RESUMEN
Three types of estrogen receptors (ER) exist in the heart, Esr1, Esr2 and the G protein-coupled estrogen receptor 1, Gper1. However, their relative importance in mediating estrogen protective action is unknown. We found that, in the male mouse ventricle, Gper1 transcripts are three- and seventeen-fold more abundant than Esr1 and Esr2 mRNAs, respectively. Analysis of the three ER knockouts (Esr1-/-, Esr2-/- and Gper1-/-) showed that only the Gper1-/- hearts lost their ability to be protected by 40 nM estrogen as measured by heart function, infarct size and mitochondrial Ca2+ overload, an index of mitochondrial permeability transition pore (mPTP) activity. Analysis of Akt, ERK1/2 and GSK-3ß salvage kinases uncovered Akt and ERK1/2 transient activation by estrogen whose phosphorylation increased during the first 5 min of non-ischemic perfusion. All these increase in phosphorylation effects were abrogated in Gper1-/-. Inhibition of MEK1/2/ERK1/2 (1 µM U0126) and PI-3K/Akt (10 µM LY294002) signaling showed that the MEK1/2/ERK1/2 pathway via GSK-3ß exclusively was responsible for cardioprotection as an addition of U0126 prevented estrogen-induced GSK-3ß increased phosphorylation, resistance to mitochondrial Ca2+-overload, functional recovery and protection against infarction. Further, inhibiting PKC translocation (1 µM chelerythrin-chloride) abolished estrogen-induced cardioprotection. These data indicate that estrogen-Gper1 acute coupling plays a key role in cardioprotection against ischemia/reperfusion injury in male mouse via a cascade involving PKC translocation, ERK1/2/GSK-3ß phosphorylation leading to the inhibition of the mPTP opening.
Asunto(s)
Cardiotónicos/uso terapéutico , Estrógenos/uso terapéutico , Sistema de Señalización de MAP Quinasas , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/enzimología , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Calcio/metabolismo , Cardiotónicos/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasa 3 beta , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Biológicos , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/genética , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
There is discrepancy regarding the duration of reperfusion required using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining to assess myocardial infarction in an isolated, perfused heart model. Several investigators prefer long-term reperfusion (120 minutes) to determine myocardial injury, while others have used a shorter duration (30-40 minutes). We investigated whether oxygen surrounding the myocardium during ischemia plays a critical role in the installation of myocardial infarction during reperfusion. Mice hearts were perfused with a Langendorff apparatus using Krebs Henseleit (KH) buffer oxygenated with 95% O2 plus 5% CO2 at 37°C. Hearts were either immersed in KH or suspended in air during 18 minutes of global ischemia in a normothermic, water-jacketed chamber. Hearts then were reperfused for 40, 60, or 90 minutes. We found that hearts immersed in KH had decreased recovery of function and increased myocardial infarct size, reaching a steady-state level after 40 minutes of reperfusion. In contrast, hearts suspended in air approached steady-state after 90 minutes of reperfusion. Thus, mitochondrial reactive oxygen species (ROS) production was much lower in air-maintained hearts than in KH-immersed hearts. To investigate whether an increase in oxygen surrounding the myocardium during ischemia might cause further damage, we bubbled the KH solution with nitrogen (KH+N2) rather than oxygen (KH+O2). With this alteration, recovery of cardiac function was improved and myocardial infarct size and mitochondrial ROS production were reduced compared with hearts immersed in KH+O2. In conclusion, short-term (40 minutes) reperfusion is sufficient to reach steady-state myocardial infarct size when hearts are immersed in physiologic solution during ischemia; however, a longer duration of reperfusion (90 minutes) is required if hearts are suspended in air. Thus, oxygen surrounding the heart during ischemia determines the extent of myocardium injury during reperfusion.
RESUMEN
The heart is an organ that requires an important energy input to ensure its contractile function. Myocardial ischemia happens when there is a deficiency of blood flow that is responsible for the passage from an aerobic to anaerobic metabolism. Myocardial ischemia results from an imbalance between inputs and the needs of nutrient and oxygen to the myocardium. The restoration of myocardial perfusion called reperfusion is a way to save the ischemic myocardium. However, although reperfusion is beneficial for the survival of the ischemic myocardium, it also induces a deleterious effect in addition to that of ischemic stress. Three decade ago, while several studies, strived to elucidate the protective effect of preconditioning, a phenomenon performed before ischemia and having a powerful protective effects against ischemia/reperfusion injury, very few have believed in the possibility of protecting the myocardium after ischemia (during reperfusion). Actually, both ischemic and pharmacological postconditioning as well as controlled reperfusion methods to protect the ischemic heart have proved effective in the reduction of damage related to ischemia/reperfusion. The possibility of protecting the myocardium during reperfusion opens a new area in the research against damage caused by ischemia/reperfusion because these methods are easily transferable in a clinic setting.
RESUMEN
The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~30nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; specifically, cytochrome c oxidase subunit 2 is distributed in clusters of ~28nm; whereas the voltage dependent anion channel 1 displays three size distributions of ~33, ~55 and ~83nm.
Asunto(s)
Microscopía Fluorescente/métodos , Mitocondrias/química , Proteínas Mitocondriales/análisis , Miocardio/química , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Intralipid (Sigma, St. Louis, MO), a brand name for the first safe fat emulsion for human use, has been shown to be cardioprotective. However, the mechanism of this protection is not known. The authors investigated the molecular mechanism(s) of Intralipid-induced cardioprotection against ischemia/reperfusion injury, particularly the role of glycogen synthase kinase-3ß (GSK-3ß) and mitochondrial permeability transition pore in this protective action. METHODS: In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (1% in ex vivo and one bolus of 20% in in vivo) or vehicle. The hemodynamic function, infarct size, threshold for the opening of mitochondrial permeability transition pore, and phosphorylation levels of protein kinase B (Akt)/extracellular signal regulating kinase (ERK)/GSK-3ß were measured. RESULTS: Administration of Intralipid at the onset of reperfusion resulted in approximately 70% reduction in infarct size in the in vivo rat model. Intralipid also significantly improved functional recovery of isolated Langendorff-perfused mouse hearts as the rate pressure product was increased from 2,999 ± 863 mmHg*beats/min in the control group to 13,676 ± 611 mmHg*beats/min (mean±SEM) and the infarct size was markedly smaller (18.3 ± 2.4% vs. 54.8 ± 2.9% in the control group, P < 0.01). The Intralipid-induced cardioprotection was fully abolished by LY294002, a specific inhibitor of PI3K, but only partially by PD98059, a specific ERK inhibitor. Intralipid also increased the phosphorylation levels of Akt/ERK1/glycogen synthase kinase-3ß by eightfold, threefold, and ninefold, respectively. The opening of mitochondrial permeability transition pore was inhibited by Intralipid because calcium retention capacity was higher in the Intralipid group (274.3 ± 8.4 nM/mg vs. 168.6 ± 9.6 nM/mg in the control group). CONCLUSIONS: Postischemic treatment with Intralipid inhibits the opening of mitochondiral permeability transition pore and protects the heart through glycogen synthase kinase-3ß via PI3K/Akt/ERK pathways.
Asunto(s)
Glucógeno Sintasa Quinasa 3/fisiología , Poscondicionamiento Isquémico , Daño por Reperfusión Miocárdica/prevención & control , Animales , Calcio/metabolismo , Cromonas/farmacología , Emulsiones/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonoides/farmacología , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolípidos/uso terapéutico , Fosforilación , Ratas , Ratas Sprague-Dawley , Aceite de Soja/uso terapéuticoRESUMEN
Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardioprotection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% O(2) and 5% CO(2)) Krebs Henseleit buffer (control), with G1 (1 microM), and G1 (1 microM) together with extracellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 microM). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37 degrees C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the Ca(2+) load required to induce mitochondria permeability transition pore (mPTP) opening. G1-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 +/- 264 vs. 2,640 +/- 334 beats mmHg(-1) min(-1), P < 0.05). The infarct size decreased significantly in G1-treated hearts (21 +/- 2 vs. 46 +/- 3%, P < 0.001), and the Ca(2+) load required to induce mPTP opening increased (2.4 +/- 0.06 vs. 1.6 +/- 0.11 microM/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 +/- 46 beats mmHg(-1) min(-1), infarct size: 53 +/- 2%, and Ca(2+) retention capacity: 1.4 +/- 0.11 microM/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotective effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway.
Asunto(s)
Mitocondrias Cardíacas/fisiología , Miocardio/patología , Daño por Reperfusión/patología , Adenosina Trifosfato/fisiología , Animales , Western Blotting , Inhibidores Enzimáticos/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Flavonoides/uso terapéutico , Cardiopatías/prevención & control , Hemodinámica/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Estrés Oxidativo/fisiología , Permeabilidad , Sustancias Protectoras , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RATIONALE: Fast-transient outward K(+) (I(to,f)) and ultrarapid delayed rectifier K(+) currents (I(K,slow), also known as I(Kur)) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results. OBJECTIVE: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K(+) currents, taking into consideration the females' estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K(+) currents underlying ventricular repolarization. METHODS AND RESULTS: Peak total K(+) current (I(K,total)) densities (pA/pF, at +40 mV) were much higher in males (48.6+/-3.0) versus females at estrus (27.2+/-2.3) but not at diestrus-2 (39.1+/-3.4). Underlying this change, I(to,f) and I(K,slow) were lower in females at estrus versus males and diestrus-2 (I(K,slow): male 21.9+/-1.8, estrus 14.6+/-0.6, diestrus-2 20.3+/-1.4; I(to,f): male 26.8+/-1.9, estrus 14.9+/-1.6, diestrus-2 22.1+/-2.1). Lower I(K,slow) in estrus was attributable to only I(K,slow)(1) reduction, without changes in I(K,slow)(2). Estrogen treatment of ovariectomized mice decreased I(K,total) (46.4+/-3.0 to 28.4+/-1.6), I(to,f) (26.6+/-1.6 to 12.8+/-1.0) and I(K,slow) (22.2+/-1.6 to 17.2+/-1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying I(to,f) and I(K,slow), respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K(+) current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval. CONCLUSION: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.
Asunto(s)
Potenciales de Acción/fisiología , Estrógenos/farmacología , Ventrículos Cardíacos/metabolismo , Potasio/metabolismo , Caracteres Sexuales , Potenciales de Acción/efectos de los fármacos , Animales , Estrógenos/metabolismo , Ciclo Estral/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ventrículos Cardíacos/citología , Proteínas de Interacción con los Canales Kv/biosíntesis , Masculino , Ratones , Canales de Potasio de la Superfamilia Shaker/biosíntesisRESUMEN
BACKGROUND: The aim of this study was to evaluate the role of the mitochondrial permeability transition pore (MPTP) in the protection achieved by post-conditioning of hearts submitted to hypothermic cardioplegia and ischemia. METHODS: Isolated rat hearts (n = 30) underwent cold cardioplegia (4 degrees C, Celsior solution) and 8 hours of ischemia at 4 degrees C, followed by a 60-minute Langendorff reperfusion. Hearts were randomly assigned to one of two groups: post-conditioning (post-C, consisting of episodes of 30-second ischemia and 30-second reperfusion at onset of reperfusion) or control (no intervention). A sham group was added for which hearts were harvested and immediately reperfused without ischemia. Coronary flow, heart rate, dP/dt and rate-pressure product were measured. Infarct size was assessed by triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH), creatine phosphokinase (CPK) and troponin I release analysis. After reperfusion, heart mitochondria were isolated and calcium overload necessary to induce MPTP opening was measured. From the onset of reperfusion, all functional parameters were significantly improved in post-C vs control hearts. RESULTS: Infarct size, measured both by TTC staining and LDH, and CPK and troponin I leakage were lower in post-C hearts (p < 0.01). Mean calcium load needed to induce MPTP opening was higher in post-C mitochondria vs controls (p < 0.01). CONCLUSIONS: Post-conditioning protects the rat heart against cold ischemia-reperfusion injury. Our data suggest that this protection involves inhibition of MPTP opening.
Asunto(s)
Proteínas de Transporte de Membrana Mitocondrial/fisiología , Infarto del Miocardio/prevención & control , Acondicionamiento Pretrasplante/métodos , Animales , Calcio/fisiología , Frío , Modelos Animales de Enfermedad , Paro Cardíaco/prevención & control , Paro Cardíaco Inducido , Isquemia/prevención & control , Masculino , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Infarto del Miocardio/fisiopatología , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
OBJECTIVE: We investigated whether phosphatidylinositol 3-kinase (PI3K) might regulate mitochondrial permeability transition pore (mPTP) opening in hearts reperfused with either low pressure or postconditioning. METHODS: Male Wistar rat hearts (n=72) were perfused according to the Langendorff technique, exposed to 30 min of ischemia, and assigned to one of the following groups: (1) reperfusion with normal pressure (NP; 100 cm H2O), (2) reperfusion with low pressure (LP; 70 cm H2O), or reperfusion with postconditioning, i.e. 3 episodes of 30 s reperfusion followed by 30 s of ischemia (PostC). Hearts received either the PI3K inhibitors wortmannin or LY294002, or vehicle at the onset of the 60 min reperfusion. Postischemic functional recovery was assessed by rate-pressure product (RPP), and irreversible injury by lactate dehydrogenase (LDH), creatine kinase (CK) and troponin I (TnI) release. Mitochondria were isolated from the reperfused myocardium, and Ca2+-induced mPTP opening was measured using a potentiometric method. RESULTS: Functional recovery was significantly improved in LP and PostC hearts with RPP averaging 13,880+/-810 (LP) and 17,130+/-900 mm Hgxbeats/min (PostC) versus 6450+/-500 mm Hgxbeats/min in NP hearts (p<0.01). LDH release averaged 230+/-30 and 145+/-15 IU/h/g of myocardial tissue in LP and PostC versus 340+/-10 IU/h/g in NP (p<0.05). Wortmannin and LY294002 prevented both RPP improvement and decrease in LDH, CK, and TnI release in LP and PostC groups. The Ca2+ load required to induce mPTP opening averaged 58+/-3 and 52+/-1 nmol/mg mitochondrial proteins in LP and PostC groups, respectively, versus 35+/-4 nmol/mg in the NP group (p<0.01). Wortmannin and LY294002 prevented the beneficial effect in both the LP and PostC groups. CONCLUSION: These results suggest that PI3K regulates the opening of the mitochondrial permeability transition pore in rat hearts reperfused with low pressure or postconditioning.