Cardiac Membrane Fraction Research Articles

Abstract 240: Strategic Distribution of the Mitochondrial Ca2+ Uniporter in Cardiac Mitochondria

Propagation of SR-derived Ca2+ signals to the mitochondrial matrix is an important tuning tool of cardiac energy metabolism (excitation-bioenergetics coupling). Effective activation of the low-affinity mitochondrial Ca2+ uniporter (mtCU) is supported by high [Ca2+] nanodomains where SR cisternae are tethered to mitochondria. Cardiac mitochondria have large inner membrane (IMM) surface with extensive cristae folding. However, recent comparative patch clamp studies found cardiac mtCU current density extremely low. We hypothesized that efficient excitation-bioenergetics coupling would require strategic positioning of the mtCU. We thus analyzed the distribution of mtCU’s pore protein, MCU in rat and mouse cardiomyocytes and cardiac membrane fractions. Immunofluorescence (IF) using the commercial Sigma antibody showed significant non-mitochondrial (nuclear, cyto/sarcoplasmic) crossreactions as verified in MCU-KO MEFs and cardiomyocytes. To isolate mitochondrial labeling, we performed IF of isolated mitochondria (mtIF) and immunogold electron microscopy of freshly isolated cardiomyocytes. mtIF showed ~50% of MCU-positive structures colocalized with RyR2, in line with a location preference to SR-mitochondria associations. This distribution bias was also reflected in the immunogold TEM analysis. Moreover, WB showed both MCU and calsequestrin being enriched in the same sub-mitochondrial membrane fraction separated on sucrose gradient. The prevalence of MCU and even more the essential MCU regulator (EMRE) was higher in the mitochondria “contaminating” the heavy SR (SR-associated mito membranes) than in the crude mitochondrial fraction. [Ca2+] activation of mitochondrial Ca2+ uptake at similar IMM potentials was as effective in suspensions of SR-associated mito membranes as in the crude mitochondrial fraction. Collectively, our data suggest a location bias of the functionally mandatory mtCU channel components MCU and EMRE toward SR-mitochondrial associations. As the bias is much stronger for EMRE, the component that is required for MCU to function as a channel, it raises the possibility that EMRE availability may be a physiological limiting factor in regional mtCU activity in the cardiac muscle mitochondria.

Abstract 19390: Sirtuin 1 Modulates Cardiac Sodium Channels in vivo

The rapid upstroke in phase 0 of the cardiac action potential is the result of the inward sodium current (I Na ) carried by the cardiac voltage-gated sodium channel Na v 1.5 (SCN5A). We recently reported that the overexpression of Silent Information Regulator 1 (Sirtuin 1, SIRT1), an NAD + dependent protein deacetylase, deacetylated SCN5A, increased its cell surface expression, and increased I Na in HEK-293 cells and rat neonatal cardiac myocytes. Overexpression of a dominant negative SIRT1 construct decreased SCN5A cell surface expression and I Na . The role of acetylation of SCN5A and its regulation by SIRT1 has not been shown in vivo, however. We engineered cardiac specific Sirt1 knockout (cSirt1KO) mice by breeding Sirt1 flox/flox with α-MHC-cre mice on a C57BL-6 background. cSirt1KO compared to littermate Sirt1 flox/flox mice showed much less expression of Sirt1 mRNA (12±6%, n=3) and protein in the heart but not in the kidney, and Scn5a immunoprecipitated from cardiac membrane fractions of the cSirt1KO mice showed an ∼2-fold increase in acetylation (Figure A). At 3 months of age, EKGs from anesthetized cSirt1KO compared to littermate Sirt1 flox/flox mice showed PR prolongation (43±3 ms, n=4, vs. 37±2 ms, n=5; p<0.02) and telemetry from ambulatory cSirt1KO mice showed high degree heart block not seen in the controls (Figure B). Thus, our results show that Scn5a is acetylated in vivo in the mouse, that the degree of acetylation is regulated by cardiac-specific expression of Sirt1, and that decreases in Sirt1 lead to conduction disease similar to those seen in Scn5a +/- mice. These findings raise the possibility that pharmacological manipulation of Sirt1 may provide a means to modulate cardiac I Na and arrhythmias.

Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short- or long-term dietary exposure.

BackgroundPrevious work showed that the functional cardiac effect of dietary alpha-linolenic acid (ALA) in rats requires a long feeding period (6 months), although a docosahexaenoic (DHA) acid-supply affects cardiac adrenergic response after 2 months. However, the total cardiac membrane n-3 polyunsaturated fatty acid (PUFA) composition remained unchanged after 2 months. This delay could be due to a specific reorganization of the different subcellular membrane PUFA profiles. This study was designed to investigate the evolution between 2 and 6 months of diet duration of the fatty acid profile in sarcolemmal (SL), mitochondrial (MI), nuclear (NU) and sarcoplasmic reticulum (SR) membrane fractions.MethodsMale Wistar rats were randomly assigned to 3 dietary groups (n = 10/diet/period), either n-3 PUFA-free diet (CTL), or ALA or DHA-rich diets. After 2 or 6 months, the subcellular cardiac membrane fractions were separated by differential centrifugations and sucrose gradients. Each membrane profile was analysed by gas chromatography (GC) after lipid extraction.ResultsAs expected the n-3 PUFA-rich diets incorporated n-3 PUFA instead of n-6 PUFA in all the subcellular fractions, which also exhibited individual specificities. The diet duration increased SFA and decreased PUFA in SL, whereas NU remained constant. The SR and MI enriched in n-3 PUFA exhibited a decreased DHA level with ageing in the DHA and CTL groups. Conversely, the n-3 PUFA level remained unchanged in the ALA group, due to a significant increase in docosapentaenoic acid (DPA). N-3 PUFA rich diets lead to a better PUFA profile in all the fractions and significantly prevent the profile modifications induced by ageing.ConclusionWith the ALA diet the n-3 PUFA content, particularly in SR and SL kept increasing between 2 and 6 months, which may partly account for the delay to achieve the modification of adrenergic response.

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T 3 increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T 3-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34–70% and 43–68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.

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.

Glyceraldehyde 3‐phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal KATP channel

Cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal K(ATP)-channel protein complex. A polypeptide of approximately 42 kDa was immunoprecipitated with an anti-SUR2A antibody from guinea-pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti-Kir6.2, anti-SUR2A and anti-GAPDH antibodies showed that GAPDH is a part of the sarcolemmal K(ATP)-channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two-hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch-clamp electrophysiology showed that GAPDH regulates K(ATP)-channel activity irrespective of high intracellular ATP, by producing 1,3-bisphosphoglycerate, a K(ATP)-channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal K(ATP)-channel protein complex, where it couples glycolysis with the K(ATP)-channel activity.

Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload

Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K + channels in a gender-dependent manner

The opening of sarcolemmal K ATP channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K ATP channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K + (K ATP) channels. RT-PCR using primers specific for K ATP channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 μM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K ATP channels which is due to decrease in levels of the SUR2A subunit.

Creatine kinase is physically associated with the cardiac ATP-sensitive K+ channel in vivo.

Cardiac sarcolemmal ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at approximately 48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.

Gender-specific difference in cardiac ATP-sensitive K+channels

OBJECTIVESThe main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K+(KATP) channels.BACKGROUNDRecently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as KATPchannels.METHODSA reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca2+imaging were performed on isolated guinea pig ventricular cardiomyocytes.RESULTSThe RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 μM), a KATPchannel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single KATPchannel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca2+loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in KATPchannel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required.CONCLUSIONSThis study has demonstrated that female tissue expresses higher levels of functional cardiac KATPchannels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.

Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100µM Ca2+; however, the interactions were interrupted in the presence of 20mM EGTA, 1µM rapamycin or 1µM FK506, suggesting that the interaction is Ca2+-dependent, and is mediated by FKBP12.6. The Ca2+-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20µM deltamethrin, 10µM cyclosporin A (CsA), or 10µM FK506 led to Ca2+ oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20µM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca2+ oscillations, probably due to the leakiness of the RyR. However, Ca2+ oscillations by rapamycin were not further affected by 10µM CsA or 10µM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca2+-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10µM ryanodine-treated cardiomyocytes, CsA failed to induce Ca2+ oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca2+ release in rat heart.

Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100 microM Ca(2+); however, the interactions were interrupted in the presence of 20 mM EGTA, 1 microM rapamycin or 1 microM FK506, suggesting that the interaction is Ca(2+)-dependent, and is mediated by FKBP12.6. The Ca(2+)-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20 microM deltamethrin, 10 microM cyclosporin A (CsA), or 10 mciroM FK506 led to Ca(2+) oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20 microM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca(2+) oscillations, probably due to the leakiness of the RyR. However, Ca(2+) oscillations by rapamycin were not further affected by 10 microM CsA or 10 mciroM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca(2+)-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10 microM ryanodine-treated cardiomyocytes, CsA failed to induce Ca(2+) oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca(2+) release in rat heart.

Dietary fish oil promotes positive inotropy and efficiency of digitalis.

Digitalis is clinically effective in heart failure, but it has an extremely narrow therapeutic index. Inclusion of fish oil (FO) in the diet may have cardioprotective effects by different mechanisms (1,2): decreased heart rate and occurrence of arrhythmias, increased mechanical activity, and ejection fraction. The aim of this study was to investigate if dietary FO with incorporation of n-3 polyunsaturated fatty acids in membranes could promote positive inotropy of ouabain. Methods. Twenty-five male Wistar rats were fed for 60 d: (i) supplemented with FO concentrate [180 mg eicosapentaenoic acid (EPA) + 120 mg docosahexaenoic acid (DHA)/g oil] at a dose of 0.5 g/kg/d (group FO), (ii) without supplementation (group C). Inotropic and toxic responses to cumulative doses of ouabain (from 100 nM to 0.3 raM) were evaluated in the isolated perfused rat heart and the relative variations in cardiac energetic metabolism, and pH were obtained using 3tp magnetic resonance spectroscopy. Activity, contribution, affinity, and expression of Na,K-ATPase isoforms were determined on purified membrane-bound Na,K-ATPase. Fatty acid composition of cardiac membrane fractions was analyzed by gas chromatography. Results. The rat is the only mammalian species with a well-characterized biphasic inotropic response to ouabain which is coupled to consecutive inhibition of the cx2- and o~[isoforms of high and low affinity for ouabain, respectively. Results show that (i) there is a twofold increase in the lowaffinity response to ouabain (0.1 mM) (% inotropy = 40 _+ 3 in group C vs. 79 -+ 7 in group FO, P < 0.001), (ii) there is a lower inhibition of ctl Na,K-ATPase activity (ICs0 = 7.2 _+ 2.0 × 10 -5 M in group C and 3.4 _+ 0.8 × 10 -4 M in group FO, P < 0.05), (iii) the higher inotropic response to ouabain is not associated with significant changes in energetics, (iv) alteration of function and energetics in response to the toxic dose of 0.3 mM is delayed in the FO group, (v) there are no differences in overall Na,K-ATPase activity or in the proportion of its isoforms, and (vi) fatty acid composition of rat hearts submitted to FO diet shows that the proportion of EPA and DHA had significantly risen. In conclusion, improving ouabain efficacy may represent an additional mechanism whereby FO exert their cardioprotective action in rats and possibly in humans. Acknowledgment. This work was supported in part by grants of the French Ministry of Health (PHRC, UF t638 and UF 1673), by MRT (grant 650994), CNRS (UMR 6612 and URA 1829), and the Assistance Publique-H6pitaux de Marseille.

Contractile Failure in Chronic Doxorubicin-induced Cardiomyopathy

The mechanisms for the progression of the anthracycline-induced cardiomyopathy to contractile failure has not been defined.In vitro, doxorubicin (DOX) appears to modify calcium-mediated excitation–contraction coupling, which depresses cardiac contractility. This study characterizes the onset of contractile failure associated with the development of DOX-induced cardiomyopathy. Rabbits were treated with DOX (1 mg/kg i.v. twice weekly, 12–18 doses; DOX-treated group) and compared with a pair-fed Control group infuse with saline vehicle. The severity of the cardiomyopathy was determined by numerically-scored histopathology. Myocardial contractility was determined in thin fiber bundles from right ventricular (RV) papillary muscles and left atria that were removed and mounted on a force transducer in oxygenated Krebs-bicarbonate buffer (pH=7.4 at 30°C) to record the amplitude (DT) and maximum rate (+dT/dt) of isometric tension. Myofibrillar and calcium loading properties were determined by the calcium and caffeine-activated tension responses respectively in chemically-permeabilized fibers. With the onset of the cardiomyopathy (score <2) DT at low frequency (0.5 Hz) was depressed (0.61±0.01 mN/mg;n=14) compared to Control (0.93±0.09 mN/mg;n=15). Contractility at higher rates (1 Hz) was not different in this DOX-treated and Control groups. Maximum calcium and caffeine-activated force and the pCa to half-maximum force of permeabilized fibers were comparable in DOX-treated and Control groups. The loss of contractility of the DOX-treated group was related to reduction in sacroplasmic reticulum calcium release channel density, as determined by Bmaxfor3H-ryanodine binding in cardiac microsomal membrane fraction. Post-rest potentiation of contractility, as well as frequency-dependent (0.25–1.5 Hz) and post-extrasystolic potentiation of contractility were preserved in the DOX-treated group.In vitro, DOX depressed post-rest potentiation of contractility. Thus, the onset of contractile failure of the DOX-induced cardiomyopathy is characterized by effects consistent with disordered calcium-mediated excitation–contraction coupling and these effects are qualitatively different thanin vitroeffects of DOX.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart

The effects of the endothelin receptor antagonist TAK-044 ( cyclo[ d-α-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]- l-alanyl- l-α-aspartyl- d-2-(2-thienyl)glycyl- l-leucyl- d-tryptop ( cyclo[ d-Asp-Pro- d-Val-Leu- d-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-1-induced responses (100 pmol/heart) with IC 50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1–30 μM) partially inhibited the endothelin-1-induced hypertension (30–40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10 μM) nor N ω-nitro- l-arginine methyl ester (100 μM) attenuated the endothelin-1-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [ 125I]endothelin-1 binding to endothelin ET A receptors with IC 50 values of 0.39 ± 0.6 and 36 ± 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET B receptor subtype (IC 50 value: 370 ± 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET A and endothelin ET B receptors, all of which are sensitive to TAK-044.

Blockade of the renin???angiotensin system in heart failure in conscious dogs

To study the different cardiac and renal hemodynamic effects of an angiotensin converting enzyme inhibitor and an angiotensin II receptor antagonist in experimental heart failure in conscious dogs. We compared the effects of the angiotensin converting enzyme inhibitor, captopril, with those of the angiotensin II (Ang II) subtype-1 receptor antagonist, losartan, on hemodynamics and hormonal changes in congestive heart failure by rapid ventricular pacing on conscious dogs. Furthermore, we characterized the Ang II receptors in canine heart, using the canine cardiac membrane fraction in heart failure. Acute intravenous administration of captopril improved the cardiac output by 19% (P < 0.01) but losartan did not, although blockade of the renin-angiotensin system by losartan (1.1 mumol/kg) or captopril (0.69 mumol/kg) induced similar changes in the plasma renin activity, norepinephrine and arginine vasopressin, and a similar decrease in mean arterial pressure (-10 mmHg). Renal blood flow was increased by either losartan or captopril. In the binding study, losartan produced a single displacement curve (IC50 = 0.25 mumol/l), while the Ang II subtype-2 (AT2) receptor antagonist PD123319 did not, indicating that the predominant Ang II receptor is type-1 (AT1) in canine heart. Neither the ratio of AT1 to AT2 receptors nor the receptor density changed with the development of heart failure. The lack of effect of losartan on cardiac output may be the result of its inability to block non-AT1 receptor-mediated Ang II activities adequately. Captopril may improve cardiac output by means of mechanisms not mediated by Ang II, such as locally increasing bradykinin.

Isolation of myocardial membrane long-chain fatty acid-binding protein: Homology with a rat membrane protein implicated in the binding or transport of long-chain fatty acids

Abnormal myocardial long-chain fatty acid uptake is suspected of being involved in certain types of heart disease, but the mechanism by which the heart takes up long-chain fatty acids remains unclear. The sulfo-N-succinimidyl derivatives of long-chain fatty acids have been reported to undergo covalent binding to a membrane protein and to irreversibly inhibit the transport of long-chain fatty acids by rat adipocytes (Harmon et al., 1991). It has been suggested that the membrane protein bound by these derivatives is a candidate transporter for long-chain fatty acids in adipocytes. However, myocardial membrane long-chain fatty acid-binding proteins have not yet been fully investigated. Rat hearts were isolated and perfused with a sulfo-N-succinimidyl derivative of tritium-labeled palmitate ([3H]SSP). Then the [3H]SSP-binding protein was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and histological autoradiography. Myocardial palmitic acid uptake was examined after pretreatment of isolated perfused rat hearts with SSP. The SSP-binding protein was isolated from bovine hearts by successive chromatography, and the amino acid sequences of lysylendopeptidase-digested peptide fragments were determined. SDS-PAGE autoradiography revealed that [3H]SSP bound to an 85-90 kDa protein derived from the myocardial microsomal fraction, and histological autoradiography demonstrated that [3H]SSP radioactivity was localized to the myocardial cell membrane. Pre-incubation with SSP inhibited palmitic acid uptake by isolated perfused rat hearts. A [3H]SSP-binding protein was also found in canine and bovine hearts, and was isolated from the bovine cardiac membrane fraction. Amino acid sequencing revealed that four peptide fragments showed strong sequence homology with rat adipocyte membrane protein, which is implicated in the binding or transport of long-chain fatty acids (Abumrad et al., 1993). We conclude that the SSP-binding protein is localized to the myocardial cell membrane and might be involved in the uptake or transport of long-chain fatty acids.

Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis.

The existence of a cardiac renin-angiotensin system, independent of the circulating renin-angiotensin system, is still controversial. We compared the tissue levels of renin-angiotensin system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. Angiotensin I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac angiotensins and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n = 11) and Ang I (n = 7) and II (n = 7) in the left and right atria were higher than in the corresponding ventricles (P < .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P < .05). The presence of these renin-angiotensin system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. Angiotensinogen levels (n = 11) in cardiac tissue were 10% to 25% of the levels in plasma, which is compatible with its diffusion from plasma into the interstitium. Like angiotensin-converting enzyme, renin was enriched in a purified cardiac membrane fraction prepared from left ventricular tissue, as compared with crude homogenate, and 12 +/- 3% (mean +/- SD, n = 6) of renin in crude homogenate was found in the cardiac membrane fraction and could be solubilized with 1% Triton X-100. Tissue levels of renin and Ang I and II in the atria and ventricles were directly correlated with plasma levels (P < .05), and in both tissue and plasma the levels were undetectably low after nephrectomy. We conclude that most if not all renin in cardiac tissue originates from the kidney. Results support the contentions that in the healthy heart, angiotensin production depends on plasma-derived renin and that plasma-derived angiotensinogen in the interstitial fluid is a potential source of cardiac angiotensins. Binding of renin to cardiac membranes may be part of a mechanism by which renin is taken up from plasma.

Triorganotin inhibition of rat cardiac adenosine triphosphatases and catecholamine binding.

Triorganotins have been reported to affect heme metabolism as well as the cardiovascular system. Our recent studies indicated that these organotins inhibit cardiac sarcoplasmic reticulum Ca(2+)-transport and cAMP-stimulated phosphorylation of specific proteins involved in Ca2+ transport, suggesting their interference with cardiac adrenergic function. The present study determines the effect of three organotins--tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT)--on rat cardiac ATPases and catecholamine binding, since these phenomena are involved in cardiac function. Cardiac membrane fraction was prepared from heart ventricles of male Sprague-Dawley rats. All three organotins inhibited cardiac Na+,K(+)-ATPase, [3H]ouabain binding, K(+)-activated p-nitrophenyl phosphatase (K(+)-PNPPase) and oligomycin-sensitive (OS) and oligomycin-insensitive (OI) Mg(2+)-ATPase in a concentration-dependent manner. K(+)-PNPPase was less sensitive to these triorganotins when compared to Na+K(+)-ATPase, suggesting that triorganotins affect the Na(+)-pump activity by acting on the Na(+)-dependent phosphorylation process. OS Mg(2+)-ATPase was more sensitive to these organotins when compared to OI Mg(2+)-ATPase, confirming their potent effect on the enzymes of oxidative phosphorylation. The order of potency is TBT greater than TET greater than TMT. TET and TMT, but not TBT, inhibited [3H]norepinephrine and [3H]dopamine binding to cardiac membranes in a concentration-dependent manner, the effect being more with TET. These results suggest that triorganotins inhibit sodium pump activity as well as ATP synthesis. Since Na+,K(+)-ATPase is involved in the active transport of catecholamines, triorganotins not only inhibited the catecholamine transport but also to some extent affected catecholamine binding, thus interfering with cardiac function.

Effects of Corn Oil Addition to the Diet on the Energy Metabolism of Heart, Liver and Kidney of Female Rats

Female rats (SIV-50, Sprague Dawley) given a diet enriched with corn oil (10% and 25% addition to rat chow, w/w) for four weeks showed changes in the energy metabolism of heart, liver and kidneys, that is, changes in oxygen consumption and uncoupling of oxidative phosphorylation of heart, liver and kidney mitochondria, concomitant to a decrease in heart mitochondrial creatine kinase activity and an increase in heart mitochondrial creatine content. The high fat diet also affected calcium binding and Na/K-, Mg- and Ca-ATPases of a cardiac myocyte membrane fraction. Lipid feeding also led to increase (biphasic and transient) of phospholipids, triglycerides and free fatty acids in the three organs studied, but in no case to hypertrophy.