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1.
Basic Res Cardiol ; 119(3): 435-451, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38499702

RESUMEN

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic-reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation-reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation-reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia-reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation-reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.


Asunto(s)
Infarto del Miocardio , Daño por Reperfusión Miocárdica , Miocardio , Mioglobina , Oxidación-Reducción , Mioglobina/metabolismo , Animales , Infarto del Miocardio/metabolismo , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/diagnóstico por imagen , Daño por Reperfusión Miocárdica/patología , Masculino , Microscopía Fluorescente , Modelos Animales de Enfermedad , Microscopía Confocal
2.
Int J Mol Sci ; 24(11)2023 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-37298493

RESUMEN

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.


Asunto(s)
Ciclosporina , Daño por Reperfusión , Animales , Ratones , Ciclosporina/farmacología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Hipoxia/metabolismo , Isquemia/metabolismo , Riñón/metabolismo , Mitocondrias/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/prevención & control , Daño por Reperfusión/metabolismo , Humanos
3.
J Hepatol ; 77(3): 710-722, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35358616

RESUMEN

BACKGROUND & AIMS: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment. METHODS: We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations. RESULTS: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index. CONCLUSIONS: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis. LAY SUMMARY: The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.


Asunto(s)
Diabetes Mellitus Tipo 2 , Hígado Graso , Resistencia a la Insulina , Hepatopatías , Animales , Calcio/metabolismo , Comunicación , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplásmico/metabolismo , Hígado Graso/etiología , Hígado Graso/metabolismo , Glucosa/metabolismo , Humanos , Hígado/metabolismo , Hepatopatías/metabolismo , Ratones , Mitocondrias/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Factores de Transcripción/metabolismo
4.
J Nutr ; 152(8): 1862-1871, 2022 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-35511216

RESUMEN

BACKGROUND: The role of hepatoportal glucose sensors is poorly understood in the context of insulin resistance. OBJECTIVES: We assessed the effects of glucose infusion in the portal vein on insulin tolerance in 2 rat models of insulin resistance, and the role of capsaicin sensitive nerves in this signal. METHODS: Male Wistar rats, 8 weeks old, weighing 250-275 g, were used. Insulin and glucose tolerance were assessed following a 4-hour infusion of either glucose or saline through catheterization in the portal vein in 3 paradigms. In experiment 1, for diet-induced insulin resistance, rats were fed either a control diet (energy content: proteins = 22.5%, carbohydrates = 64.1%, and lipids = 13.4%) or a high-fat diet (energy content: proteins = 15.3%, carbohydrates = 40.3%, and lipids =44.4%) for 4 months. In experiment 2, for centrally induced peripheral insulin resistance, catheters were inserted in the carotid artery to deliver either an emulsion of triglycerides [intralipid (IL)] or saline towards the brain for 24 hours. In experiment 3, for testing the role of capsaicin-sensitive nerves, experiment 2 was repeated following a periportal treatment with capsaicin or vehicle. RESULTS: In experiment 1, when compared to rats fed the control diet, rats fed the high-fat diet exhibited decreased insulin and glucose tolerance (P ≤ 0.05) that was restored with a glucose infusion in the portal vein (P ≤ 0.05). In experiment 2, infusion of a triglyceride emulsion towards the brain (IL rats) decreased insulin and glucose tolerance and increased hepatic endogenous production when compared to saline-infused rats (P ≤ 0.05). Glucose infusion in the portal vein in IL rats restored insulin and glucose tolerance, as well as hepatic glucose production, to controls levels (P ≤ 0.05). In experiment 3, portal infusion of glucose did not increase insulin tolerance in IL rats that received a periportal pretreatment with capsaicin. CONCLUSIONS: Stimulation of hepatoportal glucose sensors increases insulin tolerance in rat models of insulin resistance and requires the presence of capsaicin-sensitive nerves.


Asunto(s)
Resistencia a la Insulina , Insulina , Animales , Glucemia/metabolismo , Capsaicina/metabolismo , Capsaicina/farmacología , Emulsiones/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Insulina Regular Humana/farmacología , Hígado/metabolismo , Masculino , Fibras Nerviosas/metabolismo , Vena Porta/metabolismo , Ratas , Ratas Wistar , Triglicéridos/metabolismo
5.
Int J Mol Sci ; 23(16)2022 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-36012493

RESUMEN

Renal ischemia-reperfusion (IR) injury can lead to acute kidney injury, increasing the risk of developing chronic kidney disease. We hypothesized that mild therapeutic hypothermia (mTH), 34 °C, applied during ischemia could protect the function and structure of kidneys against IR injuries in mice. In vivo bilateral renal IR led to an increase in plasma urea and acute tubular necrosis at 24 h prevented by mTH. One month after unilateral IR, kidney atrophy and fibrosis were reduced by mTH. Evaluation of mitochondrial function showed that mTH protected against IR-mediated mitochondrial dysfunction at 24 h, by preserving CRC and OX-PHOS. mTH completely abrogated the IR increase of plasmatic IL-6 and IL-10 at 24 h. Acute tissue inflammation was decreased by mTH (IL-6 and IL1-ß) in as little as 2 h. Concomitantly, mTH increased TNF-α expression at 24 h. One month after IR, mTH increased TNF-α mRNA expression, and it decreased TGF-ß mRNA expression. We showed that mTH alleviates renal dysfunction and damage through a preservation of mitochondrial function and a modulated systemic and local inflammatory response at the acute phase (2-24 h). The protective effect of mTH is maintained in the long term (1 month), as it diminished renal atrophy and fibrosis, and mitigated chronic renal inflammation.


Asunto(s)
Lesión Renal Aguda , Hipotermia Inducida , Daño por Reperfusión , Lesión Renal Aguda/genética , Animales , Atrofia/patología , Fibrosis , Inflamación/metabolismo , Interleucina-6/metabolismo , Isquemia/metabolismo , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , ARN Mensajero/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Factor de Necrosis Tumoral alfa/metabolismo
6.
J Mol Cell Cardiol ; 131: 91-100, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31022374

RESUMEN

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.


Asunto(s)
Miocitos Cardíacos/metabolismo , Factor de Transcripción STAT3/metabolismo , Aminofilina/metabolismo , Animales , Atropina/metabolismo , Encéfalo/metabolismo , Línea Celular , Combinación de Medicamentos , Hígado/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Nitroglicerina/metabolismo , Fosforilación Oxidativa , Papaverina/metabolismo , Fenobarbital/metabolismo , Ratas , Transducción de Señal/fisiología
7.
Basic Res Cardiol ; 112(1): 4, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27995363

RESUMEN

Reperfusion injury is responsible for an important part of myocardial infarct establishment due notably to triggering cardiomyocytes death at the first minutes of reperfusion. AZP-531 is an optimized analog of unacylated ghrelin currently in clinical development in several metabolic diseases. We investigated a potential cardioprotective effect of AZP-531 in ischemia/reperfusion (IR) and the molecular underlying mechanism(s) involved in this protection. In vivo postconditioning with AZP-531 in C57BL6 mouse IR model decreased infarct size. Western blot analysis on areas at risk from the different mouse groups showed that AZP-531 activates Akt, ERK1-2 as well as S6 and 4EBP1, mTORC1 effectors. We also showed an inhibition of caspase 3 cleavage and Bax translocation to the mitochondria. AZP-531 also stimulated the expression of antioxidants and was capable of decreasing mitochondrial H2O2 production, contributing to the reduction of ROS accumulation. AZP-531 exhibits cardioprotective effect when administrated for postconditioning in C57BL6 mouse IR model. Treatment with AZP-531 rescued the myocardium from cell death at early reperfusion by stimulating protein synthesis, inhibiting Bax/caspase 3-induced apoptosis as well as ROS accumulation and oxidative stress-induced necrosis. AZP-531 may prove useful in the treatment of IR injury.


Asunto(s)
Ghrelina/farmacología , Poscondicionamiento Isquémico/métodos , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Animales , Western Blotting , Modelos Animales de Enfermedad , Ghrelina/análogos & derivados , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial
8.
Anesthesiology ; 123(6): 1374-84, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26460965

RESUMEN

BACKGROUND: The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition. METHODS: Adult mouse or isolated cardiomyocytes from wild-type or CypD knockout (CypD-KO) mice were subjected to ischemia or hypoxia followed by reperfusion or reoxygenation. Infarct size was assessed in vivo. Mitochondrial membrane potential and PTP opening were assessed using tetramethylrhodamine methyl ester perchlorate and calcein-cobalt fluorescence, respectively. Fluo-4 AM and rhod-2 AM staining allowed the measurement, by confocal microscopy, of Ca transient and Ca transfer from sarcoplasmic reticulum (SR) to mitochondria after caffeine stimulation. RESULTS: Both inhibition of CypD and isoflurane significantly reduced infarct size (-50 and -37%, respectively) and delayed PTP opening (+63% each). Their combination had no additive effect (n = 6/group). CypD-KO mice displayed endogenous protection against ischemia-reperfusion. Isoflurane depolarized the mitochondrial membrane (-28%, n = 5), decreased oxidative phosphorylation (-59%, n = 5), and blunted the caffeine-induced Ca transfer from SR to mitochondria (-22%, n = 7) in the cardiomyocytes of wild-type mice. Importantly, this transfer was spontaneously decreased in the cardiomyocytes of CypD-KO mice (-25%, n = 4 to 5). CONCLUSIONS: The results suggest that the partial inhibitory effect of isoflurane on respiratory complex I is insufficient to afford a synergy to CypD-induced protection. Isoflurane attenuates the Ca transfer from SR to mitochondria, which is also the prominent role of CypD, and finally prevents PTP opening.


Asunto(s)
Calcio/metabolismo , Ciclofilinas/metabolismo , Precondicionamiento Isquémico Miocárdico , Isoflurano/administración & dosificación , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Anestésicos por Inhalación/administración & dosificación , Animales , Peptidil-Prolil Isomerasa F , Complejo I de Transporte de Electrón/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo
9.
J Mol Cell Cardiol ; 68: 56-65, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24440456

RESUMEN

Roles of cardiac fibroblasts (CFs) in the regulation of myocardial structure and function have been emphasized in the last decade. Their implications in pathophysiological aspects of chronic heart diseases such as myocardial remodeling and fibrosis are now well established; however their contribution to the acute phase of ischemia-reperfusion injury still remains elusive. We hypothesized that CF may contribute to cardiomyocyte (CM) protection against ischemia-reperfusion injuries. Experiments performed on isolated neonatal rat CF and CM demonstrated that the presence of CF in co-cultures increases CM viability (58 ± 2% versus 30 ± 2% in control) against hypoxia-reoxygenation injury, in a paracrine manner. It was confirmed by a similar effect of hypoxic CF secretome alone on CM viability (51 ± 9% versus 31 ± 4% in untreated cells). These findings were corroborated by in vivo experiments in a mice model of myocardial infarction in which a 25% infarct size reduction was observed in CF secretome treated mice compared to control. Tissue inhibitor of metalloproteinases-1 (TIMPs-1) alone, abundantly detected in CF secretome, was able to decrease CM cell death (35%) and experiments with pharmacological inhibitors of PI3K/Akt and ERK1/2 pathways provided more evidence that this paracrine protection is partly mediated by these signaling pathways. In vivo experiments strengthened that TIMP-1 alone was able to decrease infarct size (37%) and were validated by depletion experiments demonstrating that CF secretome cardioprotection was abolished by TIMP-1 depletion. Our data demonstrated for the first time that CFs participate in cardioprotection during the acute phase of ischemia-reperfusion via a paracrine pathway involving TIMP-1.


Asunto(s)
Citocinas/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/fisiología , Miofibroblastos/fisiología , Animales , Supervivencia Celular , Medios de Cultivo Condicionados , Citocinas/fisiología , Ventrículos Cardíacos/patología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/metabolismo , Ratas , Ratas Wistar , Inhibidor Tisular de Metaloproteinasa-1/fisiología
10.
Arterioscler Thromb Vasc Biol ; 33(7): 1484-93, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23559630

RESUMEN

OBJECTIVE: Transintestinal cholesterol excretion (TICE) is an alternate pathway to hepatobiliary secretion. Our study aimed at identifying molecular mechanisms of TICE. APPROACH AND RESULTS: We studied TICE ex vivo in mouse and human intestinal explants, and in vivo after bile diversion and intestinal cannulation in mice. We provide the first evidence that both low-density lipoprotein (LDL) and high-density lipoprotein deliver cholesterol for TICE in human and mouse jejunal explants at the basolateral side. Proprotein convertase subtilisin kexin type 9 (PCSK9)(-/-) mice and intestinal explants show increased LDL-TICE, and acute injection of PCSK9 decreases TICE in vivo, suggesting that PCSK9 is a repressor of TICE. The acute repression was dependent on the LDL receptor (LDLR). Further, TICE was increased when mice were treated with lovastatin. These data point to an important role for LDLR in TICE. However, LDLR(-/-) mice showed increased intestinal LDL uptake, contrary to what is observed in the liver, and tended to have higher TICE. We interpret these data to suggest that there might be at least 2 mechanisms contributing to TICE; 1 involving LDL receptors and other unidentified mechanisms. Acute modulation of LDLR affects TICE, but chronic deficiency is compensated for most likely by the upregulation of the unknown mechanisms. Using mice deficient for apical multidrug active transporter ATP-binding cassette transporter B1 a and b, and its inhibitor, we show that these apical transporters contribute significantly to TICE. CONCLUSIONS: TICE is operative in human jejunal explants. It is a metabolically active process that can be acutely regulated, inversely related to cholesterolemia, and pharmacologically activated by statins.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Yeyuno/efectos de los fármacos , Lovastatina/farmacología , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Biopsia , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Humanos , Yeyuno/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proproteína Convertasa 9 , Proproteína Convertasas/deficiencia , Proproteína Convertasas/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Factores de Tiempo
11.
Nat Commun ; 14(1): 3346, 2023 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-37291092

RESUMEN

Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.


Asunto(s)
Infarto del Miocardio , Miocardio , Animales , Humanos , Ratones , Calcio/metabolismo , Homeostasis , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo
12.
Transl Res ; 249: 37-48, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35691543

RESUMEN

Uncertainty exists regarding whether cyclophilin D (CypD), a mitochondrial matrix protein that plays a key role in ischemia-reperfusion injury, can be a pharmacological target for improving outcomes after cardiac arrest (CA), especially when therapeutic hypothermia is used. Using CypD knockout mice (CypD-/-), we investigated the effects of loss of CypD on short-term and medium-term outcomes after CA. CypD-/- mice or their wild-type (WT) littermates underwent either 5 minute CA followed by resuscitation with and/or without hypothermia at 33°C-34°C (targeted temperature reached within minutes after resuscitation), or a sham procedure. Brain and cardiac injury were assessed using echocardiography, neurological scores, MRI and biomarkers. Seven day survival was compared using Kaplan-Meier estimates. The rate of restoration of spontaneous circulation was significantly higher in CypD-/- mice (with shorter cardiac massage duration) than in WT mice (P < 0.05). Loss of CypD significantly attenuated CA-induced release of troponin and S100ß protein, and limited myocardial dysfunction at 150 minutes after CA. Loss of CypD combined with hypothermia led to the best neurological and MRI scores at 24 hours and highest survival rates at 7 days compared to other groups (P < 0.05). In animals successfully resuscitated, loss of CypD had no benefits on day 7 survival while hypothermia was highly protective. Pharmacological inhibition of CypD with cyclosporine A combined with hypothermia provided similar day 7 survival than loss of CypD combined with hypothermia. CypD is a viable target to improve success of cardiopulmonary resuscitation but its inhibition is unlikely to improve long-term outcomes, unless therapeutic hypothermia is associated.


Asunto(s)
Paro Cardíaco , Hipotermia Inducida , Hipotermia , Animales , Biomarcadores , Peptidil-Prolil Isomerasa F , Ciclosporina/farmacología , Ciclosporina/uso terapéutico , Paro Cardíaco/terapia , Hipotermia/terapia , Hipotermia Inducida/métodos , Ratones , Ratones Noqueados , Troponina
13.
Front Cardiovasc Med ; 8: 660698, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33842565

RESUMEN

In front of the failure to translate from bench to bedside cardioprotective drugs against myocardial ischemia-reperfusion, research scientists are currently revising their animal models. Owing to its growing incidence nowadays, type 2 diabetes (T2D) represents one of the main risk factors of co-morbidities in myocardial infarction. However, discrepancies exist between reported animal and human studies. Our aim was here to compare the impact of diabetes on cell death after cardiac ischemia-reperfusion in a human cohort of ST-elevation myocardial infarction (STEMI) patients with a diet-induced mouse model of T2D, using a high-fat high-sucrose diet for 16 weeks (HFHSD). Interestingly, a small fraction (<14%) of patients undergoing a myocardial infarct were diabetic, but treated, and did not show a bigger infarct size when compared to non-diabetic patients. On the contrary, HFHSD mice displayed an increased infarct size after an in vivo cardiac ischemia-reperfusion, together with an increased cell death after an in vitro hypoxia-reoxygenation on isolated cardiomyocytes. To mimic the diabetic patients' medication profile, 6 weeks of oral gavage with Metformin was performed in the HFHSD mouse group. Metformin treatment of the HFHSD mice led to a similar extent of lower cell death after hypoxia-reoxygenation as in the standard diet group, compared to the HFHSD cardiomyocytes. Altogether, our data highlight that due to their potential protective effect, anti-diabetic medications should be included in pre-clinical study of cardioprotective approaches. Moreover, since diabetic patients represent only a minor fraction of the STEMI patients, diabetic animal models may not be the most suitable translatable model to humans, unlike aging that appears as a common feature of all infarcted patients.

14.
PLoS One ; 16(3): e0248554, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33765018

RESUMEN

Mitochondrial dynamics is a possible modulator of myocardial ischemia/reperfusion injuries (IRI). We previously reported that mice partially deficient in the fusion protein OPA1 exhibited higher IRI. Therefore, we investigated whether deficiency in the fission protein DRP1 encoded by Dnm1l gene would affect IRI in Dnm1l+/- mouse. After baseline characterization of the Dnm1l+/- mice heart, using echocardiography, electron microscopy, and oxygraphy, 3-month-old Dnm1l+/- and wild type (WT) mice were exposed to myocardial ischemia/reperfusion (I/R). The ischemic area-at-risk (AAR) and area of necrosis (AN) were delimited, and the infarct size was expressed by AN/AAR. Proteins involved in mitochondrial dynamics and autophagy were analyzed before and after I/R. Mitochondrial permeability transition pore (mPTP) opening sensitivity was assessed after I/R. Heart weight and left ventricular function were not significantly different in 3-, 6- and 12-month-old Dnm1l+/- mice than in WT. The cardiac DRP1 protein expression levels were 60% lower, whereas mitochondrial area and lipid degradation were significantly higher in Dnm1l+/- mice than in WT, though mitochondrial respiratory parameters and mPTP opening did not significantly differ. Following I/R, the infarct size was significantly smaller in Dnm1l+/- mice than in WT (34.6±3.1% vs. 44.5±3.3%, respectively; p<0.05) and the autophagic markers, LC3 II and P62 were significantly increased compared to baseline condition in Dnm1l+/- mice only. Altogether, data indicates that increasing fusion by means of Dnm1l deficiency was associated with protection against IRI, without alteration in cardiac or mitochondrial functions at basal conditions. This protection mechanism due to DRP1 haploinsufficiency increases the expression of autophagic markers.


Asunto(s)
Dinaminas/fisiología , Daño por Reperfusión Miocárdica/metabolismo , Animales , Dinaminas/genética , Haploinsuficiencia , Masculino , Ratones , Ratones Noqueados , Dinámicas Mitocondriales
15.
Cell Metab ; 2(5): 321-9, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16271532

RESUMEN

Protein feeding is known to decrease hunger and subsequent food intake in animals and humans. It has also been suggested that glucose appearance into portal vein, as occurring during meal assimilation, may induce comparable effects. Here, we connect these previous observations by reporting that intestinal gluconeogenesis (i.e., de novo synthesis of glucose) is induced during the postabsorptive time (following food digestion) in rats specifically fed on protein-enriched diet. This results in glucose release into portal blood, counterbalancing the lowering of glycemia resulting from intestinal glucose utilization. Comparable infusions into the portal vein of control postabsorptive rats (fed on starch-enriched diet) decrease food consumption and activate the hypothalamic nuclei regulating food intake. Similar hypothalamic activation occurs on protein feeding. All these effects are absent after denervation of the portal vein. Thus, portal sensing of intestinal gluconeogenesis may be a novel mechanism connecting the macronutrient composition of diet to food intake.


Asunto(s)
Proteínas en la Dieta , Ingestión de Alimentos , Gluconeogénesis , Glucosa-6-Fosfatasa/biosíntesis , Glutaminasa/biosíntesis , Intestino Delgado/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Animales , Conducta Animal , Carbohidratos de la Dieta , Inducción Enzimática , Glucosa/metabolismo , Hipotálamo/metabolismo , Vena Porta/inervación , Vena Porta/metabolismo , Periodo Posprandial , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Factores de Tiempo
16.
Cells ; 9(5)2020 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-32466308

RESUMEN

During myocardial infarction, dysregulation of Ca2+ homeostasis between the reticulum, mitochondria, and cytosol occurs in cardiomyocytes and leads to cell death. Ca2+ leak channels are thought to be key regulators of the reticular Ca2+ homeostasis and cell survival. The present study aimed to determine whether a particular reticular Ca2+ leak channel, the translocon, also known as translocation channel, could be a relevant target against ischemia/reperfusion-mediated heart injury. To achieve this objective, we first used an intramyocardial adenoviral strategy to express biosensors in order to assess Ca2+ variations in freshly isolated adult mouse cardiomyocytes to show that translocon is a functional reticular Ca2+ leak channel. Interestingly, translocon activation by puromycin mobilized a ryanodine receptor (RyR)-independent reticular Ca2+ pool and did not affect the excitation-concentration coupling. Second, puromycin pretreatment decreased mitochondrial Ca2+ content and slowed down the mitochondrial permeability transition pore (mPTP) opening and the rate of cytosolic Ca2+ increase during hypoxia. Finally, this translocon pre-activation also protected cardiomyocytes after in vitro hypoxia reoxygenation and reduced infarct size in mice submitted to in vivo ischemia-reperfusion. Altogether, our report emphasizes the role of translocon in cardioprotection and highlights a new paradigm in cardioprotection by functionally uncoupling the RyR-dependent Ca2+ stores and translocon-dependent Ca2+ stores.


Asunto(s)
Calcio/metabolismo , Cardiotónicos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Canales de Translocación SEC/metabolismo , Animales , Acoplamiento Excitación-Contracción , Masculino , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Puromicina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
17.
Front Physiol ; 10: 1370, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31736788

RESUMEN

BACKGROUND: Ischemic heart diseases are a major cause of death worldwide. Different animal models, including cardiac surgery, have been developed over time. Unfortunately, the surgery models have been reported to trigger an important inflammatory response that might be an effect modifier, where involved molecular processes have not been fully elucidated yet. OBJECTIVE: We sought to perform a thorough characterization of the sham effect in the myocardium and identify the interfering inflammatory reaction in order to avoid misinterpretation of the data via systems biology approaches. METHODS AND RESULTS: We combined a comprehensive analytical pipeline of mRNAseq dataset and systems biology analysis to characterize the acute phase response of mouse myocardium at 0 min, 45 min, and 24 h after surgery to better characterize the molecular processes inadvertently induced in sham animals. Our analysis showed that the surgical intervention induced 1209 differentially expressed transcripts (DETs). The clustering of positively co-regulated transcript modules at 45 min fingerprinted the activation of signalization pathways, while positively co-regulated genes at 24 h identified the recruitment of neutrophils and the differentiation of macrophages. In addition, we combined the prediction of transcription factors (TF) regulating DETs with protein-protein interaction networks built from these TFs to predict the molecular network which have induced the DETs. By mean of this retro-analysis of processes upstream gene transcription, we revealed a major role of the Il-6 pathway and further confirmed a significant increase in circulating IL-6 at 45 min after surgery. CONCLUSION: This study suggests that a strong induction of the IL-6 axis occurs in sham animals over the first 24 h and leads to the induction of inflammation and tissues' homeostasis processes.

18.
Nat Metab ; 1(10): 996-1008, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-32694842

RESUMEN

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.


Asunto(s)
Chaperoninas/metabolismo , Hipoxia/inducido químicamente , Pliegue de Proteína/efectos de los fármacos , Alanina/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , División Celular , Chaperoninas/genética , Conducta Alimentaria , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Daño por Reperfusión Miocárdica/prevención & control , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
19.
PLoS One ; 12(8): e0182358, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28796779

RESUMEN

BACKGROUND: There is experimental evidence that lethal ischemia-reperfusion injury (IRI) is largely due to mitochondrial permeability transition pore (mPTP) opening, which can be prevented by cyclosporine A (CsA). The aim of our study is to show that a higher dose of CsA (10 mg/kg) injected just before ischemia or a lower dose of CsA (3 mg/kg) injected further in advance of ischemia (1 h) protects the kidneys and improves mitochondrial function. METHODS: All mice underwent a right unilateral nephrectomy followed by 30 min clamping of the left renal artery. Mice in the control group did not receive any pharmacological treatment. Mice in the three groups treated by CsA were injected at different times and with different doses, namely 3 mg/kg 1 h or 10 min before ischemia or 10 mg/kg 10 min before ischemia. After 24 h of reperfusion, the plasma creatinine level were measured, the histological score was assessed and mitochondria were isolated to calculate the calcium retention capacity (CRC) and level of oxidative phosphorylation. RESULTS: Mortality and renal function was significantly higher in the CsA 10 mg/kg-10 min and CsA 3mg/kg-1 h groups than in the CsA 3mg/kg-10 min group. Likewise, the CRC was significantly higher in the former two groups than in the latter, suggesting that the improved renal function was due to a longer delay in the opening of the mPTP. Oxidative phosphorylation levels were also higher 24 h after reperfusion in the protected groups. CONCLUSIONS: Our results suggest that the protection afforded by CsA is likely limited by its availability. The dose and timing of the injections are therefore crucial to ensure that the treatment is effective, but these findings may prove challenging to apply in practice.


Asunto(s)
Lesión Renal Aguda/prevención & control , Inhibidores de la Calcineurina/administración & dosificación , Ciclosporina/administración & dosificación , Riñón/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial
20.
Endocrinology ; 147(5): 2432-41, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16455782

RESUMEN

The AMP-activated kinase (AMPK) is a serine threonine kinase that functions as a fuel sensor to regulate energy balance at both cellular and whole-body levels. Here we studied how hepatic AMPKalpha2 isoform affects hepatic glucose production and peripheral glucose uptake in vivo. We generated mice deleted for the AMPKalpha2 gene specifically in the liver (liveralpha2KO). Liveralpha2KO mice were glucose intolerant and hyperglycemic in the fasted state. Hyperglycemia was associated with a 50% higher endogenous glucose production than in controls as assessed in vivo. We then investigated whether this increased glucose production was sensitive to insulin. Insulin, when infused at a rate inducing physiological hyperinsulinemia, totally inhibited endogenous glucose production in liveralpha2KO mice, showing that they had normal insulin sensitivity. This was confirmed in vivo by normal insulin-induced phosphorylation of Akt and transcriptional regulation of the phosphoenolpyruvate carboxykinase, glucose-6 phosphatase, and pyruvate kinase in liver during the fasted/fed transition. Leptin and adiponectin regulate hepatic glucose production, so we then infused these adipokines into liveralpha2KO mice. Neither of these adipokines regulated hepatic glucose production in mice lacking hepatic AMPKalpha2, whereas both did so in control mice. In conclusion, we show that the hepatic AMPKalpha2 isoform is essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels in the physiological range. We also demonstrate that regulation of hepatic glucose production by leptin and adiponectin, but not insulin, requires hepatic AMPKalpha2 activity.


Asunto(s)
Adiponectina/química , Glucosa/metabolismo , Insulina/metabolismo , Leptina/química , Hígado/metabolismo , Complejos Multienzimáticos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Quinasas Activadas por AMP , Adiponectina/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Catálisis , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Glucosa-6-Fosfatasa/metabolismo , Hiperglucemia/metabolismo , Hormonas de Insectos/metabolismo , Leptina/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Modelos Estadísticos , Complejos Multienzimáticos/metabolismo , Oligopéptidos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosforilación , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Ácido Pirrolidona Carboxílico/análogos & derivados , Ácido Pirrolidona Carboxílico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Activación Transcripcional
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