RESUMEN
BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Óxido Nítrico , Ratones , Animales , Isoproterenol/farmacología , Óxido Nítrico/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cisteína/metabolismo , Ratones Endogámicos C57BL , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación , Receptores Adrenérgicos beta/metabolismo , Calcio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking ß-sarcoglycan (ß-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this ß-SG-/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in ß-SG-/- mice. Male ß-SG-/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female ß-SG-/- hearts. This protection of females was independent of ovarian hormones. Male ß-SG-/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of ß-SG-/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male ß-SG-/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male ß-SG-/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.
Asunto(s)
Antioxidantes , Cardiomiopatías , Miocardio , Estrés Oxidativo , Sarcoglicanos , Animales , Masculino , Sarcoglicanos/metabolismo , Sarcoglicanos/genética , Femenino , Cardiomiopatías/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatías/etiología , Ratones , Antioxidantes/metabolismo , Miocardio/metabolismo , Miocardio/patología , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Susceptibilidad a Enfermedades , Isoproterenol , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 1/genéticaRESUMEN
Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. NEW AND NOTEWORTHY: In this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.
Asunto(s)
Proteínas Argonautas , MicroARNs , Miocitos Cardíacos , Especies Reactivas de Oxígeno , MicroARNs/genética , MicroARNs/metabolismo , Animales , Femenino , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Masculino , Fosforilación , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Especies Reactivas de Oxígeno/metabolismo , Ratones , Mitocondrias Cardíacas/metabolismo , Calcio/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Complejo Silenciador Inducido por ARN/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Caracteres SexualesRESUMEN
The post-translational modification of intracellular proteins by O-linked ß-GlcNAc (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia-reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc, respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female WT murine hearts to ex vivo ischemia or I/R injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show that OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found that ischemia led to reduced O-GlcNAcylation and OGT-specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.
Asunto(s)
Acetilglucosamina , Corazón , Miocardio , N-Acetilglucosaminiltransferasas , Caracteres Sexuales , Transducción de Señal , Animales , Femenino , Masculino , Ratones , Acetilglucosamina/metabolismo , Corazón/fisiología , Isquemia/enzimología , Isquemia/metabolismo , Miocardio/enzimología , Miocardio/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Exposure to inorganic arsenic through drinking water is widespread and has been linked to many chronic diseases, including cardiovascular disease. Arsenic exposure has been shown to alter hypertrophic signaling in the adult heart, as well as in utero offspring development. However, the effect of arsenic on maternal cardiac remodeling during pregnancy has not been studied. As such, there is a need to understand how environmental exposure contributes to adverse pregnancy-related cardiovascular events. This study seeks to understand the impact of trivalent inorganic arsenic exposure during gestation on maternal cardiac remodeling in late pregnancy, as well as offspring outcomes. C57BL/6 J mice were exposed to 0 (control), 100 or 1000 µg/L sodium arsenite (NaAsO2) beginning at embryonic day (E) 2.5 and continuing through E17.5. Maternal heart function and size were assessed via transthoracic echocardiography, gravimetric measurement, and histology. Transcript levels of hypertrophic markers were probed via qRT-PCR and confirmed by western blot. Offspring outcomes were assessed through echocardiography and gravimetric measurement. We found that maternal heart size was smaller and transcript levels of Esr1 (estrogen receptor alpha), Pgrmc1 (progesterone receptor membrane component 1) and Pgrmc2 (progesterone receptor membrane component 2) reduced during late pregnancy with exposure to 1000 µg/L iAs vs. non-exposed pregnant controls. Both 100 and 1000 µg/L iAs also reduced transcription of Nppa (atrial natriuretic peptide). Akt protein expression was also significantly reduced after 1000 µg/L iAs exposure in the maternal heart with no change in activating phosphorylation. This significant abrogation of maternal cardiac hypertrophy suggests that arsenic exposure during pregnancy can potentially contribute to cardiovascular disease. Taken together, our findings further underscore the importance of reducing arsenic exposure during pregnancy and indicate that more research is needed to assess the impact of arsenic and other environmental exposures on the maternal heart and adverse pregnancy events.
Asunto(s)
Arsénico , Arsenitos , Enfermedades Cardiovasculares , Efectos Tardíos de la Exposición Prenatal , Humanos , Animales , Ratones , Femenino , Embarazo , Arsénico/metabolismo , Arsenitos/toxicidad , Receptores de Progesterona , Exposición Materna/efectos adversos , Remodelación Ventricular , Ratones Endogámicos C57BL , Efectos Tardíos de la Exposición Prenatal/metabolismoRESUMEN
Thousands of mammalian intracellular proteins are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.
Asunto(s)
Enfermedades Neurodegenerativas , Ratones , Animales , Anticuerpos/metabolismo , Proteoma/metabolismo , Corazón , Proteínas Contráctiles/metabolismo , Acetilglucosamina , Procesamiento Proteico-Postraduccional , Mamíferos/metabolismoRESUMEN
Arsenic exposure though drinking water is widespread and well associated with adverse cardiovascular outcomes, yet the pathophysiological mechanisms by which iAS induces these effects are largely unknown. Recently, an epidemiological study in an American population with a low burden of cardiovascular risk factors found that iAS exposure was associated with altered left ventricular geometry. Considering the possibility that iAS directly induces cardiac remodeling independently of hypertension, we investigated the impact of an environmentally relevant iAS exposure on the structure and function of male and female hearts. Adult male and female C56BL/6J mice were exposed to 615 µg/L iAS for 8 wk. Males exhibited increased systolic blood pressure via tail cuff photoplethysmography, left ventricular wall thickening via transthoracic echocardiography, and increased plasma atrial natriuretic peptide via enzyme immunoassay. RT-qPCR revealed increased myocardial RNA transcripts of Acta1, Myh7, and Nppa and decreased Myh6, providing evidence of pathological hypertrophy in the male heart. Similar changes were not detected in females, and nitric oxide-dependent mechanisms of cardioprotection in the heart appeared to remain intact. Further investigation found that Rcan1 was upregulated in male hearts and that iAS activated NFAT in HEK-293 cells via luciferase assay. Interestingly, iAS induced similar hypertrophic gene expression changes in neonatal rat ventricular myocytes, which were blocked by calcineurin inhibition, suggesting that iAS may induce pathological cardiac hypertrophy in part by targeting the calcineurin-NFAT pathway. As such, these results highlight iAS exposure as an independent cardiovascular risk factor and provide biological impetus for its removal from human consumption.NEW & NOTEWORTHY This investigation provides the first mechanistic link between an environmentally relevant dose of inorganic arsenic (iAS) and pathological hypertrophy in the heart. By demonstrating that iAS exposure may cause pathological cardiac hypertrophy not only by increasing systolic blood pressure but also by potentially activating calcineurin-nuclear factor of activated T cells and inducing fetal gene expression, these results provide novel mechanistic insight into the theat of iAS exposure to the heart, which is necessary to identify targets for medical and public health intervention.
Asunto(s)
Arsenitos/toxicidad , Hipertrofia Ventricular Izquierda/inducido químicamente , Miocitos Cardíacos/efectos de los fármacos , Compuestos de Sodio/toxicidad , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Calcineurina/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/metabolismo , Factores Sexuales , Transducción de Señal , Factores de TiempoRESUMEN
AIMS: In cardiomyocytes, there is microRNA (miR) in the mitochondria that originates from the nuclear genome and matures in the cytoplasm before translocating into the mitochondria. Overexpression of one such miR, miR-181c, can lead to heart failure by stimulating reactive oxygen species (ROS) production and increasing mitochondrial calcium level ([Ca2+]m). Mitochondrial calcium uptake 1 protein (MICU1), a regulatory protein in the mitochondrial calcium uniporter complex, plays an important role in regulating [Ca2+]m. Obesity results in miR-181c overexpression and a decrease in MICU1. We hypothesize that lowering miR-181c would protect against obesity-induced cardiac dysfunction. METHODS AND RESULTS: We used an in vivo mouse model of high-fat diet (HFD) for 18 weeks and induced high lipid load in H9c2 cells with oleate-conjugated bovine serum albumin in vitro. We tested the cardioprotective role of lowering miR-181c by using miR-181c/d-/- mice (in vivo) and AntagomiR against miR-181c (in vitro). HFD significantly upregulated heart levels of miR-181c and led to cardiac hypertrophy in wild-type mice, but not in miR-181c/d-/- mice. HFD also increased ROS production and pyruvate dehydrogenase activity (a surrogate for [Ca2+]m), but the increases were alleviated in miR-181c/d-/- mice. Moreover, miR-181c/d-/- mice fed a HFD had higher levels of MICU1 than did wild-type mice fed a HFD, attenuating the rise in [Ca2+]m. Overexpression of miR-181c in neonatal ventricular cardiomyocytes (NMVM) caused increased ROS production, which oxidized transcription factor Sp1 and led to a loss of Sp1, thereby slowing MICU1 transcription. Hence, miR-181c increases [Ca2+]m through Sp1 oxidation and downregulation of MICU1, suggesting that the cardioprotective effect of miR-181c/d-/- results from inhibition of Sp1 oxidation. CONCLUSION: This study has identified a unique nuclear-mitochondrial communication mechanism in the heart orchestrated by miR-181c. Obesity-induced overexpression of miR-181c increases [Ca2+]m via downregulation of MICU1 and leads to cardiac injury. A strategy to inhibit miR-181c in cardiomyocytes can preserve cardiac function during obesity by improving mitochondrial function. Altering miR-181c expression may provide a pharmacologic approach to improve cardiomyopathy in individuals with obesity/type 2 diabetes.
Asunto(s)
Núcleo Celular/metabolismo , MicroARNs/genética , Mitocondrias Cardíacas/metabolismo , Obesidad/genética , Obesidad/metabolismo , Disfunción Ventricular/etiología , Disfunción Ventricular/metabolismo , Animales , Biomarcadores , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ratones , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Miocitos Cardíacos/metabolismo , Obesidad/complicaciones , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción Sp1/metabolismo , Disfunción Ventricular/fisiopatologíaRESUMEN
Mitochondria have emerged as a central factor in the pathogenesis and progression of heart failure, and other cardiovascular diseases, as well, but no therapies are available to treat mitochondrial dysfunction. The National Heart, Lung, and Blood Institute convened a group of leading experts in heart failure, cardiovascular diseases, and mitochondria research in August 2018. These experts reviewed the current state of science and identified key gaps and opportunities in basic, translational, and clinical research focusing on the potential of mitochondria-based therapeutic strategies in heart failure. The workshop provided short- and long-term recommendations for moving the field toward clinical strategies for the prevention and treatment of heart failure and cardiovascular diseases by using mitochondria-based approaches.
Asunto(s)
Sistema Cardiovascular , Educación/métodos , Insuficiencia Cardíaca/terapia , Mitocondrias/fisiología , National Heart, Lung, and Blood Institute (U.S.) , Informe de Investigación , Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Sistema Cardiovascular/patología , Educación/tendencias , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/epidemiología , Humanos , National Heart, Lung, and Blood Institute (U.S.)/tendencias , Informe de Investigación/tendencias , Investigación Biomédica Traslacional/métodos , Investigación Biomédica Traslacional/tendencias , Estados Unidos/epidemiologíaRESUMEN
RATIONALE: Protein S-nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S-nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism. OBJECTIVE: Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females. METHODS AND RESULTS: Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R-/- hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R-/- female hearts, significantly reducing infarct size. CONCLUSIONS: These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Benzamidas/uso terapéutico , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Estrés Oxidativo , Pirroles/farmacología , Pirroles/uso terapéutico , Factores SexualesRESUMEN
TRIM72 is a membrane repair protein that protects against ischemia reperfusion (I/R) injury. We previously identified Cys144 (C144) on TRIM72 as a site of S-nitrosylation. To study the importance of C144, we generated a knock-in mouse with C144 mutated to a serine (TRIM72 C144S). We subjected ex vivo perfused mouse hearts to 20â¯min of ischemia followed by 90â¯min of reperfusion and observed less injury in TRIM72 C144S compared to WT hearts. Infarct size was smaller (54 vs 27% infarct size) and cardiac functional recovery (37 vs 62% RPP) was higher for the TRIM72 C144S mouse hearts. We also demonstrated that TRIM72 C144S hearts were protected against I/R injury using an in vivo LAD occlusion model. As TRIM72 has been reported to be released from muscle we tested whether C144 is involved in TRIM72 release. After I/R there was significantly less TRIM72 in the perfusate normalized to total released protein from the TRIM72 C144S compared to WT hearts, suggesting that C144 of TRIM72 regulates myocardial TRIM72 release during I/R injury. In addition to TRIM72's protective role in I/R injury, TRIM72 has also been implicated in cardiac hypertrophy and insulin resistance, and secreted TRIM72 has recently been shown to impair insulin sensitivity. However, insulin sensitivity (measured by glucose and insulin tolerance) of TRIM72 C144S mice was not impaired. Further, whole body metabolism, as measured using metabolic cages, was not different in WT vs TRIM72 C144S mice and we did not observe enhanced cardiac hypertrophy in the TRIM72 C144S mice. In agreement, protein levels of the TRIM72 ubiquitination targets insulin receptor ß, IRS1, and focal adhesion kinase were similar between WT and TRIM72 C144S hearts. Overall, these data indicate that mutation of TRIM72 C144 is protective during I/R and reduces myocardial TRIM72 release without impairing insulin sensitivity or enhancing the development of hypertrophy.
Asunto(s)
Cisteína/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Angiotensina II/farmacología , Animales , Cardiomegalia/genética , Enfermedad de la Arteria Coronaria , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Resistencia a la Insulina/genética , Ratones Endogámicos C57BL , Ratones Mutantes , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocardio/patologíaRESUMEN
Arsenic is a common contaminant in drinking water throughout the world, and recent studies support a link between inorganic arsenic (iAS) exposure and ischemic heart disease in men and women. Female hearts exhibit an estrogen-dependent reduction in susceptibility to myocardial ischemic injury compared with males, and as such, female hearts may be more susceptible to the endocrine-disrupting effects of iAS exposure. However, iAS exposure and susceptibility to ischemic heart injury have not been examined in mechanistic studies. Male and female mice (8 wk) were exposed to environmentally relevant concentrations of sodium arsenite (0, 10, 100, and 1,000 parts/billion) via drinking water for 4 wk. Pre- and postexposure echocardiography was performed, and postexposure plasma was collected for 17ß-estradiol measurement. Hearts were excised and subjected to ischemia-reperfusion (I/R) injury via Langendorff perfusion. Exposure to 1,000 parts/billion iAS led to sex-disparate effects, such that I/R injury was exacerbated in female hearts but unexpectedly attenuated in males. Assessment of echocardiographic parameters revealed statistically significant structural remodeling in iAS-treated female hearts with no change in function; males showed no change. Plasma 17ß-estradiol levels were not significantly altered by iAS in male or female mice versus nontreated controls. Although total eNOS protein levels did not change in whole heart homogenates from iAS-treated male or female mice, eNOS phosphorylation (Ser1177) was significantly elevated in iAS-treated male hearts. These results suggest that iAS exposure can induce sex-disparate effects and modulate susceptibility to ischemic heart injury by targeting distinct sex-dependent pathways. NEW & NOTEWORTHY This is the first mechanistic study examining iAS exposure on myocardial ischemia-reperfusion injury in male and female mice. Following iAS exposure, ischemia-reperfusion injury was exacerbated in female hearts but attenuated in males. iAS treatment induced statistically significant cardiac remodeling in females, with no change in males. iAS treatment also enhanced phosphorylated eNOS levels at Ser1177, but only in male hearts. These results suggest that iAS alters susceptibility to myocardial I/R injury through distinct sex-dependent pathways.
Asunto(s)
Arsenitos/toxicidad , Daño por Reperfusión Miocárdica/inducido químicamente , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/patología , Compuestos de Sodio/toxicidad , Remodelación Ventricular/efectos de los fármacos , Animales , Cardiotoxicidad , Modelos Animales de Enfermedad , Estradiol/sangre , Femenino , Preparación de Corazón Aislado , Masculino , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/sangre , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Factores SexualesRESUMEN
Premenopausal women exhibit endogenous cardioprotective signaling mechanisms that are thought to result from the beneficial effects of estrogen, which we have shown to increase protein S-nitrosylation in the heart. S-nitrosylation is a labile protein modification that increases with a number of different forms of cardioprotection, including ischemic preconditioning. Herein, we sought to identify a potential role for protein S-nitrosylation in sex-dependent cardioprotection. We utilized a Langendorff-perfused mouse heart model of ischemia-reperfusion injury with male and female hearts, and S-nitrosylation-resin-assisted capture with liquid chromatography tandem mass spectrometry to identify S-nitrosylated proteins and modification sites. Consistent with previous studies, female hearts exhibited resilience to injury with a significant increase in functional recovery compared with male hearts. In a separate set of hearts, we identified a total of 177 S-nitrosylated proteins in female hearts at baseline compared with 109 S-nitrosylated proteins in male hearts. Unique S-nitrosylated proteins in the female group included the F1FO-ATPase and cyclophilin D. We also utilized label-free peptide analysis to quantify levels of common S-nitrosylated identifications and noted that the S-nitrosylation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a was nearly 70% lower in male hearts compared with female, with no difference in expression. Furthermore, we found a significant increase in endothelial nitric oxide synthase expression, phosphorylation, and total nitric oxide production in female hearts compared with males, likely accounting for the enhanced S-nitrosylation protein levels in female hearts. In conclusion, we identified a number of novel S-nitrosylated proteins in female hearts that are likely to contribute to sex-dependent cardioprotection.
Asunto(s)
Circulación Coronaria/efectos de los fármacos , Corazón/efectos de los fármacos , Proteoma/efectos de los fármacos , S-Nitrosotioles/metabolismo , Animales , Peptidil-Prolil Isomerasa F , Ciclofilinas/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Fosforilación , Caracteres SexualesRESUMEN
Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that SNO of TRIM72 at C144 prevents the oxidation-induced degradation of TRIM72 following oxidative insult, therefore enhancing cardiomyocyte survival.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cisteína/química , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/patología , Óxido Nítrico/metabolismo , Animales , Western Blotting , Proteínas Portadoras/genética , Muerte Celular , Supervivencia Celular , Cisteína/metabolismo , Femenino , Precondicionamiento Isquémico , Masculino , Proteínas de la Membrana , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Oxidación-Reducción , ProteolisisRESUMEN
Nitric oxide is a gaseous signaling molecule that is well-known for the Nobel prize-winning research that defined nitric oxide as a physiological regulator of blood pressure in the cardiovascular system. Nitric oxide can signal via the classical pathway involving activation of guanylyl cyclase or by a post-translational modification, referred to as S-nitrosylation (SNO) that can occur on cysteine residues of proteins. As proteins with cysteine residues are common, this allows for amplification of the nitric oxide signaling. This review will focus on the possible mechanisms through which SNO can alter protein function in cardiac cells, and the role of SNO occupancy in these mechanisms. The specific mechanisms that regulate protein SNO, including redox-dependent processes, will also be discussed. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".
Asunto(s)
Corazón , S-Nitrosotioles/metabolismo , Transducción de Señal/fisiología , Animales , Humanos , Óxido Nítrico/metabolismo , Oxidación-Reducción , Procesamiento Proteico-PostraduccionalRESUMEN
Previous studies have shown a role for nitric oxide and S-nitrosylation (SNO) in postconditioning (PostC), but specific SNO proteins and sites have not been identified in the myocardium after PostC. In this study, we examined SNO signaling in PostC using a Langendorff-perfused mouse heart model. After 20 min of equilibrium perfusion and 25 min of global ischemia, PostC was applied at the beginning of reperfusion with six cycles of 10 s of reperfusion and 10 s of ischemia. The total period of reperfusion was 90 min. Compared with the ischemia-reperfusion (I/R) control, PostC significantly reduced postischemic contractile dysfunction and infarct size. PostC-induced protection was blocked by treatment with N(G)-nitro-l-arginine methyl ester (l-NAME) (10 µmol/l; a constitutive NO synthase inhibitor), but not by either ODQ (10 µmol/l, a highly selective soluble guanylyl cyclase inhibitor) or KT5823 (1 µmol/l, a specific protein kinase G inhibitor). Two biotin switch based methods, two dimensional CyDye-maleimide difference gel electrophoresis (2D CyDye-maleimide DIGE) and SNO-resin-assisted capture (SNO-RAC), were utilized to identify SNO-modified proteins and sites. Using 2D CyDye-maleimide DIGE analysis, PostC was found to cause a 25% or greater increase in SNO of a number of proteins, which was blocked by treatment with l-NAME in parallel with the loss of protection. Using SNO-RAC, we identified 77 unique proteins with SNO sites after PostC. These results suggest that NO-mediated SNO signaling is involved in PostC-induced cardioprotection and these data provide the first set of candidate SNO proteins in PostC hearts.
Asunto(s)
Óxido Nítrico/metabolismo , Proteína S/metabolismo , Daño por Reperfusión/metabolismo , Animales , Carbazoles/farmacología , Poscondicionamiento Isquémico , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , NG-Nitroarginina Metil Éster/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: S-nitrosylation (SNO) is a reversible, thiol-based protein modification that plays an important role in the myocardium by protecting critical cysteine residues from oxidation. However, little is known with regard to the percentage of a given protein that is modified by SNO (ie, SNO occupancy). Current methods allow for the relative quantification of SNO levels, but not for the determination of SNO occupancy. OBJECTIVE: To develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium. METHODS AND RESULTS: We developed a differential cysteinereactive tandem mass tag (cysTMT) labeling procedure for the measurement of SNO occupancy. To validate this cysTMT labeling method, we treated whole-heart homogenates with the S-nitrosylating agent S-nitrosoglutathione and determined maximal SNO occupancy. We also examined SNO occupancy under more physiological conditions and observed that SNO occupancy is low for most protein targets at baseline. Following ischemic preconditioning, SNO occupancy increased to an intermediate level compared to baseline and Snitrosoglutathione treatment, and this is consistent with the ability of SNO to protect against cysteine oxidation. CONCLUSIONS: This novel cysTMT labeling approach provides a method for examining SNO occupancy in the myocardium. Using this approach, we demonstrated that IPC-induced SNO occupancy levels are sufficient to protect against oxidation.
Asunto(s)
Precondicionamiento Isquémico/métodos , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , S-Nitrosoglutatión/metabolismo , Espectrometría de Masas en Tándem/métodos , Animales , Cisteína/análisis , Cisteína/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/química , Oxidación-Reducción , Perfusión , S-Nitrosoglutatión/análisis , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismoRESUMEN
RATIONALE: Redox modifications play an important role in many cellular processes, including cell death. Ischemic preconditioning (IPC) has been shown to involve redox signaling. Protein S-nitrosylation (SNO) is increased following myocardial IPC, and SNO is thought to provide cardioprotection, in part, by reducing cysteine oxidation during ischemia/reperfusion (IR) injury. OBJECTIVE: To test the hypothesis that SNO provides cardioprotection, in part, by shielding against cysteine oxidation following IR injury. METHODS AND RESULTS: We developed a new method to measure protein oxidation using resin-assisted capture (Ox-RAC), which is similar to the SNO-RAC method used in the quantification of SNO. Langendorff-perfused hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR, IPC/reperfusion) and homogenized. Each sample was divided into 2 equal aliquots, and the SNO-RAC/Ox-RAC procedure was performed to simultaneously analyze SNO and oxidation. We identified 31 different SNO proteins with IPC, 27 of which showed increased SNO compared to baseline. Of the proteins that showed significantly increased SNO with IPC, 76% showed decreased oxidation or no oxidation following ischemia and early reperfusion (IPC-IR) at the same site when compared to IR alone; for non-SNO proteins, oxidation was reduced by only 50%. We further demonstrated that IPC-induced protein SNO is quickly reversible. CONCLUSIONS: These results support the hypothesis that IPC-induced protein SNO provides cardioprotection by shielding cysteine residues from reactive oxygen species-induced oxidation during IR injury. Therefore, the level of protein SNO plays a critical role in IR injury, where ROS production is increased.
Asunto(s)
Precondicionamiento Isquémico Miocárdico/métodos , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Proteínas/metabolismo , Resinas Sintéticas/metabolismo , S-Nitrosotioles/metabolismo , Animales , Cisteína/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Masculino , Ratones , Modelos Animales , Daño por Reperfusión Miocárdica/prevención & control , Oxidación-ReducciónRESUMEN
During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. Here we show that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of perfused mouse hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive â¼180 KDa Opa1 complex, distinct from the â¼270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1-/- cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1-/- cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during cardiac ischemia-reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independent of mitochondrial fusion.
Asunto(s)
Enfermedad de la Arteria Coronaria , Daño por Reperfusión Miocárdica , Atrofia Óptica Autosómica Dominante , Animales , Ratones , Muerte Celular , Cisteína/metabolismo , Peróxido de Hidrógeno , Daño por Reperfusión Miocárdica/metabolismo , Atrofia Óptica Autosómica Dominante/metabolismo , Estrés OxidativoRESUMEN
Exposure to inorganic arsenic through drinking water is widespread and has been linked to many chronic diseases, including cardiovascular disease. Arsenic exposure has been shown to alter hypertrophic signaling in the adult heart, as well as in-utero offspring development. However, the effect of arsenic on maternal cardiac remodeling during pregnancy has not been studied. As such, there is a need to understand how environmental exposure contributes to adverse pregnancy-related cardiovascular events. This study seeks to understand the impact of trivalent inorganic arsenic exposure during gestation on maternal cardiac remodeling in late pregnancy, as well as offspring outcomes. C57BL/6J mice were exposed to 0 (control), 100 or 1000 µg/L sodium arsenite (NaAsO 2 ) beginning at embryonic day (E) 2.5 and continuing through E17.5. Maternal heart function and size were assessed via transthoracic echocardiography, gravimetric measurement, and histology. Transcript levels of hypertrophic markers were probed via qRT-PCR and confirmed by western blot. Offspring outcomes were assessed through echocardiography and gravimetric measurement. We found that exposure to 1000 µg/L iAs abrogated normal physiologic growth of the maternal heart during late pregnancy and reduced transcript levels of estrogen receptor alpha (ERα), progesterone receptor membrane component 1 (Pgrmc1) and progesterone receptor membrane component 2 (Pgrmc2). Both 100 and 1000 µg/L iAs also reduced transcription of protein kinase B (Akt) and atrial natriuretic peptide (ANP). Akt protein expression was also significantly reduced after 1000 µg/L iAs exposure in the maternal heart with no change in activating phosphorylation. This significant abrogation of maternal cardiac hypertrophy suggests that arsenic exposure during pregnancy can potentially contribute to cardiovascular disease. Taken together, our findings further underscore the importance of reducing arsenic exposure during pregnancy and indicate that more research is needed to assess the impact of arsenic and other environmental exposures on the maternal heart and adverse pregnancy events.