RESUMEN
The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca(i)(2+)) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca(i)(2+) cycling, myocardial Ca(i)(2+)-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with beta-agonist isoproterenol and the sarcoplasmic reticular Ca(2+)-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca(2+) handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. beta-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca(i)(2+)-level and a decrease in Ca(2+)-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca(i)(2+) handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.
Asunto(s)
Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , Corazón/fisiopatología , Hemodinámica , Proteínas Musculares/fisiología , Factores de Edad , Animales , Western Blotting , Índice de Masa Corporal , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Insuficiencia Cardíaca/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Indoles/farmacología , Isoproterenol/farmacología , Proteínas con Dominio LIM , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tasa de SupervivenciaRESUMEN
The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP-ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca(2+)(i)) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca(2+)(i) concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca(2+)(i) level (control, 111-/+20 vs DM, 221-/+35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na(+)/Ca(2+) exchanger. In response to beta-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca(2+)(i) handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling.
Asunto(s)
Calcio/metabolismo , Cardiomiopatías/metabolismo , Diabetes Mellitus Experimental/complicaciones , Angiopatías Diabéticas/metabolismo , Miocardio/metabolismo , Animales , Calcio/análisis , ATPasas Transportadoras de Calcio/análisis , ATPasas Transportadoras de Calcio/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/fisiopatología , Diabetes Mellitus Experimental/fisiopatología , Angiopatías Diabéticas/etiología , Angiopatías Diabéticas/fisiopatología , Hemodinámica , Masculino , Contracción Miocárdica , Miocardio/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/análisis , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
Reactive oxygen and nitrogen species are overproduced in the cardiovascular system in response to the exposure to doxorubicin, a cardiotoxic anticancer compound. Oxidant-induced cell injury involves the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and pharmacological inhibition of PARP has recently been shown to improve myocardial contractility in doxorubicin-induced heart failure models. The current investigation, by utilizing an isolated perfused heart system capable of beat-to-beat intracellular calcium recording, addressed the following questions: (1) is intracellular calcium handling altered in hearts of rats after 6-week doxorubicin treatment, under baseline conditions, and in response to oxidative stress induced by hydrogen peroxide exposure in vitro; and (2) does pharmacological inhibition of PARP with the phenanthridinone-based PARP inhibitor PJ34 affect the changes in myocardial mechanical performance and calcium handling in doxorubicin-treated hearts under normal conditions and in response to oxidative stress. The results showed a marked elevation in intracellular calcium in the doxorubicin-treated hearts which was normalized by pharmacological inhibition of PARP. PARP inhibition also prevented the myocardial contractile disturbances and calcium overload that developed in response to hydrogen peroxide in the doxorubicin-treated hearts. We conclude that PARP activation contributes to the development of the disturbances in cellular calcium handling that develop in the myocardium in response to prolonged doxorubicin exposure.
Asunto(s)
Calcio/metabolismo , Doxorrubicina/toxicidad , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Insuficiencia Cardíaca/inducido químicamente , Masculino , Estrés Oxidativo , Ratas , Ratas Sprague-DawleyRESUMEN
Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca2+ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 degrees C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca2+i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45+/-11 vs. 64+/-22 mmHg; developed pressure: 72+/-12 vs. 41+/-20 mmHg; maximum rate of pressure increase (+dP/dtmax): 1,513+/-305 vs. 938+/-500 mmHg/s; maximum rate of pressure decrease (-dP/dtmax): -1,354+/-304 vs. -806+/-403 mmHg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca2+] ([Ca2+]i) after reinstallation of flow. The dynamic parameters of the Ca2+i transients, i.e. the maximum rate of increase/decrease (+/-dCa2+i/dtmax) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca2+i load, and most likely, preserved Ca2+ sensitivity of the myocardial contractile machinery.
Asunto(s)
Calcio/metabolismo , Corazón/fisiología , Respuesta al Choque Térmico/fisiología , Precondicionamiento Isquémico , Daño por Reperfusión/prevención & control , Animales , Diástole/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Masculino , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Presión Ventricular/fisiologíaRESUMEN
Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that beta-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the beta-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca(2+)](i) levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca(2+)](i) transient the kinetic parameters of the sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca(2+)](i) transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca(2+)](i) amplitude and rate of rise and decline of [Ca(2+)](i) as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under beta-adrenoceptor stimulation. Upon beta-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca(2+)](i) amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+)-release channel in response to beta-adrenoceptor challenge. Alterations in Ca(2+)(i) handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.
Asunto(s)
Calcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
The main aim of this study was to assess the kinetics of intracellular free calcium (Ca(2+)i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca(2+)i transient. Changes in mechanical performance and degradation of membrane phospholipids--estimated by tissue arachidonic acid content--were correlated with Ca(2+)i levels of the heart. The fluorescence ratio technique was applied to estimate Ca(2+)i. The disappearance of mechanical activity of the heart preceded that of the Ca(2+)i transient in the first 2 min of ischemia. The slope of upstroke of the Ca(2+)i transient, reflecting Ca2+ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca2+ sequestration, expressed a significant prolongation (105 +/- 17 vs. 149 +/- 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca(2+)i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca(2+)i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca(2+)i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca(2+)i handling and sustained depression of mechanical performance.
Asunto(s)
Calcio/metabolismo , Miocardio/patología , Animales , Ácido Araquidónico/metabolismo , Diástole , Ventrículos Cardíacos/metabolismo , Iones , Isquemia , Masculino , Daño por Reperfusión Miocárdica , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión , Espectrometría de Fluorescencia , Factores de Tiempo , Presión VentricularRESUMEN
Animal experimentation is terminated by the euthanasia procedure in order to avoid pain and minimize suffering. Very little is known about the real time physiological changes taking place in the brain of animals during the euthanasia. Since there is no way to evaluate the suffering of animals under euthanasia, it is assumed that objective physiological changes taking place could serve as a good way to compare various types of euthanasia procedures. In the present study we compared the effect of euthanasia induced by i. v. injection of concentrated KCL to that of Taxan T-61 (a standard mixture used by veterinarians). The responses of the cat brain were evaluated by monitoring the hemodynamic (CBF), metabolic (NADH redox state), electrical (EcoG) and extracellular ion levels, as an indicator to the ionic homeostasis.
Asunto(s)
Amidas/farmacología , Encéfalo/efectos de los fármacos , Eutanasia , Dimensión del Dolor/métodos , Dolor/metabolismo , Cloruro de Potasio/farmacología , Compuestos de Amonio Cuaternario/farmacología , Tetracaína/farmacología , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Dióxido de Carbono/sangre , Gatos , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Combinación de Medicamentos , Electroencefalografía , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Iones/metabolismo , NAD/metabolismo , Dolor/inducido químicamente , Dolor/fisiopatología , Potasio/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatologíaRESUMEN
OBJECTIVE: It has been well established that oxytocin (OXT) increases intracellular free calcium ([Ca(2+)](i)) by targeting both intracellular and extracellular stores, but the mechanisms involved in the increase through activation with prostaglandin F(2alpha) (PGF(2alpha)) are still incompletely understood. This study was designed to elucidate the source(s) of increased [Ca(2+)](i) in response to PGF(2alpha) (10(-6) M) or OXT (10(-8) M) administration in the near-term rat myometrium. METHODS: The animals were divided into an in vitro group (n= 8), where the developed tension of uterine strips was assessed, and an in vivo group (n= 5), where a lobe of the uterus with intact innervation and circulation was loaded with the fluorescent indicator Indo-1 AM to assess [Ca(2+)](i). RESULTS: PGF(2alpha) and OXT induced a 30.1% and 35.9%, respectively, increase in developed tension in the potassium chloride-depolarized myometrial strips. Nifedipine reduced the PGF(2alpha) and OXT increased tension by 65.8% and 49.4%, respectively. In vivo, both PGF(2alpha) and OXT increased [Ca(2+)](i) in the potassium chloride-depolarized uterine muscle by 35.7% and 44.6%, respectively, increases similar to the rises in tension in vitro. Nifedipine reduced these effects of PGF(2alpha) and OXT by 45.3% and 39.6%. CONCLUSION: These findings indicate that in near-term myometrium the source of increased [Ca(2+)](i) after administration of PGF(2alpha), similar to OXT, is both extracellular and intracellular.