RESUMO
Postmenopausal diabetic women are at higher risk to develop cardiovascular diseases (CVD) compared with nondiabetic women. Alterations in cardiac cellular metabolism caused by changes in sirtuins are one of the main causes of CVD in postmenopausal diabetic women. Several studies have demonstrated the beneficial actions of the G protein-coupled estrogen receptor (GPER) in postmenopausal diabetic CVD. However, the molecular mechanisms by which GPER has a cardioprotective effect are still not well understood. In this study, we used an ovariectomized (OVX) type-two diabetic (T2D) rat model induced by high-fat diet/streptozotocin to investigate the effect of G-1 (GPER-agonist) on sirtuins, and their downstream pathways involved in regulation of cardiac metabolism and function. Animals were divided into five groups: Sham-Control, T2D, OVX+T2D, OVX+T2D+Vehicle, and OVX+T2D+G-1. G-1 was administrated for six weeks. At the end, hemodynamic factors were measured, and protein levels of sirtuins, AMP-activated protein kinase (AMPK), and uncoupling protein 2 (UCP2) were determined by Western blot analysis. In addition, cardiac levels of oxidative stress biomarkers were measured. The findings showed that T2D led to left ventricular dysfunction and signs of oxidative stress in the myocardium, which were accompanied by decreased protein levels of Sirt1/2/3/6, p-AMPK, and UCP2 in the heart. Moreover, the induction of the menopausal state exacerbated these changes. In contrast, treatment with G-1 ameliorated the hemodynamic changes associated with ovariectomy by increasing Sirt1/3, p-AMPK, UCP2, and improving oxidative status. The results provide evidence of the cardioprotective effects of GPER operating through Sirt1/3, p-AMPK, and UCP2, thereby improving cardiac function. Our results suggest that increasing Sirt1/3 levels may offer new therapeutic approaches for postmenopausal diabetic CVD.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Disfunção Ventricular Esquerda , Animais , Feminino , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Estrogênios/farmacologia , Pós-Menopausa/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sirtuína 1/metabolismo , Proteína Desacopladora 2 , Disfunção Ventricular Esquerda/metabolismoRESUMO
BACKGROUND: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO2MAX and poor muscle fitness. However, the role of Ahnak1 in the aging process remained unknown. Here, we investigated the age-related role of Ahnak1 in murine exercise capacity, mitochondrial function, and contractile function of cardiac and skeletal muscles. METHODS: We employed 15- to 16-month-old female and male Ahnak1-knockout (Ahnak1-KO) and wild-type (WT) mice and performed morphometric, biochemical, and bioenergetics assays to evaluate the effects of Ahnak1 on exercise capacity and mitochondrial morphology and function in cardiomyocytes and tibialis anterior (TA) muscle. A human left ventricular (LV) cardiomyocyte cell line (AC16) was used to investigate the direct role of Ahnak1 in cardiomyocytes. RESULTS: We found that the level of Ahnak1 protein is significantly up-regulated with age in the murine LV (1.9-fold) and TA (1.8-fold) tissues. The suppression of Ahnak1 was associated with improved exercise tolerance, as all aged adult Ahnak1-KO mice (100%) successfully completed the running programme, whereas approximately 31% male and 8% female WT mice could maintain the required running speed and distance. Transmission electron microscopic studies showed that LV and TA tissue specimens of aged adult Ahnak1-KO of both sexes have significantly more enlarged/elongated mitochondria and less small mitochondria compared with WT littermates (P < 0.01 and P < 0.001, respectively) at basal level. Further, we observed a shift in mitochondrial fission/fusion balance towards fusion in cardiomyocytes and TA muscle from aged adult Ahnak1-KO mice. The maximal and reserve respiratory capacities were significantly higher in cardiomyocytes from aged adult Ahnak1-KO mice compared with the WT counterparts (P < 0.05 and P < 0.01, respectively). Cardiomyocyte contractility and fatigue resistance of TA muscles were significantly increased in Ahnak1-KO mice of both sexes, compared with the WT groups. In vitro studies using AC16 cells have confirmed that the alteration of mitochondrial function is indeed a direct effect of Ahnak1. Finally, we presented Ahnak1 as a novel cardiac mitochondrial membrane-associated protein. CONCLUSIONS: Our data suggest that Ahnak1 is involved in age-related cardiac and skeletal muscle dysfunction and could therefore serve as a promising therapeutical target.
Assuntos
Mitocôndrias , Músculo Esquelético , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Dinâmica Mitocondrial , Contração Muscular , Músculo Esquelético/metabolismoRESUMO
Numerous epidemiological, clinical, and animal studies showed that cardiac function and manifestation of cardiovascular diseases (CVDs) are different between males and females. The underlying reasons for these sex differences are definitely multifactorial, but major evidence points to a causal role of the sex steroid hormone 17ß-estradiol (E2) and its receptors (ER) in the physiology and pathophysiology of the heart. Interestingly, it has been shown that cardiac calcium (Ca2+) ion channels and mitochondrial function are regulated in a sex-specific manner. Accurate mitochondrial function and Ca2+ signaling are of utmost importance for adequate heart function and crucial to maintaining the cardiovascular health. Due to the highly sensitive nature of these processes in the heart, this review article highlights the current knowledge regarding sex dimorphisms in the heart implicating the importance of E2 and ERs in the regulation of cardiac mitochondrial function and Ca2+ ion channels, thus the contractility. In particular, we provide an overview of in-vitro and in-vivo studies using either E2 deficiency; ER deficiency or selective ER activation, which suggest that E2 and ERs are strongly involved in these processes. In this context, this review also discusses the divergent E2-responses resulting from the activation of different ER subtypes in these processes. Detailed understanding of the E2 and ER-mediated molecular and cellular mechanisms in the heart under physiological and pathological conditions may help to design more specifically targeted drugs for the management of CVDs in men and women.
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Aims: Sex differences in cardiac fibrosis point to the regulatory role of 17ß-Estradiol (E2) in cardiac fibroblasts (CF). We, therefore, asked whether male and female CF in rodent and human models are differentially susceptible to E2, and whether this is related to sex-specific activation of estrogen receptor alpha (ERα) and beta (ERß). Methods and results: In female rat CF (rCF), 24 h E2-treatment (10-8 M) led to a significant down-regulation of collagen I and III expression, whereas both collagens were up-regulated in male rCF. E2-induced sex-specific collagen regulation was also detected in human CF, indicating that this regulation is conserved across species. Using specific ERα- and ERß-agonists (10-7 M) for 24 h, we identified ERα as repressive and ERß as inducing factor in female and male rCF, respectively. In addition, E2-induced ERα phosphorylation at Ser118 only in female rCF, whereas Ser105 phosphorylation of ERß was exclusively found in male rCF. Further, in female rCF we found both ER bound to the collagen I and III promoters using chromatin immunoprecipitation assays. In contrast, in male rCF only ERß bound to both promoters. In engineered connective tissues (ECT) from rCF, collagen I and III mRNA were down-regulated in female ECT and up-regulated in male ECT by E2. This was accompanied by an impaired condensation of female ECT, whereas male ECT showed an increased condensation and stiffness upon E2-treatment, analysed by rheological measurements. Finally, we confirmed the E2-effect on both collagens in an in vivo mouse model with ovariectomy for E2 depletion, E2 substitution, and pressure overload by transverse aortic constriction. Conclusion: The mechanism underlying the sex-specific regulation of collagen I and III in the heart appears to involve E2-mediated differential ERα and ERß signaling in CFs.
Assuntos
Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Estradiol/análogos & derivados , Estrogênios/farmacologia , Fibroblastos/efeitos dos fármacos , Cardiopatias/metabolismo , Miocárdio/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Idoso , Animais , Sítios de Ligação , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Modelos Animais de Doenças , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Cardiopatias/genética , Cardiopatias/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miocárdio/patologia , Fosforilação , Regiões Promotoras Genéticas , Ratos Wistar , Receptores de Estrogênio/metabolismo , Fatores Sexuais , Transdução de Sinais , Regulação para CimaRESUMO
Epidemiological, clinical and animal studies revealed that sex differences exist in the manifestation and outcome of cardiovascular disease (CVD). The underlying molecular mechanisms implicated in these sex differences are not fully understood. The reasons for sex differences in CVD are definitely multifactorial, but major evidence points to the contribution of sex steroid hormone, 17ß-estradiol (E2), and its receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß). In this review, we summarize past and present studies that implicate E2 and ER as important determinants of sexual dimorphism in the physiology and pathophysiology of the heart. In particular, we give an overview of studies aimed to reveal the role of E2 and ER in the physiology of the observed sex differences in CVD using ER knock-out mice. Finally, we discuss recent findings from novel transgenic mouse models, which have provided new information on the sexual dimorphic roles of ER specifically in cardiomyocytes under pathological conditions.
Assuntos
Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Estradiol/análise , Estradiol/genética , Estrogênios/análise , Estrogênios/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Caracteres SexuaisRESUMO
Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17ß-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.
Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica/genética , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/biossíntese , Animais , Western Blotting , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Reação em Cadeia da Polimerase , Técnicas do Sistema de Duplo-HíbridoRESUMO
ERß (oestrogen receptor ß) activation has been shown to be cardioprotective, but the cell types and mechanisms involved are not understood. To investigate whether ERß restricted to cardiomyocytes contributes to the observed cardioprotection, we tested the effects of cardiomyocyte-specific ERß-OE (ERß overexpression) on survival, cardiac remodelling and function after MI (myocardial infarction) and studied the molecular pathways potentially involved. Female and male mice with cardiomyocyte-specific ERß-OE and WT (wild-type) littermates were subjected to chronic anterior coronary artery ligation or sham surgery. Two weeks after MI, ERß-OE mice showed improved survival (100% and 83% compared with 76% and 58% in WT females and males respectively). ERß-OE was associated with attenuated LV (left ventricular) dilatation, smaller increase in heart weight, less lung congestion at similar MI size, and improved systolic and diastolic function in both sexes. We identified two potential pathways for ERß-mediated myocardial protection. First, male and female ERß-OE mice had a lower reduction of SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a) expression after MI, suggesting less reduction in diastolic Ca(2+)-reuptake into the sarcoplasmic reticulum post-MI. Secondly, male ERß-OE revealed attenuated cardiac fibrosis in the remote LV tissue and expression of fibrosis markers collagen I and III, periostin and miR-21. Cardiomyocyte-specific ERß-OE improved survival associated with reduced maladaptive remodelling, improved cardiac function and less heart failure development after MI in both sexes. These effects seem to be related, at least in part, to a better maintenance of Ca(2+) cycling in both sexes and a lower induction of cardiac fibrosis in males after MI.
Assuntos
Receptor beta de Estrogênio/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Biomarcadores/metabolismo , Diástole/fisiologia , Receptor beta de Estrogênio/fisiologia , Feminino , Fibrose , Masculino , Camundongos Transgênicos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores Sexuais , Sístole/fisiologia , Ultrassonografia , Remodelação Ventricular/fisiologiaRESUMO
Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17ß-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERß-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal "female advantage".
Assuntos
Remodelamento Atrial/efeitos dos fármacos , Estradiol/farmacologia , Complexos Multiproteicos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Estradiol/metabolismo , Feminino , Coração/efeitos dos fármacos , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Companheira de mTOR Insensível à Rapamicina , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
INTRODUCTION: Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) may be suitable for myocardial repair. While their functional and structural properties have been extensively investigated, their response to ischemia-like conditions has not yet been clearly defined. METHODS: iPS-CMs were differentiated and enriched from murine induced pluripotent stem cells expressing enhanced green fluorescent protein (eGFP) and puromycin resistance genes under the control of an α-myosin heavy chain (α-MHC) promoter. iPS-CMs maturity and function were characterized by microscopy, real-time PCR, calcium transient recordings, electrophysiology, and mitochondrial function assays, and compared to those from neonatal murine cardiomyocytes. iPS-CMs as well as neonatal murine cardiomyocytes were exposed for 3 hours to hypoxia (1% O2) and glucose/serum deprivation, and viability, apoptosis markers, reactive oxygen species, mitochondrial membrane potential and intracellular stress signaling cascades were investigated. Then, the iPS-CMs response to mesenchymal stromal cell-conditioned medium was determined. RESULTS: iPS-CMs displayed key morphological and functional properties that were comparable to those of neonatal cardiomyocytes, but several parameters indicated an earlier iPS-CMs maturation stage. During hypoxia and glucose/serum deprivation, iPS-CMs exhibited a significantly higher proportion of poly-caspase-active, 7-aminoactinomycin D-positive and TUNEL-positive cells than neonatal cardiomyocytes. The average mitochondrial membrane potential was reduced in "ischemic" iPS-CMs but remained unchanged in neonatal cardiomyocytes; reactive oxygen species production was only increased in "ischemic" iPS-CMs, and oxidoreductase activity in iPS-CMs dropped more rapidly than in neonatal cardiomyocytes. In iPS-CMs, hypoxia and glucose/serum deprivation led to upregulation of Hsp70 transcripts and decreased STAT3 phosphorylation and total PKCε protein expression. Treatment with mesenchymal stromal cell-conditioned medium preserved oxidoreductase activity and restored pSTAT3 and PKCε levels. CONCLUSION: iPS-CMs appear to be particularly sensitive to hypoxia and nutrient deprivation. Counteracting the ischemic susceptibility of iPS-CMs with mesenchymal stromal cell-conditioned medium may help enhance their survival and efficacy in cell-based approaches for myocardial repair.
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Hipóxia Celular , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Quinase C-épsilon/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Experimental studies showed that 17ß-estradiol (E2) and activated Estrogen Receptors (ER) protect the heart from ischemic injury. However, the underlying molecular mechanisms are not well understood. To investigate the role of ER-alpha (ERα) in cardiomyocytes in the setting of myocardial ischemia, we generated transgenic mice with cardiomyocyte-specific overexpression of ERα (ERα-OE) and subjected them to Myocardial Infarction (MI). At the basal level, female and male ERα-OE mice showed increased Left Ventricular (LV) mass, LV volume and cardiomyocyte length. Two weeks after MI, LV volume was significantly increased and LV wall thickness decreased in female and male WT-mice and male ERα-OE, but not in female ERα-OE mice. ERα-OE enhanced expression of angiogenesis and lymphangiogenesis markers (Vegf, Lyve-1), and neovascularization in the peri-infarct area in both sexes. However, attenuated level of fibrosis and higher phosphorylation of JNK signaling pathway could be detected only in female ERα-OE after MI. In conclusion, our study indicates that ERα protects female mouse cardiomyocytes from the sequelae of ischemia through induction of neovascularization in a paracrine fashion and impaired fibrosis, which together may contribute to the attenuation of cardiac remodelling.
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The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 µm/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 µm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 µm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3±3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1(E56G)≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.
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Coração/fisiologia , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Animais , Módulo de Elasticidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Proteínas Motores Moleculares/ultraestrutura , Cadeias Leves de Miosina/ultraestrutura , Relação Estrutura-Atividade , Resistência à Tração/fisiologiaRESUMO
Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.
Assuntos
Actinas/metabolismo , Miosinas Atriais/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosinas Ventriculares/metabolismo , Actinas/química , Actinas/genética , Miosinas Atriais/química , Miosinas Atriais/genética , Dicroísmo Circular , Humanos , Contração Miocárdica , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Miosinas Ventriculares/química , Miosinas Ventriculares/genéticaRESUMO
AIMS: Oestrogen receptor alpha (ERα) and beta (ERß) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action. METHODS AND RESULTS: We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERß-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERß-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERß deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERß-deleted mice. CONCLUSION: The sex-specific response of the heart to exercise is modulated by ERß. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERß.
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Cardiomegalia/etiologia , Receptor beta de Estrogênio/fisiologia , Condicionamento Físico Animal , Adaptação Fisiológica , Animais , Células Cultivadas , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores de Estrogênio/fisiologia , Caracteres Sexuais , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: 17ß-Oestradiol (E2) and its receptors (ERα and ERß) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. METHODS AND RESULTS: Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. CONCLUSION: We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.
Assuntos
Fator Natriurético Atrial/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Miócitos Cardíacos/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Successful reduction of body weight (BW) is often followed by recidivism to obesity. BW-changes including BW-loss and -regain is associated with marked alterations in energy expenditure (EE) and adipose tissue (AT) metabolism. Since these processes are sex-specifically controlled, we investigated sexual dimorphisms in metabolic processes during BW-dynamics (gain-loss-regain). RESEARCH DESIGN: Obesity was induced in C57BL/6J male (m) and female (f) mice by 15 weeks high-fat diet (HFD) feeding. Subsequently BW was reduced (-20%) by caloric restriction (CR) followed by adaptive feeding, and a regain-phase. Measurement of EE, body composition, blood/organ sampling were performed after each feeding period. Lipolysis was analyzed ex-vivo in gonadal AT. RESULTS: Male mice exhibited accelerated BW-gain compared to females (relative BW-gain m:140.5±3.2%; f:103.7±6.5%; p<0.001). In consonance, lean mass-specific EE was significantly higher in females compared to males during BW-gain. Under CR female mice reached their target-BW significantly faster than male mice (m:12.2 days; f:7.6 days; p<0.001) accompanied by a sustained sex-difference in EE. In addition, female mice predominantly downsized gonadal AT whereas the relation between gonadal and total body fat was not altered in males. Accordingly, only females exhibited an increased rate of forskolin-stimulated lipolysis in AT associated with significantly higher glycerol concentrations, lower RER-values, and increased AT expression of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Analysis of AT lipolysis in estrogen receptor alpha (ERα)-deficient mice revealed a reduced lipolytic rate in the absence of ERα exclusively in females. Finally, re-feeding caused BW-regain faster in males than in females. CONCLUSION: The present study shows sex-specific dynamics during BW-gain-loss-regain. Female mice responded to CR with an increase in lipolytic activity, and augmented lipid-oxidation leading to more efficient weight loss. These processes likely involve ERα-dependent signaling in AT and sexual dimorphic regulation of genes involved in lipid metabolism.
Assuntos
Tecido Adiposo/metabolismo , Peso Corporal , Lipólise , Animais , Dieta Hiperlipídica , Receptor alfa de Estrogênio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Fatores Sexuais , Aumento de Peso , Redução de PesoRESUMO
BACKGROUND: In patients with aortic stenosis, pressure overload induces cardiac hypertrophy and fibrosis. Female sex and estrogens influence cardiac remodeling and fibrosis in animal models and in men. Sex differences and their molecular mechanisms in hypertrophy regression after aortic valve replacement have not yet been studied. METHODS AND RESULTS: We prospectively obtained preoperative and early postoperative echocardiography in 92 patients, 53 women and 39 men, undergoing aortic valve replacement for isolated aortic stenosis. We analyzed in a subgroup of 10 patients matrix gene expression in left ventricular (LV) biopsies. In addition, we determined the effect of 17ß-estradiol on collagen synthesis in isolated rat cardiac fibroblasts. Preoperatively, women and men had similar ejection fraction. Similar percentages of women and men had increased LV diameters (37% and 38%). Women more frequently exhibited LV hypertrophy than men (women: 86%; men: 56%; P<0.01). Postoperatively, increased LV diameters persisted in 34% of men but only in 12% of women (P<0.023). LV hypertrophy reversed more frequently in women than in men, leading to a similar prevalence of LV hypertrophy after surgery (women: 45%; men: 36%). In surgical biopsies, men had significantly higher collagen I and III and matrix metalloproteinase 2 gene expression than women. In isolated rat cardiac fibroblasts, 17ß-estradiol significantly increased collagen I and III gene expressions in male cells but decreased it in female cells. CONCLUSIONS: Women adapt to pressure overload differently from men. Less fibrosis before surgery may enable faster regression after surgery.
Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Estradiol , Estrogênios , Proteínas da Matriz Extracelular/biossíntese , Fibroblastos/metabolismo , Hipertrofia Ventricular Esquerda , Proteínas Musculares/biossíntese , Próteses e Implantes , Caracteres Sexuais , Idoso , Idoso de 80 Anos ou mais , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/complicações , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/cirurgia , Células Cultivadas , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Fibroblastos/patologia , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Ratos , Ratos Wistar , Fatores SexuaisRESUMO
BACKGROUND: Although circulating levels of sexual hormones in elderly men and women are low and quite similar, the adaptation of the elderly heart to stress differs between the sexes. We have hypothesized that the effects of sexual hormones in the heart may differ in men and women. Here, we assessed whether 17ß-oestradiol regulates gene expression in the human heart in a sex-dependent manner. We selected the progesterone receptor as a well studied 17ß-oestradiol target that may be pathologically linked to cardiac remodelling. METHODS: In order to assess the ex vivo effects of 17ß-oestradiol in intact human cardiac tissues, we developed a 24-h model for the culture of human atrial myocardium. We verified tissue viability after 24 h in culture with two standard assays to determine the degree of apoptosis and metabolic activity of cardiac tissues. Progesterone receptor mRNA and protein level were measured after 24-h treatment of tissues with 17ß-oestradiol. Statistical analysis was performed by the Mann-Whitney U test and two-way ANOVA. RESULTS: We established a tissue culture model that allows for the study of viable human cardiac tissue over a 24-h period. After 24 h, cultured cardiac tissues revealed low apoptosis, retained their metabolic activity and, therefore, remained viable. Treatment with 17ß-oestradiol led to an induction of the progesterone receptor mRNA level in female (P = 0.001) but not in male tissues. Similarly, there was an increase in the level of progesterone receptor protein in female tissues (P = 0.03), while a decreasing trend was observed in male tissues (P = 0.079) exposed to 17ß-oestradiol. CONCLUSIONS: Our novel finding may offer a molecular explanation for the sex-specific differences observed in cardiac remodelling. The culture model we established for human cardiac tissue will facilitate the study of cellular processes in health and disease and will be of use for pharmacological testing.
RESUMO
AIMS: Female sex and sex hormones contribute to cardiac remodelling. 17beta-estradiol (E2) is involved in the modulation of extracellular matrix composition and function. Here, we analysed the effect of E2 on matrix metalloproteinase (MMP)-2 gene expression and studied the underlying molecular mechanisms in rat cardiac fibroblasts and in a human fibroblast cell line. METHODS AND RESULTS: In adult rat cardiac fibroblasts, E2 significantly decreased MMP-2 gene expression in an estrogen receptor (ER)-dependent manner. Transient transfection experiments of human MMP-2 (hMMP-2) promoter deletion constructs in a human fibroblast cell line revealed a regulatory region between -324 and -260 bp that is involved in E2/ERalpha-mediated repression of hMMP-2 gene transcription. Electrophoretic mobility shift assays (EMSA) and supershift analysis demonstrated the binding of transcription factor Elk-1 within this promoter region. Elk-1 was phosphorylated by E2 via the mitogen-activated protein kinase (MAPK) signalling pathway as shown by western blotting. Treatment of cells with the MAPK inhibitor PD98059 blocked the E2-dependent repression of hMMP-2 promoter activity as well as the endogenous MMP-2 mRNA levels in both human fibroblast cells and rat cardiac fibroblasts. CONCLUSION: E2 inhibits MMP-2 expression via the ER and the MAPK pathway in rat cardiac fibroblasts and in a human fibroblast cell line. These mechanisms may contribute to sex-specific differences in fibrotic processes that are observed in human heart and other diseases.
Assuntos
Estradiol/metabolismo , Estrogênios/metabolismo , Fibroblastos/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 2 da Matriz/genética , Miocárdio/citologia , Animais , Linhagem Celular , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Inibidores de Metaloproteinases de Matriz , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Remodelação Ventricular/fisiologia , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but the regulation of ER expression in the human heart has not yet been analyzed. In various cell lines and tissues, multiple human estrogen receptor alpha (hERalpha) mRNA isoforms are transcribed from distinct promoters and differ in their 5'-untranslated regions. Using PCR-based strategies, we show that in the human heart the ERalpha mRNA is transcribed from multiple promoters, namely, A, B, C, and F, of which the F-promoter is most frequently used variant. Transient transfection reporter assays in a human cardiac myocyte cell line (AC16) with F-promoter deletion constructs demonstrated a negative regulatory region within this promoter. Site-directed mutagenesis and electrophoretic mobility shift assays indicated that NF-kappaB binds to this region. An inhibition of NF-kappaB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-kappaB expression by tumor necrosis factor-alpha reduced the expression of ERalpha, indicating that the NF-kappaB pathway inhibits expression of ERalpha in human cardiomyocytes. Finally, 17beta-estradiol induced the transcriptional activity of hERalpha promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERalpha expression via activation and subsequent binding of NF-kappaB to the ERalpha F-promoter, and 17beta-estradiol/hERalpha may antagonize the inhibitory effect of NF-kappaB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-kappaB.