RESUMEN
Sarcoplasmic reticulum (SR) is a specialized tubular network, which not only maintains the intracellular concentration of Ca2+ at a low level but is also known to release and accumulate Ca2+ for the occurrence of cardiac contraction and relaxation, respectively. This subcellular organelle is composed of several phospholipids and different Ca2+-cycling, Ca2+-binding and regulatory proteins, which work in a coordinated manner to determine its function in cardiomyocytes. Some of the major proteins in the cardiac SR membrane include Ca2+-pump ATPase (SERCA2), Ca2+-release protein (ryanodine receptor), calsequestrin (Ca2+-binding protein) and phospholamban (regulatory protein). The phosphorylation of SR Ca2+-cycling proteins by protein kinase A or Ca2+-calmodulin kinase (directly or indirectly) has been demonstrated to augment SR Ca2+-release and Ca2+-uptake activities and promote cardiac contraction and relaxation functions. The activation of phospholipases and proteases as well as changes in different gene expressions under different pathological conditions have been shown to alter the SR composition and produce Ca2+-handling abnormalities in cardiomyocytes for the development of cardiac dysfunction. The post-translational modifications of SR Ca2+ cycling proteins by processes such as oxidation, nitrosylation, glycosylation, lipidation, acetylation, sumoylation, and O GlcNacylation have also been reported to affect the SR Ca2+ release and uptake activities as well as cardiac contractile activity. The SR function in the heart is also influenced in association with changes in cardiac performance by several hormones including thyroid hormones and adiponectin as well as by exercise-training. On the basis of such observations, it is suggested that both Ca2+-cycling and regulatory proteins in the SR membranes are intimately involved in determining the status of cardiac function and are thus excellent targets for drug development for the treatment of heart disease.
Asunto(s)
Calcio , Retículo Sarcoplasmático , Calcio/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Relación Estructura-ActividadRESUMEN
Phosphatidylethanolamine N-methylation was examined in cardiac subcellular membranes after inducing chronic experimental diabetes in rats (65 mg streptozotocin/kg, i.v.). The incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine in diabetic sarcolemma was significantly depressed at all three catalytic sites (I, II, and III) of the methyltransferase system. An increase in methyl group incorporation was evident at site I without any changes at sites II and III in diabetic sarcoplasmic reticulum and mitochondria. Similar changes were also seen for the individual N-methylated lipids (monomethyl-, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site in all cardiac membranes from diabetic animals. These alterations in N-methylation were reversible by a 14-d insulin therapy to the diabetic animals. In the presence of 10 microM ATP and 0.1 microM Ca2+, N-methylation was maximally activated at site I in both control and diabetic sarcolemma and sarcoplasmic reticulum, but not in mitochondria. Incubation of cardiac membranes with of S-adenosyl-L-methionine showed that Ca2(+)-stimulated ATPase activities in both sarcolemma and sarcoplasmic reticulum were augmented; however, the activation of diabetic sarcolemma was lesser and that of diabetic sarcoplasmic reticulum was greater in comparison with the control preparations. These results identify alterations in phosphatidylethanolamine N-methylation in subcellular membranes from diabetic heart, and it is suggested that these defects may be crucial in the development of cardiac dysfunction in chronic diabetes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Metiltransferasas/metabolismo , Miocardio/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Calcio/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Masculino , Metilación , Mitocondrias Cardíacas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa , Ratas , Ratas Endogámicas , S-Adenosilmetionina/metabolismoRESUMEN
The transmethylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) was studied in rat heart sarcolemmal membrane. Kinetically, three apparent Km values for S-adenosyl-L-methionine (AdoMet) were obtained when the total [3H]methyl groups incorporation into the phospholipids was examined in the presence of 0.01-250 microM AdoMet. A first methyltransferase active site having a very low Km (0.1 microM) for AdoMet showed a partial requirement for Mg2+ and an optimum pH of 8.0 with a major formation of phosphatidyl-N-monomethylethanolamine (PMME). Both Ca2+ and K+ were inhibitory to this site. A second active site with a Km of 3.6 microM showed an optimum pH of 7.0 with predominant formation of phosphatidyl-N,N-dimethylethanolamine (PDME) and no Mg2+ requirement; in addition, transmethylation activity was also observed over a broad alkaline pH range (9-11) with an optimum at pH 10.5. This site was insensitive to Ca2+ but was stimulated by Na+, while K+ had an inhibitory effect. A third active site with a Km of 119 microM showed an optimum pH of 10.5 with major formation of PC and no Mg2+ requirement. This site was also insensitive to Ca2+ but markedly inhibited by both K+ and Na+. Under optimal conditions, the activities of all three methyltransferase sites were linear for at least 30 min of incubation and the sensitivity to the inhibitory effect of S-adenosyl-L-homocysteine was different for each site. Addition of exogenous PMME and PDME as substrates enhanced the synthesis of the corresponding methylated products by 3-5-fold and 3-8-fold, respectively. In contrast, exogenous PE failed to increase methyltransferase activity. These results provide evidence for the existence of three distinct methyltransferase active sites in rat heart sarcolemma.
Asunto(s)
Metiltransferasas/metabolismo , Miocardio/enzimología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Sarcolema/enzimología , Animales , Calcio/farmacología , Cinética , Magnesio/farmacología , Masculino , Fosfatidiletanolamina N-Metiltransferasa , Potasio/farmacología , Ratas , Ratas Endogámicas , S-Adenosilmetionina/metabolismo , Sodio/farmacologíaRESUMEN
Although cardiac myofibrillar ATPase activity has been shown to be depressed during the development of diabetic heart dysfunction, the mechanisms of this alteration are not fully understood. Since phosphorylation of troponin I (TnI) is known to decrease the myofibrillar ATPase activity, the present study was undertaken to examine the TnI phosphorylation capacity in the diabetic heart homogenate. For this purpose rats were made diabetic by injecting streptozotocin (65 mg/kg; i.v.) and the hearts were removed 8 wk later. Some 6 wk diabetic animals were injected with insulin (3 U/day) for 2 wk. TnI content in the heart homogenate was measured by immunoblot assay, the mRNA abundance for TnI gene was determined by Northern blot analysis and the in vitro phosphorylation level of TnI was estimated by the ratio of phosphorylated TnI and total (phosphorylated and unphosphorylated) TnI. No significant changes in TnI content and gene expression of TnI were observed in right and left ventricles from the diabetic rats. However, the phosphorylation of TnI was higher (approximately 40%) in the diabetic hearts; this change was reversible upon insulin treatment. These results regarding TnI phosphorylation measured under in vitro conditions suggest that increased phosphorylation of TnI may contribute toward the depression in cardiac myofibrillar ATPase activity in chronic diabetes.
Asunto(s)
Miocardio/metabolismo , Troponina/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Expresión Génica , Masculino , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Troponina/genética , Troponina IRESUMEN
The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5'-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5'-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same Km for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in Km for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5'-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.
Asunto(s)
Miocardio/enzimología , Nucleotidasas/aislamiento & purificación , Fosfolipasas/metabolismo , Sarcolema/enzimología , Fosfolipasas de Tipo C/metabolismo , 5'-Nucleotidasa , Animales , Cinética , Lípidos de la Membrana , Nucleotidasas/metabolismo , Fosfatidilinositoles , Ratas , Solubilidad , Especificidad por SustratoRESUMEN
The phospholipase D (PL D), which catalyzes the formation of phosphatidic acid (PA), was studied in rat myocardium using 14C-labelled phosphatidylcholine (PC) as an exogenous substrate. Subcellular distribution experiments indicated the presence of PL D in particulate fractions only. Different procedures for the isolation of purified cardiac subcellular organelles showed the presence of PL D in sarcolemma (SL), sarcoplasmic reticulum (SR) and mitochondria with 14-, 11- and 5-fold enrichment when compared to the homogenate value, respectively. The activity of SL PL D was observed over a narrow acid pH range with an optimum at 6.5, and it showed a high specificity for PC while phosphatidylethanolamine and phosphatidylinositol showed a low rate of hydrolysis. Under optimal conditions, PA formation was linear for a 90-min period of incubation and the reaction rate was constant for 10 to 100 micrograms SL protein in the assay medium. The SR PL D displayed properties similar to those seen with the SL PL D. In membrane fractions PL D was also found to catalyze a transphosphatidylation reaction for the synthesis of phosphatidylglycerol. Assessment of the intramembranal levels of radioactive 1,2-diacylglycerol (DAG) in the absence or presence of KF suggested the presence of an active PA phosphohydrolase activity. This study indicates that a PC-specific PL D activity is localized in different membrane systems of the myocardium and may be associated with PA phosphohydrolase to act in a coordinated manner. The functional significance of PL D-dependent formation of PA in cardiac membranes is discussed.
Asunto(s)
Ventrículos Cardíacos/enzimología , Miocardio/enzimología , Fosfolipasa D/metabolismo , Animales , Diglicéridos/metabolismo , Hidrólisis , Masculino , Mitocondrias/enzimología , Fosfatidilgliceroles/metabolismo , Ratas , Ratas Endogámicas , Sarcolema/enzimología , Retículo Sarcoplasmático/enzimología , Especificidad por SustratoRESUMEN
We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.
Asunto(s)
Miocardio/enzimología , Fosfolipasas de Tipo C/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Radicales Libres , Glutatión/farmacología , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/farmacología , Sarcolema/enzimología , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.
Asunto(s)
Miocardio/enzimología , Nucleotidasas/metabolismo , Sarcolema/enzimología , Alameticina/farmacología , Animales , Fraccionamiento Celular/métodos , Activación Enzimática , Neuraminidasa/metabolismo , Polietilenglicoles/farmacología , Ratas , Solubilidad , Tripsina/metabolismoRESUMEN
In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions (O2-) and hydrogen peroxide (H2O2), an activated species of oxygen, was found to decrease Ca(2+)-stimulated ATPase activity, increase Mg(2+)-ATPase activity and reduce sulfhydryl (SH) group contents in myofibrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both H2O2 and hypochlorous acid (HOCl), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H2O2 and HOCl were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), inhibitors of SH groups, also produced effects similar to those seen with X + XO. Dithiothreitol (DTT), a well known sulfhydryl-reducing agent, prevented the actions of X + XO, H2O2, HOCl, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may be mediated by the oxidation of SH groups.
Asunto(s)
Proteínas Contráctiles/metabolismo , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Oxígeno/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Ditiotreitol/farmacología , Radicales Libres , Peróxido de Hidrógeno/farmacología , Ácido Hipocloroso/farmacología , Técnicas In Vitro , Cinética , Masculino , Ratas , Ratas Endogámicas , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismo , Xantina , Xantina Oxidasa/metabolismo , Xantinas/metabolismoRESUMEN
Incubation of purified cardiac sarcolemmal vesicles (SL) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine (PE), increased the Ca2+-stimulated ATPase and ATP-dependent Ca2+ accumulation activities. Quantitative analysis of the methylated phospholipids revealed that maximal increase of Ca2+-pump activities was associated with predominant synthesis and intramembranal accumulation of phosphatidyl-N,N-dimethylethanolamine. The stimulation of SL Ca2+-pump activities was prevented by inhibitors of PE N-methylation such as S-adenosyl-L-homocysteine and methyl acetimidate hydrochloride. The results suggest a possible role of PE N-methylation in the regulation of Ca2+-transport across the heart SL membrane.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Miocardio/enzimología , Fosfatidiletanolaminas/metabolismo , S-Adenosilmetionina/farmacología , Sarcolema/enzimología , Animales , Cinética , Masculino , Metilación , Metiltransferasas/metabolismo , Fosfatidil-N-Metiletanolamina N-Metiltransferasa , Ratas , Ratas Endogámicas , Sarcolema/efectos de los fármacosRESUMEN
This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Miocardio/metabolismo , Fosfatidiletanolaminas/metabolismo , Superóxidos/farmacología , Animales , Calcio/metabolismo , Radicales Libres , Masculino , Metilación , Ratas , Ratas Endogámicas , Retículo Sarcoplasmático/metabolismo , Xantina , Xantina Oxidasa/farmacología , Xantinas/farmacologíaRESUMEN
The adenylate cyclase activity and phospholipid composition were determined in rat heart sarcolemma after treating the membranes with a phosphatidylinositol-specific phospholipase C. Complete hydrolysis of phosphatidylinositol in sarcolemma was associated with a marked loss of the basal adenylate cyclase activity. The recombination of the supernatant with the phosphatidylinositol-depleted membranes was found to reactivate the adenylate cyclase activity. The soluble component(s) in the supernatant, which restored the adenylate cyclase activity, was thermolabile and precipitated by ammonium sulfate. Extensive hydrolysis of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in sarcolemma with a Clostridium welchii phospholipase C treatment did not affect the basal adenylate cyclase activity. These results suggest that phosphatidylinositol anchors component(s) essential for the expression of basal adenylate cyclase activity to the myocardial cell membrane.
Asunto(s)
Adenilil Ciclasas/metabolismo , Miocardio/enzimología , Fosfatidilinositoles/fisiología , Sarcolema/enzimología , Animales , Membrana Celular/metabolismo , Clostridium/enzimología , Fosfatidilinositoles/metabolismo , Fosfolípidos/análisis , Ratas , Staphylococcus aureus/enzimología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Although lysophosphatidylcholine (lyso-PtdCho) accumulates in the sarcolemmal (SL) membrane and alters its function during myocardial ischemia and diabetic cardiomyopathy, the effects of lyso-PtdCho on SL signalling processes have not yet been investigated. The present study was carried out to examine the actions of lyso-PtdCho on the rat heart SL membrane enzymes involved in the phosphoinositide pathway. Different lyso-PtdCho species (10 to 200 microM) inhibited the activities of both phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase in the SL membrane in a concentration-dependent manner. The inhibitory potency of lyso-PtdCho compounds for phosphatidylinositol kinase was lyso-PtdCho plasmalogen > 1-oleoyl-lyso-PtdCho > 1-stearoyl-lyso-PtdCho > 1-palmitoyl-lyso-PtdCho, and that for phosphatidylinositol-4-phosphate kinase was lyso-PtdCho plasmalogen > 1-oleoyl-lyso-PtdCho > 1-palmitoyl-lyso-PtdCho > 1-stearoyl-lyso-PtdCho. The inhibitory effect of lyso-PtdCho on phosphatidylinositol-4-phosphate kinase was greater than that on phosphatidylinositol kinase. Lyso-PtdCho structural analogues, such as phosphatidylcholine, lysophosphatidic acid, lysophosphatidylethanolamine, L-alpha-glycerophosphate, oleate and phosphorylcholine, did not affect the phosphoinositide kinases, suggesting that the intact structure of lyso-PtdCho was required for the inhibition of the kinases. The detrimental action of lyso-PtdCho on PtdIns kinase was potentiated by acidosis. Unlike Ca2+, ATP (0.1 and 4 mM) increased lyso-PtdCho-induced deactivation of the kinases. Both enzyme activities were found to be depressed in the ischemic-reperfused or diabetic hearts. None of the tested lyso-PtdCho species altered phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis by SL phospholipase C. These results indicate that accumulation of lyso-PtdCho in the SL membrane under pathological conditions may diminish the availability of the PtdIns(4,5)P2 substrate for the production of second messengers by receptor-linked phospholipase C.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Corazón/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Sarcolema/enzimología , Fosfolipasas de Tipo C/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/farmacología , Diabetes Mellitus Experimental/metabolismo , Lisofosfatidilcolinas/química , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/enzimología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Plasmalógenos/farmacología , Ratas , Ratas Sprague-Dawley , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
In view of the depressed sarcoplasmic reticulum (SR) Ca2+-pump and Ca2+-release activities in the diabetic heart and the critical role of phosphorylation in regulating the SR function, we examined the status of Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA)-mediated phosphorylations in the diabetic heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (65 mg/kg i.v.), and the animals were killed 6 weeks later for assessment of the ventricular SR function. Depressed cardiac performance and SR Ca2+-uptake and -release activities in diabetic animals were accompanied by a significant decrease in the level of SR Ca2+-cycling proteins, such as ryanodine receptor, Ca2+-pump ATPase, and phospholamban. On the other hand, the CaMK- and PKA-mediated phosphorylations of these Ca2+-cycling proteins, the endogenous SR CaMK and PKA activities, and the endogenous SR and cytosolic phosphatase activities were increased in the diabetic heart. Treatment of 3-week diabetic animals with insulin partially or fully prevented the diabetes-induced changes in cardiac performance, SR Ca2+-uptake and -release activites, and SR protein content, whereas the diabetes-induced changes in SR CaMK- and PKA-mediated phosphorylations and activities, as well as phosphatase activities, were not significantly affected. These results suggest that the reduced content of the Ca2+-cycling proteins, unlike alterations in PKA and phosphatase activities, appear to be the major defect underlying SR dysfunction in the diabetic heart.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Corazón/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/fisiología , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Masculino , Miocardio/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
It has been suggested that the occurrence of an intracellular Ca2+ overload may result in the development of diabetic cardiomyopathy, which is associated with depletion of high-energy phosphate stores and a derangement of ultrastructure and cardiac dysfunction. Accordingly, the effects of verapamil, a Ca2+ antagonist, on cardiac function, ultrastructure, and high-energy phosphate stores in the myocardium were evaluated in rats made diabetic by an intravenous injection of streptozocin (65 mg/kg). Four weeks after the induction of diabetes, the animals were treated with three doses (2, 4, or 8 mg.kg-1.day-1) of verapamil for 4 wk until they were used for the measurement of different parameters. Untreated diabetic animals had slower heart rates, depressed rate of contraction and rate of relaxation, lower peak left ventricular systolic pressure, and elevated left ventricular diastolic pressure. All of these changes were significantly improved in diabetic rats receiving verapamil treatment. The beneficial effects of verapamil were more evident with higher doses (8 mg.kg-1.day-1) than with the lower doses (2 mg.kg-1.day-1). The diabetic animals also showed alterations in myocardial high-energy phosphate stores and exhibited evidence of ultrastructural damage; these abnormalities were improved by verapamil treatment without affecting their hyperglycemic status. Our results demonstrate that verapamil is capable of preventing diabetes-induced myocardial changes and support the involvement of Ca2+ in the cardiac pathology during diabetes.
Asunto(s)
Cardiomiopatías/prevención & control , Diabetes Mellitus Experimental/complicaciones , Verapamilo/uso terapéutico , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/patología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Frecuencia Cardíaca , Masculino , Microscopía Electrónica , Contracción Miocárdica , Miocardio/metabolismo , Miocardio/patología , Miofibrillas/patología , Presión , Ratas , Ratas Endogámicas , Sarcómeros/patologíaRESUMEN
OBJECTIVES: We examined the possible effect of neuropeptide Y in modulating central sympathetic activity after myocardial infarction in rats. BACKGROUND: Previous studies have shown the coexistence of neuropeptide Y and norepinephrine in the brain and a possible functional interaction between the two. Neuropeptide Y inhibits the release of norepinephrine at the presynaptic level and can be considered to act as a neuromodulator. METHODS: Two groups of rats were examined in this study-an experimental group, defined as those rats undergoing left coronary artery ligation, and a sham group without coronary artery ligation, serving as the control group. The animal in both groups underwent microdialysis in the paraventricular nucleus at 2, 4 and 8 weeks after operation. Microdialysis samples were collected with and without injecting neuropeptide Y in the paraventricular nucleus. The concentration of norepinephrine was determined by injecting purified microdialysate samples during high performance liquid chromatography. To explore the receptor's possible role, autoradiographic localization of neuropeptide Y receptors in the paraventricular nucleus was also carried out in the experimental and sham groups. RESULTS: The concentration of norepinephrine measured in the samples was decreased by 50% with neuropeptide Y in 2- and 4-week old rats after infarction, but by only 20% (p < 0.05) in 8-week old rats after infraction. The diminished inhibitory effects of neuropeptide Y on norepinephrine release was associated with increased sympathetic activity, as reflected by plasma norepinephrine; 8-week old rats after infarction had almost a 100% (p < 0.05) increase in their plasma norepinephrine level compared with the sham group. Autoradiography revealed a significant decrease in density of neuropeptide Y receptors in the paraventricular nucleus in 8-week old rats after infarction (p < 0.05). CONCLUSIONS: The data presented in this report suggest that the reduction of the inhibitory activation of neuropeptide Y on sympathetic release may contribute to elevated norepinephrine levels after myocardial infarction.
Asunto(s)
Infarto del Miocardio/fisiopatología , Neuropéptido Y/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Autorradiografía , Masculino , Microdiálisis , Norepinefrina/biosíntesis , Núcleo Hipotalámico Paraventricular/química , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/análisisRESUMEN
Although ischemia reperfusion has been shown to depress gene expression of the sarcoplasmic reticulum (SR) proteins, such as the ryanodine receptor, Ca2+-pump ATPase, phospholamban, and calsequestrin in the heart, the mechanisms of these changes are not understood. Given the occurrence of hypoxia and the lack of glucose during the ischemic phase, we investigated the effects of these factors on the cardiac SR gene expression. Isolated rat hearts perfused in the absence of oxygen and/or glucose for 30 min showed an increase in the expression of SR genes. However, perfusion of hearts for 60 min with normal oxygenated medium after 30 min of lack of both oxygen and glucose depressed the transcript levels for the SR proteins; these changes did not occur when hearts were deprived of either oxygen or glucose. The effect of intracellular Ca2+-overload, which occurs during reperfusion, was studied by using hearts perfused for 5 min with Ca2+-free medium and then reperfused for 30 min. Ca2+-depletion/repletion induced a dramatic decrease in the transcript levels of the SR genes. These results suggest that the lack of both oxygen and glucose during ischemia are necessary for reperfusion-induced depression in SR gene expression, possibly due to the occurrence of intracellular Ca2+-overload.
Asunto(s)
Glucosa/farmacología , Miocardio/metabolismo , Oxígeno/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , ATPasas Transportadoras de Calcio/genética , Calsecuestrina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hipoxia , Técnicas In Vitro , Isquemia Miocárdica , Reperfusión Miocárdica , Perfusión , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismoRESUMEN
BACKGROUND: Myocardial ischemia-reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia-reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocyte damage. These changes are considered to be the consequence of imbalance between the formation of oxidants and the availability of endogenous antioxidants in the heart. OBSERVATIONS: An increase in the formation of reactive oxygen species during ischemia-reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Although several experimental studies as well as clinical trials have demonstrated the cardioprotective effects of antioxidants, some studies have failed to substantiate the results. Nonetheless, it is becoming evident that some of the endogenous antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase act as a primary defense mechanism whereas the others including vitamin E may play a secondary role for attenuating the ischemia-reperfusion injury. The importance of various endogenous antioxidants in suppressing oxidative stress is evident from the depression in their activities and the inhibition of cardiac alterations which they produce during ischemia-reperfusion injury. The effects of an antioxidant thiol containing compound, N-acetylcysteine, and ischemic preconditioning were shown to be similar in preventing changes in the ischemic-reperfused hearts. CONCLUSIONS: The available evidence support the role of oxidative stress in ischemia-reperfusion injury and emphasize the importance of antioxidant mechanisms in cardioprotection.
Asunto(s)
Antioxidantes/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Estrés Oxidativo , Acetilcisteína/metabolismo , Animales , Apoptosis , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Humanos , Precondicionamiento Isquémico Miocárdico , Contracción Miocárdica , Superóxido Dismutasa/metabolismo , Vitamina E/metabolismoRESUMEN
Heart dysfunction in chronic diabetes has been observed to be associated with depressed myofibrillar adenosine triphosphatase activities as well as abnormalities in the sarcoplasmic reticular and sarcolemmal calcium transport processes. The evidence has been presented to show that alterations in the expression of myosin isozymes and regulatory proteins as well as myosin phosphorylation contribute to the development of myofibrillar remodeling in the diabetic heart. Defects in sarcoplasmic reticular and sarcolemmal calcium transport appear to be due to the accumulation of lipid metabolites in the membrane. Different agents, such as calcium-antagonists, beta-adrenoceptor blockers, angiotensin converting enzyme inhibitors, metabolic interventions and antioxidants, have been reported to exert beneficial effects in preventing subcellular remodeling and cardiac dysfunction in chronic diabetes. Clinical and experimental investigations have suggested that increased sympathetic activity, activated cardiac renin-angiotensin system, myocardial ischemia/functional hypoxia and elevated levels of glucose for a prolonged period, due to insulin deficiency, result in oxidative stress. It is proposed that oxidative stress associated with a deficit in the status of the antioxidant defense system may play a critical role in subcellular remodeling, calcium-handling abnormalities and subsequent diabetic cardiomyopathy.
Asunto(s)
Cardiomiopatías/etiología , Complicaciones de la Diabetes , Corazón/fisiopatología , Miocardio/ultraestructura , Calcio/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Humanos , Insulina/metabolismo , Contracción Miocárdica , Miofibrillas/metabolismo , Estrés Oxidativo , Sistema Renina-Angiotensina , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Sistema Nervioso Simpático/fisiopatología , Hormonas Tiroideas/deficienciaRESUMEN
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), PLCdelta(1) isoenzyme activity in the SL membrane was elevated, with a concomitant increase in its protein content, whereas PLCbeta(1) and gamma(1) isoenzyme activities were depressed despite the increase in their protein levels. 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 Ca(2+) overload in the failing cardiomyocyte.