RESUMEN

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Cardiolipinas/biosíntesis , Grupo Citocromo c/metabolismo , Miocardio/metabolismo , Ácido Palmítico/toxicidad , Animales , Animales Recién Nacidos , Células Cultivadas , Espectrometría de Masas , Miocardio/citología , Miocardio/enzimología , Ratas , Ratas Sprague-Dawley

RESUMEN

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H2O2 production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (nonapoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H2O2 synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.

Asunto(s)

Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Miocardio/metabolismo , Ácido Palmítico/farmacología , Transducción de Señal , Animales , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Ceramidas/metabolismo , Grupo Citocromo c/metabolismo , ADN/análisis , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Miocardio/patología , Ácido Oléico/farmacología , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley

RESUMEN

Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (-301/-289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta) gene defines regulatory regions in the -391/+80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart.

Asunto(s)

Carnitina O-Palmitoiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Mitocondrias Cardíacas/enzimología , Miocardio/enzimología , Proteínas Nucleares/metabolismo , Podofilino/análogos & derivados , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Animales Recién Nacidos , Células Cultivadas , Factor de Transcripción GATA4 , Genes Reporteros , Humanos , Isoenzimas/genética , Luciferasas/genética , Miocardio/citología , Especificidad de Órganos , Podofilino/metabolismo , Podofilotoxina/análogos & derivados , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Respuesta Sérica , Transfección , Dedos de Zinc

RESUMEN

After cardiac ischemia, long-chain fatty acids, such as palmitate, increase in plasma and heart. Palmitate has previously been shown to cause apoptosis in cardiac myocytes. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. Phosphatidylserine translocation and caspase 3-like activity confirmed the apoptotic action of palmitate. Cytosolic cytochrome c was detected at 8 h and plateaued at 12 h. The mitochondrial membrane potential (DeltaPsi) in tetramethylrhodamine ethyl ester-loaded cardiac myocytes decreased significantly in individual mitochondria by 8 h. This loss was heterogeneous, with a few energized mitochondria per myocyte remaining at 24 h. Total ATP levels remained high at 16 h. The DeltaPsi loss was delayed by cyclosporin A, a mitochondrial permeability transition inhibitor. Mitochondrial swelling accompanied changes in DeltaPsi. Carnitine palmitoyltransferase I activity fell at 16 h; this decline was accompanied by ceramide increases that paralleled decreased complex III activity. We conclude that carnitine palmitoyltransferase I inhibition, ceramide accumulation, and complex III inhibition are downstream events in cardiac apoptosis mediated by palmitate and occur independent of events leading to caspase 3-like activation.

Asunto(s)

Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Palmitatos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Ciclosporina/farmacología , Grupo Citocromo c/metabolismo , Citosol/metabolismo , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/ultraestructura , Dilatación Mitocondrial/efectos de los fármacos , Miocardio/citología , Palmitatos/farmacología , Fosfatidilserinas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos

RESUMEN

During ischemia and reperfusion, increased palmitate oxidation is associated with diminished function of the myocardium. Palmitate, but not oleate, has been implicated in the induction of apoptosis in isolated neonatal rat ventricular myocytes. We report that extended incubation (20 h) of cultured neonatal rat cardiomyocytes, in the presence of palmitate, causes a decrease in the ability of these cells to oxidize fatty acids, an increase in cellular malonyl-CoA and a decrease in the activity of 5' AMP-activated protein kinase (AMPK) compared to myocytes incubated in the presence of oleate. While palmitate decreases the oxidative metabolism of fatty acids, it increases the formation of intracellular triglyceride and ceramide. Increased ceramide formation is associated with an increase in apoptosis in many cell systems and we also observe an increase in caspase-3 like activity and DNA-laddering in these cells. At the onset of cardiac failure, a switch in myocardial substrate utilization from fatty acids to glucose occurs. Our data suggest that decreased palmitate oxidation in cardiac myocytes in culture may signal the initiation of programmed cell death and ceramide elevation previously documented in ischemic, reperfused hearts.

Asunto(s)

Ácidos Grasos/metabolismo , Miocardio/metabolismo , Palmitatos/metabolismo , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Ceramidas/metabolismo , Malonil Coenzima A/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Miocardio/citología , Palmitatos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Triglicéridos/metabolismo

RESUMEN

Electrically stimulated pacing of cultured cardiomyocytes serves as an experimentally convenient and physiologically relevant in vitro model of cardiac hypertrophy. Electrical pacing triggers a signaling cascade that results in the activation of the muscle-specific Adss1 gene and the repression of the nonmuscle Adss2 isoform. Activation of the Adss1 gene involves the calcineurin-mediated dephosphorylation of NFAT3, allowing its translocation to the nucleus, where it can directly participate in Adss1 gene activation. Mutational studies show that an NFAT binding site located in the Adss1 5'-flanking region is essential for this activation. Electrical pacing also results in the increased synthesis of GATA4, another critical cardiac transcription factor required for Adss1 gene expression. MEF2C also produces transactivation of the Adss1 gene reporter in control and paced cardiac myocytes. Using the Adss1 gene as a model, these studies are the first to demonstrate that electrical pacing activates the calcineurin/NFAT3 and GATA4 pathways as a means of regulating cardiac gene expression.

Asunto(s)

Adenilosuccinato Sintasa/genética , Proteínas de Unión al ADN/metabolismo , Estimulación Eléctrica , Regulación Enzimológica de la Expresión Génica , Miocardio/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Northern Blotting , Calcineurina/metabolismo , Estimulación Cardíaca Artificial , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Factor de Transcripción GATA4 , Regulación de la Expresión Génica , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos/metabolismo , Factores de Transcripción NFATC , Isoformas de Proteínas/metabolismo , Ratas , Factores de Tiempo , Factores de Transcripción/genética , Activación Transcripcional , Transfección , Regulación hacia Arriba

RESUMEN

Carnitine (1, 3-hydroxy-4-trimethylammoniobutyrate) is important in mammalian tissue as a carrier of acyl groups. In order to explore the binding requirements of the carnitine acyltransferases for carnitine, we designed conformationally defined cyclohexyl carnitine analogues. These diastereomers contain the required gauche conformation between the trimethylammonium and hydroxy groups but vary the conformation between the hydroxy and carboxylic acid groups. Here we describe the synthesis and biological activity of the all-trans diastereomer (2), which was prepared by the ring opening of trans-methyl 2,3-epoxycylohexanecarboxylate with NaN3. Racemic 2 was a competitive inhibitor of neonatal rat cardiac myocyte CPT-1 (K(i) 0.5 mM for racemic 2; K(m) 0.2 mM for L-carnitine) and a noncompetitive inhibitor of neonatal rat cardiac myocyte CPT-2 (K(i) 0.67 mM). These results suggest that 2 represents the bound conformation of carnitine for CPT-1.

Asunto(s)

Carnitina O-Palmitoiltransferasa/metabolismo , Ácidos Ciclohexanocarboxílicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Compuestos de Amonio Cuaternario/síntesis química , Animales , Células Cultivadas , Columbidae , Ácidos Ciclohexanocarboxílicos/química , Ácidos Ciclohexanocarboxílicos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Espectroscopía de Resonancia Magnética , Conformación Molecular , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Ratas

RESUMEN

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

Asunto(s)

Calcio/metabolismo , Citocinas/farmacología , Corazón/efectos de los fármacos , Óxido Nítrico/fisiología , Nucleótidos Cíclicos/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Inhibidores Enzimáticos/farmacología , Miocardio/citología , Miocardio/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , omega-N-Metilarginina/farmacología

RESUMEN

Activation of cytochrome c (cyt c) transcription in electrically stimulated neonatal rat cardiac myocytes is preceded by transient expression of the activating protein-1 family of transcription factors, c-Fos, c-Jun, and JunB, as well as nuclear respiratory factor-1 (NRF-1). Mutations in either the NRF-1 or in the two cyclic AMP response elements on the cyt c promoter significantly reduce cyt c promoter activation produced either by electrical stimulation (Xia, Y., Buja, L. M., Scarpulla, R. C., and McMillin, J. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11399-11404) or by transfection of c-jun into nonpaced cardiac myocytes. Electrical stimulation of cardiac myocytes activates the c-Jun N-terminal kinase (McDonough, P. M., Hanford, D. S., Sprenkle, A. B., Mellon, N. R., and Glembotski, C. C. (1997) J. Biol. Chem. 272, 24046-24053) so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun were cotransfected. Physical association of NRF-1 protein with the NRF-1 enhancer element and of c-Jun with the cyclic AMP response element binding sites on the cyt c promoter was demonstrated by gel shift competition assays and by antibody super shifts. This is the first demonstration that induction of NRF-1 and c-Jun by pacing of cardiac myocytes directly mediates cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.

Asunto(s)

Grupo Citocromo c/genética , Proteínas de Unión al ADN/metabolismo , Miocardio/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transactivadores/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Proteínas de Unión al ADN/genética , Estimulación Eléctrica , Miocardio/citología , Factor 1 Relacionado con NF-E2 , Factor Nuclear 1 de Respiración , Factores Nucleares de Respiración , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transactivadores/genética , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional

RESUMEN

Fatty acids are the preferred substrate of ischemic, reperfused myocardium and may account for the decreased cardiac efficiency during aerobic recovery. Neonatal cardiac myocytes in culture respond to hypoxia/serum- and glucose-free medium by a slow decline in ATP which reverses upon oxygenation. This model was employed to examine whether carnitine palmitoyltransferase I (CPT-I) modulates high rates of beta-oxidation following oxygen deprivation. After 5 h of hypoxia, ATP levels decline to 30% control values and CPT-I activity is significantly stimulated in hypoxic myocytes with no alteration in cellular carnitine content or in the release of the mitochondrial matrix marker, citrate synthase. This stimulation was attributed to an increase in the affinity of hypoxic CPT-I for carnitine, suggesting that the liver CPT-I isoform is more dominant following hypoxia. However, there was no alteration in hypoxic CPT-I inhibition by malonyl-CoA. DNP-etomoxiryl-CoA, a specific inhibitor of the liver CPT-I isoform, uncovered identical Michaelis kinetics of the muscle isoform in control and hypoxic myocytes with activation of the liver isoform. Northern blotting did not reveal any change in the relative abundance of mRNA for the liver vs. the muscle CPT-I isoforms. The tyrosine phosphatase inhibitor, pervanadate, reversed the hypoxia-induced activation of CPT-I and returned the affinity of cardiac CPT-I for carnitine to control. Reoxygenation was also associated with a return of CPT-I activity to control levels. The data demonstrate that CPT-I is activated upon ATP depletion. Lower enzyme activities are present in control and reoxygenated cells where ATP is abundant or when phosphatases are inhibited. This is the first suggestion that phosphorylation may modulate the activity of the liver CPT-I isoform in heart.

Asunto(s)

Carnitina O-Palmitoiltransferasa/metabolismo , Isoenzimas/metabolismo , Hígado/enzimología , Miocardio/enzimología , Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Activación Enzimática , Cinética , Malonil Coenzima A/metabolismo , Consumo de Oxígeno , Ratas

RESUMEN

Carnitine palmitoyltransferase I (CPT-I) is a key enzyme involved in the regulation of fatty acid oxidation. CPT-IA and CPT-IB are isoforms of carnitine palmitoyltransferase I, of which CPT-IA is expressed in liver, kidney, fibroblasts, and heart and CPT-IB is expressed in skeletal muscle, heart, brown and white adipocytes, and testes. Although the genomic DNA sequence of human CPT-IB is available, the transcription start site and upstream regulatory sequences are not known. For rat CPT-IB, only the cDNA sequence has been published. We have cloned the entire rat CPT-IB gene from a Lambda fix II rat kidney genomic library. The genomic structure contains 19 exons, with the transcription start site for CPT-IB located in a short first exon, which is a 13-bp extension to the previously published cDNA 5' sequence. The coding sequence is identical with the rat muscle cDNA. The rat CPT-IB gene contains 18 introns and 19 exons, the latter 18 exons showing 85% homology to the human CPT-IB cDNA. CPT-IB maps to rat chromosome 7 at band q34. A putative promoter region was identified to within 391 bp of the transcription start site. The muscle specificity of the 5' flanking region was verified by comparison of luciferase expression to that of beta-galactosidase in cardiac myocytes and in HepG2 cells.

Asunto(s)

Carnitina O-Palmitoiltransferasa/genética , Mapeo Cromosómico , Músculo Esquelético/enzimología , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Genes Reporteros , Humanos , Hibridación in Situ , Isoenzimas/genética , Datos de Secuencia Molecular , Miocardio/citología , Miocardio/enzimología , Reacción en Cadena de la Polimerasa , Ratas , Análisis de Secuencia de ADN , Transcripción Genética , Transfección

RESUMEN

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed sequentially by c-jun (0.5-3 hr), JunB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), and muscle-specific CPT-I (48-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, beta-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Asunto(s)

Corazón/fisiología , Mitocondrias Cardíacas/fisiología , Miocardio/citología , Transcripción Genética , Animales , Animales Recién Nacidos , Carnitina O-Palmitoiltransferasa/biosíntesis , Diferenciación Celular , División Celular , Células Cultivadas , Grupo Citocromo c/biosíntesis , Estimulación Eléctrica , Complejo IV de Transporte de Electrones/biosíntesis , Isoenzimas/biosíntesis , Cinética , Hígado/metabolismo , Músculos/metabolismo , Miocardio/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Ratas , Transfección

RESUMEN

This study investigates the hypothesis that inflammatory cytokines, interleukin (IL)-1alpha IL-1beta, and tumor necrosis factor (TNF), influence cardiac function by affecting calcium homeostasis and that this effect is mediated by the beta-adrenergic-adenylate cyclase system. After 4 days in culture, neonatal rat ventricular myocytes were treated with cytokines (10 ng/ml) for short (2 h) or longer (18 h) times. Myocyte calcium, contractility, and adenylate cyclase were measured under each condition. Anticipated stepwise increases in adenylate cyclase and intracellular calcium were found in controls (non-cytokine-treated) with 10(-7) M isoproterenol, 10(-7) M isoproterenol + 0.1 mM guanosine triphosphate, and 10(-9) M forskolin. Cells in the presence of cytokine for 2 h show increased basal calcium levels but no changes in adenylate cyclase activities, and isoproterenol fails to elevate adenylate cyclase levels or affect contractile shortening. After long-term treatment with IL-1beta or TNF, but not IL-1alpha, the significantly elevated levels of basal systolic calcium remain, and isoproterenol increases adenylate cyclase activity, unlike after short exposure. Forskolin maximally activates adenylate cyclase following both short- and long-term incubation, but the stepwise increase in activity is blunted following prolonged exposure. Thus short-term cytokine treatment blocks the adrenergic receptor-mediated increases in adenosine 3',5'-cyclic monophosphate, dissociating adenylate cyclase activation from cytokine-mediated increases in cell calcium, whereas longer treatment apparently produces direct affects on adenylate cyclase. Time-dependent differences in contractile response were found with IL-1alpha at 2 h and TNF at 18 h, implying that myofibrillar responsiveness to increased cytoplasmic calcium is dependent on both cytokine species and exposure time.

Asunto(s)

Adenilil Ciclasas/metabolismo , Calcio/metabolismo , Corazón/efectos de los fármacos , Interleucina-1/farmacología , Miocardio/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Células Cultivadas , Colforsina/farmacología , Guanosina Trifosfato/farmacología , Corazón/fisiología , Ventrículos Cardíacos , Isoproterenol/farmacología , Cinética , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Ratas , Factores de Tiempo

RESUMEN

Cytokines exert autocrine and paracrine effects on the heart, some of which may be mediated by inducible nitric oxide synthase (i-NOS) expression. We studied the effects of cytokine-mediated NO synthesis on cell injury in the presence of deoxyglucose (DOG) and cyanide (CV)(20 mM DOG and 2 mM CN) for up to 3 hours and during recovery (18 hours). The influence of heat shock protein-70 on the extent of myocyte damage was also assessed. IL-1 beta and gamma-IFN act synergistically to enhance NO synthesis by cardiac myocytes. When these cytokines are present, the rate of ATP depletion after DOG and CN is significantly greater than in their absence. When IL-1 beta and gamma-IFN are added with the NOS inhibitor, L-monomethyl-L-arginine (L-NMMA), or when a cytokine that does not produce NO (TNF-alpha) is present, the rate of ATP depletion is no different from the rate seen with DOG and CN alone. After recovery for 18 hours, myocytes that were exposed to IL-1 beta and gamma-IFN release more lactic dehydrogenase and have significantly lower levels of ATP. L-NMMA decreases lactic dehydrogenase release and maintains ATP at levels similar to metabolically inhibited cells in the absence of these cytokines. Consistent with the decreased recovery in ATP with cells incubated with DOG and CN plus IL-1 beta and gamma-IFN is a decrease in cytochrome oxidase activity. Decreases in cellular ATP correspond to increased levels of heat shock protein-70 measured in myocytes after 18 hours of recovery after metabolic inhibition in the presence of IL-1 beta and gamma-IFN. In contrast, prior induction of heat shock protein-70 reduces the rate of ATP depletion in myocytes treated with DOG and CN and maintains ATP at levels that are significantly higher than those seen in non-heat-shocked cells. Recovery of cells exposed to heat shock is also greater, as seen by decreased lactic dehydrogenase and citrate synthase release. The heat-shocked myocytes contain significantly more glycogen than the cells that were not heat shocked. The increased cellular glycogen is likely responsible for the greater lactate production and slower rates of ATP depletion in the heat-shocked, metabolically inhibited cells. Cell survival under conditions of metabolic inhibition is closely related to cellular ATP preservation.

Asunto(s)

Adenosina Trifosfato/metabolismo , Citocinas/toxicidad , Metabolismo Energético/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Corazón/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Óxido Nítrico Sintasa/biosíntesis , omega-N-Metilarginina/farmacología , Animales , Animales Recién Nacidos , Citrato (si)-Sintasa/metabolismo , Cianuros/farmacología , Desoxiglucosa/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Glucógeno/metabolismo , Interferón gamma/farmacología , L-Lactato Deshidrogenasa/análisis , Óxido Nítrico/biosíntesis , Ratas , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

The mitochondrial carnitine palmitoyltransferase (CPT) system is composed of two proteins, CPT-I and -II, which, together with carnitine acylcarnitine translocase, are involved in the transport of fatty acids into the mitochondrial matrix for beta-oxidation. In the liver, CPT-I and its inhibition by malonyl-CoA are sensitive to hormonal (10(-9) M) levels of insulin; however, a similar effect of insulin on heart CPT is controversial. In cultured neonatal rat cardiac myocytes, tissue culture concentrations (1.7 microM) of insulin increase CPT and cytochrome oxidase activities as well as mitochondrial protein synthesis, suggesting that a growth mechanism may be involved. Because insulin at high concentrations may interact with the insulin-like growth factor (IGF-I) receptor, the consequences of insulin's action on heart cells in culture may be mediated through the IGF pathway. Consistent with an IGF-mediated pathway for the effect of insulin, incorporation of radioactivity into immunoprecipitated CPT-II from insulin-treated cardiac myocytes is dramatically increased over control cells. The amount of immunoreactive CPT-I is also increased in insulin-treated cells. Moreover, an IGF-I analogue that inhibits the autophosphorylation of the IGF-I receptor blunts the insulin-mediated increase in CPT-I and -II activities by > 70%. At low physiologically relevant concentrations (10 ng/ml), IGF-I significantly increases the activities of both CPT-I and -II, and the IGF-I analogue eliminates the IGF-I response. This is the first study to suggest involvement of the IGF-I pathway in the regulation of mitochondrial CPT synthesis and activities in the heart.

Asunto(s)

Carnitina O-Palmitoiltransferasa/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Miocardio/enzimología , Animales , Animales Recién Nacidos , Células Cultivadas , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Factor I del Crecimiento Similar a la Insulina/farmacología , Miocardio/citología , Ratas

RESUMEN

Electrical stimulation of neonatal rat cardiac myocytes in culture produces increases in myocyte size (hypertrophy) and organization of actin into myofibrillar arrays. The maturation of the cells is associated with enhanced contractile parameters and cellular calcium content. The numbers and intensity of cellular mitochondrial profiles increase, as measured by scanning laser confocal microscopy. Consistent with the hypertrophic response is increased cellular content of beta-myosin heavy chain and cytochrome oxidase subunit Va messages, as well as increases in cytochrome oxidase activity in the stimulated cardiac myocytes. Myocyte contractile capacity is associated with increased expression of the muscle carnitine palmitoyltransferase (CPT-I) isoform as measured by Northern analysis, immunoblotting, and altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated cells. The data suggest that a switch from the liver isoform of CPT-I, prominent in the neonatal rat heart, to the muscle CPT-I which predominates in adult rat heart, takes place in the neonatal cardiac myocytes over the same time period as the hypertrophic-mediated changes in myofibrillar assembly and increased contractile activity.

Asunto(s)

Carnitina O-Palmitoiltransferasa/análisis , Isoenzimas/análisis , Contracción Miocárdica , Miocardio/enzimología , Animales , Animales Recién Nacidos , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/genética , Células Cultivadas , Estimulación Eléctrica , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Ratas

RESUMEN

The presence of a 280,000 M(r) isoform of acetyl-CoA carboxylase (ACC-280) in the cardiac myocyte suggests that heart muscle is capable of malonyl-CoA synthesis. Cellular factors which regulate activity of ACC-280 are unknown. We have employed a neonatal rat cardiac myocyte culture (where the majority of ACC is present as ACC-280) to examine the effects of hypoxia and decreased cellular ATP on the activity of ACC in the cells. The myocyte culture has the following advantages over similar studies in the intact rat heart: the presence of a pure population of myocytes and the ability to measure cytosolic ACC free from contamination by mitochondrial carboxylases. ACC activity in cultured cardiac myocytes is completely dependent on the presence of citrate (A0.5=3.8 mM). Under control conditions, the cytosolic citrate concentration in situ is determined to be less than 1 mM. With 5 h of hypoxia, cytosolic ATP decreases from 9.85 +/- 0.23 to 2.83 +/- 0.25 mM and cytosolic AMP increases from undetectable levels to 40 +/- 0.4 microM. With hypoxia, a significant portion of the total ACC activity is now expressed in the absence of citrate and the amount of activity which is stimulated by 10 mM citrate is significantly less (1,268 +/- 0.106 nmol/4 x 10(5) cells) than is seen under control conditions (3.042 +/- 0.048). There are no significant changes in the total amount of cellular protein on the plates after 5 h of hypoxia. Consistent with net ACC activation in hypoxia, malonyl-CoA levels increase in the cells by 7 h of hypoxia. Decreased radioactive phosphate content of immunopurified ACC-280 after 5 h of hypoxia is consistent with net dephosphorylation of ACC-280 and increased citrate-independent activity.

Asunto(s)

Acetil-CoA Carboxilasa/metabolismo , Hipoxia de la Célula/fisiología , Citratos/metabolismo , Miocardio/metabolismo , Acetil-CoA Carboxilasa/química , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Ácido Cítrico , Citosol/metabolismo , Malonil Coenzima A/metabolismo , Peso Molecular , Fosforilación , Ratas

RESUMEN

Carnitine palmitoyltransferase-I (CPT-I) inhibitors improve postischemic myocardial function either by decreasing muscle long-chain acylcarnitines (LCAC) during ischemia or by increasing oxidation of alternate substrates such as glucose during reperfusion. These possibilities were evaluated using oxfenicine, a CPT-I inhibitor, and alternate substrates that bypass carnitine-dependent metabolism. Isolated rat hearts subjected to 20 min of ischemia followed by 40 min of reperfusion with 1.8 mM palmitate as exogenous substrate recovered little function during reperfusion. Hearts made ischemic and reperfused with palmitate and 2.4 mM hexanoate as exogenous substrates had significantly improved reperfusion function compared to palmitate-perfused hearts. Addition of 2 mM oxfenicine to palmitate-hexanoate-perfused hearts gave an additional small improvement in reperfusion function. At the end of ischemia, the LCAC content of hearts perfused with palmitate or hexanoate and palmitate was identical. Palmitate-, hexanoate, and oxfenicine-perfused hearts had significantly decreased LCAC content at the end of ischemia compared with hexanoate-palmitate-perfused hearts. Therefore, depressed reperfusion function in long-chain fatty acid-perfused hearts can be ameliorated by alternate substrates, including medium-chain fatty acids. LCAC accumulation during ischemia apparently plays only a minor role in the postischemic dysfunction of long-chain fatty acid-perfused hearts.

Asunto(s)

Carnitina/metabolismo , Metabolismo Energético/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Reperfusión Miocárdica , Miocardio/metabolismo , Ácidos Palmíticos/farmacología , Acilación , Adenosina Trifosfato/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Caproatos/farmacología , Carnitina/análogos & derivados , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Glucosa/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Isquemia Miocárdica/metabolismo , Ácido Palmítico , Fosfatos/metabolismo , Fosfocreatina/metabolismo , Ratas , Ratas Sprague-Dawley , Función Ventricular Izquierda/efectos de los fármacos

RESUMEN

Insulin increases the synthesis of mitochondrial proteins in the isolated perfused heart and total cell protein synthesis in neonatal cardiac myocytes. Since carnitine-dependent fatty acid oxidation is modulated by insulin in a variety of tissues, the effects of 1.7 microM insulin on the mitochondrial enzyme(s), carnitine palmitoyltransferase (malonyl-CoA-sensitive CPT-I and the matrix-facing CPT-II), were studied in neonatal rat cardiac myocytes cultured in the absence of serum. Following incubation in serum-free medium, there is a four-fold increase in the I50 of CPT-I for malonyl-CoA (3.8 microM) compared to cells cultured in serum-free medium to which insulin has been added (I50 = 0.8 microM). CPT-I activity in the insulin-supplemented, serum-free cultures is 57% higher (P < 0.002) than CPT-I activity in cells cultured in the absence of insulin; CPT-II activity is also significantly increased (P < 0.01) in the presence of insulin. Since CPT-II is an inner membrane protein, the CPT response to insulin may be coordinately regulated with other mitochondrial activities. Similar to CPT, cytochrome oxidase activity of cardiac myocytes in serum-free medium is increased 33% by insulin. Consistent with this finding, both CPT-II and cytochrome oxidase mRNA expression is elevated over control in the presence of insulin. CPT-II activity increases significantly only at very high insulin concentrations (1.7 microM), suggesting a role for insulin-like growth factor pathway. When myocytes are cultured in the presence of 1.7 microM insulin and then transferred to an insulin-free medium, subsequent addition of insulin does not stimulate uptake of deoxyglucose. These results suggest that the response of CPT to insulin is mediated by insulin-like growth factor activity and not by cellular glucose availability. The response of CPT to insulin does not appear to be mediated by the protein kinase C pathway since CPT-II activity is not reduced by the protein kinase C inhibitor, chelerythrine. Insulin significantly increases protein synthesis in the neonatal cardiac myocyte and in isolated mitochondria by increasing incorporation of labelled amino acid into total myocyte and/or mitochondrial protein. The degradation rate of radiolabelled protein in cardiac myocytes cultured in the presence of insulin is not different from that of insulin-deprived cells. The data suggest that insulin can affect the activity and expression of mitochondrial proteins, e.g., CPT, through the insulin-like growth factor-I pathway in neonatal cardiac myocytes.

Asunto(s)

Carnitina O-Palmitoiltransferasa/metabolismo , Insulina/farmacología , Miocardio/enzimología , Animales , Animales Recién Nacidos , Transporte Biológico/efectos de los fármacos , Carnitina O-Palmitoiltransferasa/biosíntesis , Células Cultivadas , Medio de Cultivo Libre de Suero , Sondas de ADN , Desoxiglucosa/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Isoenzimas/metabolismo , Cinética , Malonil Coenzima A/metabolismo , ARN Ribosómico 18S/biosíntesis , Ratas , Especificidad por Sustrato , Factores de Tiempo

RESUMEN

The selective inhibition of individual carnitine acyltransferases may be useful in the therapy of diabetes and heart disease. Aminocarnitine (3) is a weak competitive inhibitor (K(i) = 4.0 mM) for carnitine acetyltransferase (CAT), although the N-acetyl derivative 4 is about 165 times more potent (K(i) = 0.024 mM) than 3. Compound 3 is also a potent competitive inhibitor for carnitine palmitoyltransferases 1 and 2 (CPT-1 and CPT-2) (IC50 for CPT-2 = 805 nM). We synthesized 3-amino-5,5-dimethylhexanoic acid (7) and its N-acetyl derivative (8) as isosteric analogs of 3 and 4 that lack the quaternary ammonium positive charge. Like 3 and 4, compounds 7 and 8 were competitive inhibitors of CAT with significantly different potencies, but in this case, 8 (K(i) = 25 mM) was 10 times less potent than 7 (K(i) = 2.5 mM). R-(-)-7 and S-(+)-7 were stereoselective inhibitors of CAT (K(i) = 1.9 and 9.2 mM, respectively). Racemic 7 was a weak competitive inhibitor of CPT-2 (K(i) = 20 mM) and had no effect on CPT-1. These results are consistent with differences among the carnitine-binding sites on carnitine acyl-transferases that may be useful in selective inhibitor design. Furthermore, the data suggest that the quaternary ammonium positive charge of carnitine may be important for the proper orientation of carnitine and its analogs in the binding site.

Asunto(s)

Aminocaproatos/síntesis química , Carnitina Aciltransferasas/antagonistas & inhibidores , Aminocaproatos/farmacología , Animales , Unión Competitiva , Carnitina Aciltransferasas/metabolismo , Columbidae , Cinética , Estereoisomerismo