RESUMO
Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods:Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.
Assuntos
Cardiotônicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Cardiotônicos/uso terapêutico , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Humanos , Preparação de Coração Isolado , Masculino , Microvasos/citologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Nitroarginina/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca²âº uptake. Accordingly, uncoupling of the organelles or blocking Ca²âº transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca²âº transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.
Assuntos
Retículo Endoplasmático/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Estresse Fisiológico , Antibacterianos/farmacologia , Apoptose/fisiologia , Cálcio/metabolismo , Respiração Celular , Inibidores Enzimáticos/farmacologia , Células HeLa , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Potencial da Membrana Mitocondrial , Consumo de Oxigênio/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVES: We sought to define the role of interstitial fibrosis in the proarrhythmic phenotype of failing ventricular myocardium. BACKGROUND: Multiple cellular events that occur during pathological remodeling of the failing ventricle are implicated in the genesis of ventricular tachycardia (VT), including interstitial fibrosis. Recent studies suggest that ventricular fibrosis is reversible, and current anti-remodeling therapies attenuate ventricular fibrosis. However, the role of interstitial fibrosis in the proarrhythmic phenotype of failing ventricular myocardium is currently not well defined. METHODS: Class II histone deacetylases (HDACs) have been implicated in promoting collagen biosynthesis. As these enzymes are inhibited by protein kinase D1 (PKD1), we studied mice with cardiomyocyte-specific transgenic over-expression of a constitutively active mutant of PKD1 (caPKD). caPKD mice were compared with animals in which cardiomyopathy was induced by severe thoracic aortic banding (sTAB). Hearts were analyzed by echocardiographic and electrocardiographic means. Interstitial fibrosis was assessed by histology and quantified biochemically. Ventricular arrhythmias were induced by closed-chest, intracardiac pacing. RESULTS: Similar degrees of hypertrophic growth, systolic dysfunction and mortality were observed in the two models. In sTAB mice, robust ventricular fibrosis was readily detected, but myocardial collagen content was significantly reduced in caPKD mice. As expected, VT was readily inducible by programmed stimulation in sTAB mice and VT was less inducible in caPKD mice. Surprisingly, episodes of VT manifested longer cycle lengths and longer duration in caPKD mice. CONCLUSION: Attenuated ventricular fibrosis is associated with reduced VT inducibility, increased VT duration, and significantly longer arrhythmia cycle length.
Assuntos
Cardiomiopatia Dilatada/complicações , Insuficiência Cardíaca/etiologia , Miocárdio/patologia , Taquicardia Ventricular/etiologia , Remodelação Ventricular , Potenciais de Ação , Animais , Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Técnicas Eletrofisiológicas Cardíacas , Fibrose , Genótipo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Miocárdio/enzimologia , Fenótipo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Taquicardia Ventricular/enzimologia , Taquicardia Ventricular/genética , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Taquicardia Ventricular/prevenção & controle , Regulação para CimaRESUMO
The L-type Ca(2+) channel (LTCC) is the major mediator of Ca(2+) influx in cardiomyocytes, leading to both mechanical contraction and activation of signaling cascades. Among these Ca(2+)-activated cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. Coimmunoprecipitations from heart extracts and pulldowns using heterologously expressed proteins provided evidence for direct binding of calcineurin at both the N and C termini of alpha(1)1.2. At the C terminus, calcineurin bound specifically at amino acids 1943 to 1971, adjacent to a well-characterized protein kinase (PK)A/PKC/PKG phospho-acceptor site Ser1928. In vitro assays demonstrated that calcineurin can dephosphorylate alpha(1)1.2. Channel function was increased in voltage-clamp recordings of I(Ca,L) from cultured cardiomyocytes expressing constitutively active calcineurin, consistent with previous observations in cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin pharmacologically or with specific peptides decreased I(Ca,L). These data reveal direct physical interaction between the LTCC and calcineurin in heart. Furthermore, they demonstrate that calcineurin induces robust increases in I(Ca,L) and highlight calcineurin as a key modulator of pathological electrical remodeling in cardiac hypertrophy.
Assuntos
Calcineurina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Animais , Sítios de Ligação , Cardiomegalia , Células Cultivadas , Técnicas Eletrofisiológicas Cardíacas , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Fosforilação , Proteínas Quinases/metabolismo , RatosRESUMO
OBJECTIVE: To investigate the effect of Korean red ginseng total saponins (GTS) on ovarian morphology and nerve growth factor (NGF) expression in the ovaries, pituitary, and hippocampus. DESIGN: Polycystic ovary (PCO) rat model induced by estradiol valerate (EV). SETTING: University research laboratory. PATIENT(S): Thirty sexually mature female Sprague-Dawley rats weighing 190-210 g. INTERVENTION(S): Female Sprague-Dawley rats (190-210 g) were separated into three groups: EV control (n = 10), EV plus GTS (n = 10), and oil control (n = 10). MAIN OUTCOME MEASURE(S): Ovarian morphology and NGF protein expression. RESULT(S): Polycystic ovary was fully developed in rats with a single intramuscular injection of EV. Increased expression of NGF was noted in the ovaries and the brain of rats with PCO. GTS administration attenuated NGF expression in the ovaries but not in the brain. CONCLUSION(S): Our findings suggest a role for GTS in the regulation of NGF expression in female rats with PCO.
Assuntos
Infertilidade Feminina/metabolismo , Panax/química , Síndrome do Ovário Policístico/metabolismo , Saponinas/farmacologia , Animais , Encéfalo/metabolismo , Feminino , Infertilidade Feminina/tratamento farmacológico , Coreia (Geográfico) , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/biossíntese , Ovário/metabolismo , Síndrome do Ovário Policístico/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Saponinas/uso terapêuticoRESUMO
Future research in endometriosis must focus on pathogenesis studies in the baboon model, the early interactions between endometrial and peritoneal cells in the pelvic cavity at the time of menstruation, and potential differences between eutopic endometrium and myometrium in women with and without endometriosis. More integration is needed between the areas of epidemiology and genetics. Pelvic inflammation in women with endometriosis could be the target for new diagnostic and therapeutic approaches. Important questions remain regarding the relationship between endometriosis and environmental factors. Systemic and extrapelvic manifestations of endometriosis must be analyzed carefully, and better tools are needed to measure quality of life in women with chronic pain caused by endometriosis. Most current evidence supports a causal relationship between endometriosis and subfertility, and the spontaneous progressive nature of endometriosis has been demonstrated in 30% to 60% of patients. Recurrence of endometriosis after classic medical and surgical therapy is a major and underestimated problem, especially in women with advanced disease. Integrated clinical and research teams are needed that combine expert medical, surgical, and holistic care with state-of-the-art research expertise in immunology, endocrinology, and genetics to discover new diagnostic methods and medical treatments for endometriosis.