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1.
Circulation ; 140(7): 566-579, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31177839

RESUMO

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

2.
Nature ; 568(7752): 351-356, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30971818

RESUMO

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

3.
Circulation ; 139(20): 2342-2357, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-30818997

RESUMO

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

4.
Circ Res ; 122(11): 1545-1554, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29669712

RESUMO

RATIONALE: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. OBJECTIVE: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. METHODS AND RESULTS: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78. CONCLUSIONS: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

5.
J Mol Cell Cardiol ; 118: 110-121, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29518398

RESUMO

AIMS: Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. METHODS AND RESULTS: Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. CONCLUSION: Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

6.
Circulation ; 137(24): 2613-2634, 2018 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-29437120

RESUMO

BACKGROUND: Myocardium irreversibly injured by ischemic stress must be efficiently repaired to maintain tissue integrity and contractile performance. Macrophages play critical roles in this process. These cells transform across a spectrum of phenotypes to accomplish diverse functions ranging from mediating the initial inflammatory responses that clear damaged tissue to subsequent reparative functions that help rebuild replacement tissue. Although macrophage transformation is crucial to myocardial repair, events governing this transformation are poorly understood. METHODS: Here, we set out to determine whether innate immune responses triggered by cytoplasmic DNA play a role. RESULTS: We report that ischemic myocardial injury, along with the resulting release of nucleic acids, activates the recently described cyclic GMP-AMP synthase-stimulator of interferon genes pathway. Animals lacking cyclic GMP-AMP synthase display significantly improved early survival after myocardial infarction and diminished pathological remodeling, including ventricular rupture, enhanced angiogenesis, and preserved ventricular contractile function. Furthermore, cyclic GMP-AMP synthase loss of function abolishes the induction of key inflammatory programs such as inducible nitric oxide synthase and promotes the transformation of macrophages to a reparative phenotype, which results in enhanced repair and improved hemodynamic performance. CONCLUSIONS: These results reveal, for the first time, that the cytosolic DNA receptor cyclic GMP-AMP synthase functions during cardiac ischemia as a pattern recognition receptor in the sterile immune response. Furthermore, we report that this pathway governs macrophage transformation, thereby regulating postinjury cardiac repair. Because modulators of this pathway are currently in clinical use, our findings raise the prospect of new treatment options to combat ischemic heart disease and its progression to heart failure.

7.
Am J Physiol Heart Circ Physiol ; 313(6): H1119-H1129, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28822967

RESUMO

Hypertension is one of the most important risk factors of heart failure. In response to high blood pressure, the left ventricle manifests hypertrophic growth to ameliorate wall stress, which may progress into decompensation and trigger pathological cardiac remodeling. Despite the clinical importance, the temporal dynamics of pathological cardiac growth remain elusive. Here, we took advantage of the puromycin labeling approach to measure the relative rates of protein synthesis as a way to delineate the temporal regulation of cardiac hypertrophic growth. We first identified the optimal treatment conditions for puromycin in neonatal rat ventricular myocyte culture. We went on to demonstrate that myocyte growth reached its peak rate after 8-10 h of growth stimulation. At the in vivo level, with the use of an acute surgical model of pressure-overload stress, we observed the maximal growth rate to occur at day 7 after surgery. Moreover, RNA sequencing analysis supports that the most profound transcriptomic changes occur during the early phase of hypertrophic growth. Our results therefore suggest that cardiac myocytes mount an immediate growth response in reply to pressure overload followed by a gradual return to basal levels of protein synthesis, highlighting the temporal dynamics of pathological cardiac hypertrophic growth.NEW & NOTEWORTHY We determined the optimal conditions of puromycin incorporation in cardiac myocyte culture. We took advantage of this approach to identify the growth dynamics of cardiac myocytes in vitro. We went further to discover the protein synthesis rate in vivo, which provides novel insights about cardiac temporal growth dynamics in response to pressure overload.


Assuntos
Aorta Torácica/fisiopatologia , Pressão Arterial , Cardiomegalia/patologia , Proliferação de Células , Miócitos Cardíacos/patologia , Animais , Animais Recém-Nascidos , Aorta Torácica/cirurgia , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Constrição , Modelos Animais de Doenças , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Biossíntese de Proteínas , Puromicina/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo
8.
Sci Signal ; 9(422): ra34, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27048565

RESUMO

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs), a group of chromatin remodeling enzymes, has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that, as part of mTOR complex 1 (mTORC1), drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2), an mTOR inhibitor, was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes, loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity, and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease.


Assuntos
Cardiomegalia/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Cardiomegalia/genética , Linhagem Celular , Células Cultivadas , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptídeos Cíclicos/farmacologia , Interferência de RNA , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
9.
Nature ; 481(7382): 511-5, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22258505

RESUMO

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.


Assuntos
Autofagia/fisiologia , Glucose/metabolismo , Homeostase , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Adiponectina/sangue , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína Beclina-1 , Células Cultivadas , Gorduras na Dieta/efeitos adversos , Privação de Alimentos/fisiologia , Técnicas de Introdução de Genes , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/prevenção & controle , Teste de Tolerância a Glucose , Homeostase/efeitos dos fármacos , Leptina/sangue , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mutação , Miocárdio/citologia , Fosforilação/genética , Resistência Física/genética , Resistência Física/fisiologia , Esforço Físico/genética , Esforço Físico/fisiologia , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Corrida/fisiologia
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