmal membrane involved in excitation-contraction (EC) coupling. Several proteins directly involved in EC coupling have been shown to reside either in the transverse tubular membrane or in closely associated structures. With the use of immunofluorescence microscopy, we have found that G(S) and adenylyl cyclase, key elements in the beta-adrenergic signal transduction cascade, are essentially homogeneously distributed throughout the transverse tubular network of isolated rat ventricular myocytes. G(S), in particular, was much more abundant within the transverse tubular membrane than in the peripheral sarcolemma. Furthermore, both proteins are also present in the intercalated disk region. The location of these elements of the cAMP-signaling cascade within a few micrometers of every inotropic target suggests that control and action of this second messenger are quite local. Furthermore, a similar distribution is likely for negatively inotropic receptor systems that oppose G(S)-linked receptors at the level of adenylyl cyclase. Thus, in addition to their role in EC coupling, transverse tubules appear to be the primary site for signaling by inotropic agents.

Pyridoxal 5′-Phosphate is an ATP-Receptor Antagonist in Freshly Isolated Rat Cardiomyocytes

Although extracellular ATP is considered to exert a positive inotropic action on the myocardium through purinoceptors, very little information is available regarding interventions which may modify the actions of ATP on the heart. We report here that pyridoxal 5′-phosphate (PLP), an active form of vitamin B6, shows antagonism towards ATP-induced positive inotropic effect in isolated perfused rat hearts, ATP-induced increase in [Ca2+] in freshly isolated adult cardiomyocytes and ATP-binding in cardiac sarcolemma; ED50for PLP in each of these cases varied from 10–15μm . PLP (5–50μm) was observed to antagonize the positive inotropic effect of ATP but did not modify the action of isoproterenol in the isolated perfused heart. Preincubation of cardiomyocytes with 1–50μm PLP prevented the ATP-induced increase in [Ca2+]iin a concentration-dependent manner but showed no effect on the KCl-induced increase in [Ca2+]i. Creatine phosphate and Na2HPO4as well as vitamin B6-related compounds, such as pyridoxine, pyridoxal, 4-deoxypyridoxine and isonicotinic acid hydrazide showed no effect on the ATP-induced increase in [Ca2+]iin cardiomyocytes. Furthermore, different concentrations of PLP (1–50μm) were shown to inhibit the specific ATPγS binding at both the high and low affinity sites in the cardiac sarcolemmal membrane; adrenoceptor and Ca2+-channel inhibitors did not affect the ATP-binding. It is concluded that PLP may antagonize the actions of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. Furthermore, it is suggested that PLP may serve as a valuable tool for monitoring the role of purinoceptors in cellular function.