RESUMEN
We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 µM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Etoxzolamida/farmacología , Corazón/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Miocardio/metabolismo , Animales , Benzofenantridinas/farmacología , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Preparación de Corazón Aislado , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica , Proteína Quinasa C/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas Mitocondriales/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Preparación de Corazón Aislado , Masculino , Mitocondrias/efectos de los fármacos , Infarto del Miocardio/complicaciones , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cultivo Primario de Células , Inhibidores de Proteasoma/uso terapéutico , Ratas , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Hypothyroidism is considered a cardiac risk factor, but there is controversial evidence about its effects on coronary disease. The aim of this work was to evaluate the influence of hypothyroidism in rat hearts exposed to 2 degrees of stunning due to ischemia and reperfusion (I/R) as well as the underlying mechanisms. Hypothyroid (HypoT) rats were obtained by drinking 0.02% methimazole during 15 days. Isolated hearts were perfused and introduced in a flow calorimeter to measure contractile performance (P), total heat rate (Ht), and muscle economy (P/Ht). Hearts were exposed to 2 models of I/R, moderate and severe (respectively 20 or 30 minutes I/45 minutes R). Moreover, free cytosolic and mitochondrial calcium changes were measured by confocal fluorometry on cardiomyocytes. Comparison to euthyroid (EuT) hearts was done. Hypothyroidism was cardioprotective, but HypoT hearts were more sensitive than EuT hearts to the preischemic blockade of mitochondrial transporters mNCX and mKATP channels. Moreover, the postischemic recovery of P and P/Ht in HypoT hearts was strongly reduced by inhibition of the cellular pathways of PI3K/Akt and protein kinase C (PKC), and it was increased by nitric oxide synthase (NOS) inhibition. However, physiological concentrations of adrenaline reduced the cardioprotection of HypoT, but oral treatment with 20 mg/kg/day carvedilol prevented it. Results show that hypothyroidism reduces the mitochondrial Ca2+ overload during I/R by mKATP channel activation and Ca2+ extrusion through mNCX, while the PI3K/Akt and PKC pathways are involved in that cardioprotection. Contrarily, NOS activation and adrenaline blunt such cardioprotection, but carvedilol prevented the adrenergic dysfunction. These results would explain why hypothyroidism is a clinical risk factor in angor patients under adrenergic exacerbation but reduced the incidence of acute episodes of coronary syndrome in hospitalized patients. Results suggest that a treatment with carvedilol could be a potential therapeutic agent to prevent cardiac postischemic dysfunction in hypothyroid patients.
Asunto(s)
Metabolismo Energético , Hipotiroidismo/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Femenino , Frecuencia Cardíaca , Hipotiroidismo/patología , Hipotiroidismo/fisiopatología , Preparación de Corazón Aislado , Masculino , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Contracción Miocárdica , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Óxido Nítrico Sintasa/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Canales de Potasio/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Abstract Background: D-limonene (DL) is a monoterpene and is the major component in the essential oil of citrus fruit. It presents antihyperglycemic and vasodilatation activities. Objectives: This study evaluated the cardiovascular effects and potential antiarrhythmic of DL in rats. Methods: Hemodynamic and electrocardiographic (ECG) parameters were measured in male Wistar rats, which under anesthesia had been cannulated in the abdominal aorta and lower vena cava and had electrodes subcutaneously implanted. In the in vitro approach, the heart was removed and perfused using the Langendorff technique. The significance level adopted was 5% (p < 0.05). Results: DL, in doses of 10, 20, and 40 mg/kg (i.v), produced intense and persistent bradycardia associated with hypotension. Bradycardia with prolonged QTc was observed in the ECG in vivo recording. In the in vivo model of arrhythmia induced by Bay K8644, DL (10 mg/kg) decreased the arrhythmia score from 15.33 ± 3.52 to 4.0 ± 2.64 u.a (p < 0.05, n = 4). In isolated perfused hearts, DL (10-3 M) promoted significant reductions in heart rate (from 228.6 ± 8.5 ms to 196.0 ± 9.3 bpm; p < 0.05) and left ventricular development pressure (from 25.2 ± 3.4 to 5.9 ± 1.8 mmHg; n = 5, p < 0.05). Conclusions: DL produces bradycardia and antiarrhythmic activity in rat heart.
Resumo Fundamento: O D-limoneno (DL) é um monoterpeno e o principal componente do óleo essencial de frutas cítricas. Ele apresenta atividades anti-hiperglicêmicas e vasodilatadoras. Objetivos: Este estudo avaliou os efeitos cardiovasculares e antiarrítmicos potenciais do DL em ratos. Métodos: Os parâmetros hemodinâmicos e eletrocardiográficos (ECG) foram mensurados em ratos Wistar machos que, sob anestesia, tiveram a aorta abdominal e a veia cava inferior canuladas e receberam eletrodos implantados subcutaneamente. Na abordagem in vitro, o coração foi removido e perfundido utilizando a técnica de Langendorff. O nível de significância adotado foi de 5% (p < 0,05). Resultados: DL, nas doses de 10, 20 e 40 mg/kg (i.v), produziu bradicardia intensa e persistente associada à hipotensão. A bradicardia com QTc prolongado foi observada no registro in vivo do ECG. No modelo in vivo de arritmia induzida por Bay K8644, DL (10 mg / kg) houve diminuição do escore da arritmia de 15,33 ± 3,52 para 4,0 ± 2,64 u.a (p < 0,05, n = 4). Em corações perfundidos isolados, o DL (10-3 M) promoveu reduções significativas na frequência cardíaca (de 228,6 ± 8,5 ms para 196,0 ± 9,3 bpm; p < 0,05) e na pressão desenvolvida do ventrículo esquerdo (de 25,2 ± 3,4 para 5,9 ± 1,8 mmHg; n = 5, p < 0,05). Conclusões: O DL produz bradicardia e atividade antiarrítmica no coração de ratos.
Asunto(s)
Animales , Masculino , Arritmias Cardíacas/tratamiento farmacológico , Bradicardia/tratamiento farmacológico , Limoneno/uso terapéutico , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/inducido químicamente , Presión Sanguínea/efectos de los fármacos , Bradicardia/diagnóstico , Ratas Wistar , Presión Ventricular/efectos de los fármacos , Modelos Animales , Electrocardiografía , Preparación de Corazón Aislado , Limoneno/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipotensión , Antiarrítmicos/farmacologíaRESUMEN
BACKGROUND: D-limonene (DL) is a monoterpene and is the major component in the essential oil of citrus fruit. It presents antihyperglycemic and vasodilatation activities. OBJECTIVES: This study evaluated the cardiovascular effects and potential antiarrhythmic of DL in rats. METHODS: Hemodynamic and electrocardiographic (ECG) parameters were measured in male Wistar rats, which under anesthesia had been cannulated in the abdominal aorta and lower vena cava and had electrodes subcutaneously implanted. In the in vitro approach, the heart was removed and perfused using the Langendorff technique. The significance level adopted was 5% (p < 0.05). RESULTS: DL, in doses of 10, 20, and 40 mg/kg (i.v), produced intense and persistent bradycardia associated with hypotension. Bradycardia with prolonged QTc was observed in the ECG in vivo recording. In the in vivo model of arrhythmia induced by Bay K8644, DL (10 mg/kg) decreased the arrhythmia score from 15.33 ± 3.52 to 4.0 ± 2.64 u.a (p < 0.05, n = 4). In isolated perfused hearts, DL (10-3 M) promoted significant reductions in heart rate (from 228.6 ± 8.5 ms to 196.0 ± 9.3 bpm; p < 0.05) and left ventricular development pressure (from 25.2 ± 3.4 to 5.9 ± 1.8 mmHg; n = 5, p < 0.05). CONCLUSIONS: DL produces bradycardia and antiarrhythmic activity in rat heart.
Asunto(s)
Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Bradicardia/tratamiento farmacológico , Limoneno/uso terapéutico , Animales , Antiarrítmicos/farmacología , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/diagnóstico , Presión Sanguínea/efectos de los fármacos , Bradicardia/diagnóstico , Electrocardiografía , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipotensión , Preparación de Corazón Aislado , Limoneno/farmacología , Masculino , Modelos Animales , Ratas Wistar , Presión Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Information on the role of intermittent fasting (IF) on pathologic cardiac remodeling is scarce. We compared the effects of IF before and after myocardial infarction (MI) on rat cardiac remodeling and survival. METHODS: Wistar rats were intermittently fasted (food available every other day) or fed ad libitum for 12 weeks and then divided into three groups: AL - fed ad libitum; AL/IF - fed AL before MI and IF after MI; and IF - fed IF before and after MI. Echocardiogram was performed before MI and 2 and 12 weeks after surgery. Isolated hearts were evaluated in Langendorff preparations. RESULTS: Before surgery, body weight (BW) was lower in IF than AL. Final BW was lower in AL/IF and IF than AL. Perioperative mortality did not change between AL (31.3%) and IF (27.3%). Total mortality was lower in IF than AL. Before surgery, echocardiographic parameters did not differ between groups. Two weeks after surgery, MI size did not differ between groups. Twelve weeks after MI, left ventricular (LV) diastolic posterior wall thickness was lower in AL/IF and IF than AL. The percentage of variation of echocardiographic parameters between twelve and two weeks showed that MI size decreased in all groups and the reduction was higher in IF than AL/IF. In Langendorff preparations, LV volume at zero end-diastolic pressure (V0; AL: 0.41 ± 0.05; AL/IF: 0.34 ± 0.06; IF: 0.28 ± 0.05 mL) and at 25 mmHg end-diastolic pressure (V25; AL: 0.61 ± 0.05; AL/IF: 0.54 ± 0.07; IF: 0.44 ± 0.06 mL) was lower in AL/IF and IF than AL and V25 was lower in IF than AL/IF. V0/BW ratio was lower in IF than AL and LV weight/V0 ratio was higher in IF than AL. Myocyte diameter was lower in AL/IF and IF than AL (AL: 17.3 ± 1.70; AL/IF: 15.1 ± 2.21; IF: 13.4 ± 1.49 µm). Myocardial hydroxyproline concentration and gene expression of ANP, Serca 2a, and α- and ß-myosin heavy chain did not differ between groups. CONCLUSION: Intermittent fasting initiated before or after MI reduces myocyte hypertrophy and LV dilation. Myocardial fibrosis and fetal gene expression are not modulated by feeding regimens. Benefit is more evident when intermittent fasting is initiated before rather than after MI.
Asunto(s)
Restricción Calórica , Ayuno , Infarto del Miocardio/dietoterapia , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Modelos Animales de Enfermedad , Fibrosis , Preparación de Corazón Aislado , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Ratas Wistar , Factores de Tiempo , Pérdida de PesoRESUMEN
Synchronization theory predicts that if an oscillator interacts with a rhythmical external force, then it should react to a rhythmical force by adjusting its frequency. Furthermore, noise is present in nature, and it affects the nervous and cardiovascular systems. In this paper, we analyze the heart as an oscillator, where noisy periodic electrical stimulation can be regarded as an external forcing. This study aimed to investigate, from an experimental point of view, whether noise can induce synchronization of higher order in the mechanical heart response. A Langendorff heart preparation was used to obtain two variables of the mechanical response, intensity of contractile force and heart rate. The experiments show frequency locking in the electrical stimulation-contractile response coupling with and without noise induced. The role of noise in the response of effector organs invites further investigation.
Asunto(s)
Frecuencia Cardíaca/fisiología , Corazón/fisiología , Contracción Miocárdica/fisiología , Ruido , Potenciales de Acción/fisiología , Animales , Simulación por Computador , Estimulación Eléctrica , Preparación de Corazón Aislado , Masculino , Ratones , Modelos CardiovascularesRESUMEN
The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110â¯min of perfusion; 2) Ischemic control (IC): 30â¯min of global ischemia and 60â¯min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10â¯min of R; 4) SBâ¯+â¯a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca2+ response (CaR), Ca2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Simportadores de Sodio-Bicarbonato/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anticuerpos/farmacología , Preparación de Corazón Aislado/métodos , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
AIMS: Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. METHODS AND RESULTS: We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. CONCLUSION: Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.
Asunto(s)
Arritmias Cardíacas/enzimología , Proteínas de Unión al Calcio/deficiencia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Mitocondrias Cardíacas/enzimología , Infarto del Miocardio/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocitos Cardíacos/enzimología , Potenciales de Acción , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Señalización del Calcio , Proteínas de Unión al Calcio/genética , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Frecuencia Cardíaca , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/patología , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/patología , Fosforilación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
BACKGROUND: Recent studies from our laboratory show the cardioprotective action of benzolamide (BZ, carbonic anhydrase inhibitor) against ischemia-reperfusion injury. However, the mechanisms involved have not been fully elucidated. OBJECTIVE: To examine the participation of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) in the effects of BZ in a model of regional ischemia. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40â¯min of coronary artery occlusion followed by 60â¯min of reperfusion (IC). Other hearts received BZ during the first 10â¯min of reperfusion in absence or presence of L-NAME, NOS inhibitor. The infarct size (IS) and the post-ischemic recovery of myocardial function were measured. Oxidative/nitrosative damage were assessed by reduced glutathione (GSH) content, thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine levels. The expression of phosphorylated forms of Akt, p38MAPK and eNOS, and the concentration of inducible nitric oxide synthase (iNOS) were also determined. RESULTS: BZ significantly decreased IS (6.2⯱â¯0.5% vs. 34⯱â¯4%), improved post-ischemic contractility, preserved GSH levels and diminished TBARS and 3-nitrotyrosine. In IC hearts, P-Akt, P-p38MAPK and P-eNOS decreased and iNOS increased. After BZ addition the levels of P-kinases and P-eNOS increased and iNOS decreased. Except for P-Akt, P-p38MAPK and iNOS, the effects of BZ were abolished by L-NAME. CONCLUSIONS: Our data demonstrate that the treatment with BZ at the onset of reperfusion was effective to reduce cell death, contractile dysfunction and oxidative/nitrosative damage produced by coronary artery occlusion. These BZ-mediated beneficial actions appear mediated by eNOS/NO-dependent pathways.
Asunto(s)
Benzolamida/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animales , Preparación de Corazón Aislado , Masculino , Ratas , Ratas WistarRESUMEN
PURPOSE: Protecting the heart from ischaemia-reperfusion (IR) injury is a major goal in patients presenting with an acute myocardial infarction. Pyroptosis is a novel form of cell death in which caspase 1 is activated and cleaves interleukin 1ß. VX-785 is a highly selective, prodrug caspase 1 inhibitor that is also clinically available. It has been shown to be protective against acute IR in vivo rat model, and therefore might be a promising possibility for future cardioprotective therapy. However, it is not known whether protection by VX-765 involves the reperfusion injury salvage kinase (RISK) pathway. We therefore investigated whether VX-765 protects the isolated, perfused rat heart via the PI3K/Akt pathway and whether protection was additive with ischaemic preconditioning (IPC). METHODS: Langendorff-perfused rat hearts were subject to ischaemia and reperfusion injury in the presence of 30 µM VX-765, with precedent IPC, or the combination of VX-765 and IPC. RESULTS: VX-765 reduced infarct size (28 vs 48% control; P < 0.05) to a similar extent as IPC (30%; P < 0.05). The PI3 kinase inhibitor, wortmannin, abolished the protective effect of VX-765. Importantly in the model used, we were unable to show additive protection with VX-765 + IPC. CONCLUSIONS: The caspase 1 inhibitor, VX-765, was able to reduce myocardial infarction in a model of IR injury. However, the addition of IPC did not demonstrate any further protection.
Asunto(s)
Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Dipéptidos/farmacología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/enzimología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , para-Aminobenzoatos/farmacología , Animales , Citoprotección , Modelos Animales de Enfermedad , Preparación de Corazón Aislado , Masculino , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Piroptosis/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
The response to ischemia/reperfusion and the effects of ischemic post-conditioning (IPC) are sex-dependent, but the mechanisms have not been clarified. Male (M) and female (F) rat hearts isolated and perfused using the Langendorff technique were subject to 30 min of global ischemia (GI) and 60 min reperfusion (R). In IPC hearts, three cycles of 30-sec GI/30-sec R were applied at the beginning of R. Infarct size and myocardial function were assessed. Superoxide production, antioxidant systems, and expressions of phosphorylated forms of serine/threonine kinase (Akt), glycogen synthase kinase 3ß (GSK-3ß), protein kinase C ε (PKCε), endothelial nitric oxide synthase (eNOS), and apoptosis were measured. In the basal state, superoxide production and apoptosis were lower, and antioxidant systems and phospho-kinase expressions were higher in F rather than in M hearts. After ischemia-reperfusion, infarct size was less in F hearts, and post-ischemic recovery of myocardial function was higher in F rather than in M hearts. Superoxide production, phospho-kinase activity, phospho-eNOS, and apoptosis increased in both sexes while antioxidants decreased in both sexes. After IPC, infarct size, superoxide production, and apoptosis decreased and phospho-eNOS increased in F and M hearts but phospho-kinase expressions and post-ischemic recovery of myocardial function improved only in M hearts. These results show that Akt/GSK-3ß/PKCε/eNOS-dependent pathways-mediated superoxide production and apoptosis appear as important factors involved in the observed gender differences.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Poscondicionamiento Isquémico/métodos , Isquemia Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Antioxidantes/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Modelos Animales de Enfermedad , Femenino , Preparación de Corazón Aislado , Masculino , Isquemia Miocárdica/enzimología , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Estrés Oxidativo , Fosforilación , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factores Sexuales , Transducción de Señal , Factores de Tiempo , Función Ventricular IzquierdaRESUMEN
Ischaemic preconditioning (IPC) provides myocardial resistance to ischaemia/reperfusion (I/R) injuries. The protection afforded by IPC is not limited to the target tissue but extends to remote tissues, suggesting a mechanism mediated by humoral factors. The aim of the present study was to identify the humoral factors that are responsible for the cardioprotection induced by the coronary effluent transferred from IPC to naïve hearts. Isolated rat hearts were submitted to IPC (three cycles of 5 min I/R) before 30-min global ischaemia and 60-min reperfusion. The coronary effluent (Efl_IPC) collected during IPC was fractionated by ultrafiltration in different molecular weight ranges (<3, 3-5, 5-10, 10-30, 30-50, and >50 kDa) and evaluated for cardioprotective effects by perfusion before I/R in naïve hearts. Only the <3, 5-10 and <10 kDa fractions of hydrophobic eluate reduced I/R injuries. The cardioprotective effect of the 5-10 fraction was blocked by KATP channel blockers and a PKC inhibitor. An Efl_IPC proteomic analysis revealed 14 cytoprotection-related proteins in 4-12 kDa peptides. HSP10 perfusion protected the heart against I/R injuries. These data provide insights into the mechanisms of cardioprotection in humoral factors released by IPC. Cardioprotection is afforded by hydrophobic peptides in the 4-12 kDa size range, which activate pathways that are dependent on PKC and KATP. Fourteen 4-12 kDa peptides were identified, suggesting a potential therapeutic role for these molecules in ischaemic diseases. One of these, HSP10, identified by mass spectrometry, reduced I/R injuries and may be a potential candidate as a therapeutic target.
Asunto(s)
Chaperonina 10/metabolismo , Precondicionamiento Isquémico Miocárdico , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Función Ventricular Izquierda , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Preparación de Corazón Aislado , Canales KATP/metabolismo , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Proteína Quinasa C/metabolismo , Proteómica/métodos , Ratas Wistar , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray , Volumen Sistólico , Espectrometría de Masas en Tándem , Factores de Tiempo , Presión VentricularAsunto(s)
Agonistas Adrenérgicos beta/farmacología , Insuficiencia Cardíaca/inducido químicamente , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Lactancia/fisiología , Leptina/administración & dosificación , Animales , Femenino , Preparación de Corazón Aislado , Embarazo , Ratas , Ratas Wistar , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
NEW FINDINGS: What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca(2+) handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3'-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia-reperfusion. Rats were injected s.c. daily with 20 µg kg(-1) triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca(2+) from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na(+) ] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium-calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by ciclosporin, suggesting a dependance on activation of the mitochondrial permeability transition pore. Blockade of the mitochondrial Ca(2+) uniporter with Ru360 reduced P/Ht and PICR to â¼10% in both HpT and EuT hearts. Blockade of mitochondrial K(+) channels with 5-hydroxydecanoate increased LVEDP and reduced PICR and P/Ht in HpT hearts, while it only increased LVEDP in EuT hearts. The results suggest that hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channels. Both HpT and EuT hearts showed a similar and critical role of the uniporter. The HpT hearts have a slow sarcoplasmic reticulum Ca(2+) loss and low mitochondrial Ca(2+) uptake.
Asunto(s)
Metabolismo Energético , Hiperparatiroidismo/metabolismo , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Aturdimiento Miocárdico/prevención & control , Miocitos Cardíacos/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Señalización del Calcio , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Femenino , Hiperparatiroidismo/inducido químicamente , Hiperparatiroidismo/fisiopatología , Preparación de Corazón Aislado , Masculino , Moduladores del Transporte de Membrana/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Contracción Miocárdica , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Aturdimiento Miocárdico/metabolismo , Aturdimiento Miocárdico/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas Wistar , Recuperación de la Función , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , Factores de Tiempo , Triyodotironina , Función Ventricular Izquierda , Presión VentricularRESUMEN
Estudios clínicos y epidemiológicos sugieren que el danazol ha sido considerado como un factor de riesgo para desarrollar hipertensión. Para proporcionar información adicional acerca de este fenómeno, en este trabajo fue caracterizado el efecto inducido por el danazol y el hemisuccinato de danazol sobre la presión de perfusión y la resistencia vascular en corazón aislado de rata a flujo constante (modelo de Langendorff). Los resultados, mostraron que; 1) el hemisuccinato de danazol [10-9 M] incrementa la presión de perfusión en comparación con el danazol [10-9 M]; 2) los efectos del derivado de danazol [10-9 M - 10-4 M] sobre la presión de perfusión fueron inhibidos por flutamida [10-6 M]; 3) la nifedipina [10-6 M], bloqueó los efectos ejercidos por el hemisuccinato de danazol [10-9 M -10-4 M] sobre la presión de perfusión y 4) el efecto del derivado de danazol [10-9 M - 10-4 M] sobre la presión de perfusión en presencia del montelukast [10-6 M] fue inhibido significativamente (p=0,008). En conclusión, los efectos inducidos por el danazol y hemisuccinato de danazol sobre la presión de perfusión y la resistencia vascular podrían depender de su estructura química. Este fenómeno podría involucrar la interacción del receptor de andrógenos e indirectamente la activación de la síntesis de leucotrienos D4 (LTD4) y consecuentemente inducir variaciones en la presión de perfusión.
Epidemiological and clinical studies suggest that danazol has been considered a risk factor for hypertension development. In order to provide additional information about this phenomenon, the effect induced by both danazol and hemisuccinate of danazol on perfusion pressure and vascular resistance was characterized in isolated rat heart at constant flow (Langendorff model) and it was evaluated in this work.The results showed that; 1) hemisuccinate of danazol [10-9 M] increases perfusion pressure and vascular resistance in comparison with danazol [10-9 M]; 2) the effects of danazol-derivative [10-9 M - 10-4 M] on perfusion pressure were inhibited by flutamide [10-6 M]; 3) nifedipine [10-6 M] blockaded the effects exerted by hemisuccinate of danazol [10-9 M -10-4 M] on perfusion pressure; and 4) the effect of danazol-derivative [10-9 M - 10-4 M] on perfusion pressure in presence of montelukast [10-6 M] was significantly inhibited (p=0.008). In conclusion, the effects induced by both danazol and hemisuccinate of danazol on perfusion pressure and vascular resistance could depend on their chemical structure. This phenomenon could involve the interaction of androgene steroid-receptor and indirect activation of leukotriene D4 (LTD4) synthesis and consequently, induce variations in the perfusion pressure.