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1.
J Mol Cell Cardiol ; 145: 59-73, 2020 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-32553594

RESUMO

Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.

2.
Diabetes Care ; 43(5): 1033-1040, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32161048

RESUMO

OBJECTIVE: To assess whether the risk of subsequent lower-limb amputations and death following an initial toe amputation among individuals with diabetes has changed over time and varies by demographic characteristics and geographic region. RESEARCH DESIGN AND METHODS: Using Veterans Health Administration (VHA) electronic medical records from 1 October 2004 to 30 September 2016, we determined risk of subsequent ipsilateral minor and major amputation within 1 year after an initial toe/ray amputation among veterans with diabetes. To assess changes in the annual rate of subsequent amputation over time, we estimated age-adjusted incidence of minor and major subsequent ipsilateral amputation for each year, separately for African Americans (AAs) and whites. Geographic variation was assessed across VHA markets (n = 89) using log-linear Poisson regression models adjusting for age and ethnoracial category. RESULTS: Among 17,786 individuals who had an initial toe amputation, 34% had another amputation on the same limb within 1 year, including 10% who had a major ipsilateral amputation. Median time to subsequent ipsilateral amputation (minor or major) was 36 days. One-year risk of subsequent major amputation decreased over time, but risk of subsequent minor amputation did not. Risk of subsequent major ipsilateral amputation was higher in AAs than whites. After adjusting for age and ethnoracial category, 1-year risk of major subsequent amputation varied fivefold across VHA markets. CONCLUSIONS: Nearly one-third of individuals require reamputation following an initial toe amputation, although risks of subsequent major ipsilateral amputation have decreased over time. Nevertheless, risks remain particularly high for AAs and vary substantially geographically.

3.
Circ Res ; 126(7): 907-922, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32081062

RESUMO

RATIONALE: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. OBJECTIVE: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. CONCLUSIONS: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

4.
Arch Toxicol ; 93(4): 1051-1065, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30810770

RESUMO

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

5.
ASAIO J ; 65(7): 744-752, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30153196

RESUMO

Lack of an ideal patch material for cardiac repairs continues to challenge congenital heart surgeons. The current materials are unable to grow and result in scarring, contraction, and arrhythmias. An acellular extracellular matrix (ECM) patch derived from porcine small intestinal submucosa has demonstrated remodeling potential when used to repair various tissues. This study investigated the in vivo electrophysiologic, mechanical, and histological properties of an ECM patch used to repair a right-ventricular (RV) wall defect in a growing ovine model. A full-thickness, 2 × 2 cm RV defect was created in 11 juvenile sheep and repaired with an ECM patch. Longitudinal RV three-dimensional-electrical mapping, magnetic resonance imaging (MRI), and histological analysis were performed at 3, 6, 9, and 12 months. Three-dimensional mapping demonstrated consistent conduction across the patch with little to no difference in voltage, but conduction velocity was consistently less than native myocardium. Magnetic resonance imaging revealed changing strain properties of the patch which by 9-12 months resembled native tissue. Histologic analysis at 3 months demonstrates cardiomyocyte degeneration and partial replacement via proliferation of connective tissue cells that were predominately fibroblasts and smooth muscle cells. There was marked neovascularization and an absence of calcification at 12 months. Over time, the ECM patch remained viable with stable muscle at the edges. In growing sheep, an ECM patch becomes a viable tissue and remains so up to at least a year. Although ECM demonstrates some functional aspects of remodeling to native myocardium, histologically it remained immature.

6.
Circ Res ; 123(12): 1285-1297, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30566042

RESUMO

RATIONALE: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. OBJECTIVE: TGFß (transforming growth factor-ß) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFß signaling during chronic Mybpc340kDa expression. METHODS AND RESULTS: To specifically block TGFß signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFß receptor II (TßRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. CONCLUSIONS: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFß signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.


Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/genética , Miócitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Transdução de Sinais , Animais , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Camundongos , Mutação , Receptor do Fator de Crescimento Transformador beta Tipo II/genética
7.
J Am Heart Assoc ; 7(20): e010013, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30371263

RESUMO

Background Transforming growth factor beta ( TGF -ß) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte-specific expression of a mutant αB-crystallin (Cry ABR 120G), which causes human desmin-related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so-called myofibroblast state and TGF -ß binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast-based TGF -ß signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte-based etiology. Methods and Results Against the background of cardiomyocyte-restricted expression of Cry ABR 120G, we have partially ablated TGF -ß signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry ABR 120G production. Transgenic Cry ABR 120G mice were crossed with mice containing a floxed allele of TGF -ß receptor 2 ( Tgfbr2 f/f). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry ABR 120G mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF -ß signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry ABR 120G. Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast-targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.


Assuntos
Miocárdio/patologia , Miofibroblastos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Análise de Variância , Animais , Cardiomiopatias/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/fisiologia , Fibrose/etiologia , Cardiopatias/patologia , Masculino , Camundongos Transgênicos , Distrofias Musculares/patologia , Miócitos Cardíacos/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Transdução de Sinais/fisiologia , Cadeia B de alfa-Cristalina/metabolismo
8.
J Am Heart Assoc ; 7(20): e009775, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30371279

RESUMO

Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1-/-). Methods and Results Sigmar1-/- hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a ß1-adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 -/- hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 -/- hearts compared with wild-type hearts. Ultrastructural analysis of Sigmar1-/- cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1-/- hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1-/- hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1-/- hearts compared with wild-type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.


Assuntos
Cardiopatias/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , Dinâmica Mitocondrial/fisiologia , Receptores sigma/metabolismo , Trifosfato de Adenosina/metabolismo , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Biomarcadores/metabolismo , Respiração Celular/fisiologia , Dobutamina/farmacocinética , Ecocardiografia , Transporte de Elétrons/fisiologia , Metabolismo Energético/fisiologia , Feminino , Fibrose/fisiopatologia , Hemodinâmica/fisiologia , Masculino , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias Cardíacas/ultraestrutura , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Contração Miocárdica/fisiologia , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/ultraestrutura , Consumo de Oxigênio/fisiologia
9.
Proc Natl Acad Sci U S A ; 115(44): 11238-11243, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30322937

RESUMO

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human ß-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999-1006.]. Since then, more than 300 mutations in the ß-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or ß-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a ß-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force-velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in ß-cardiac myosin.


Assuntos
Cardiomiopatia Hipertrófica/genética , Contração Miocárdica/genética , Miofibrilas/genética , Cadeias Pesadas de Miosina/genética , Miosinas/genética , Mutação Puntual/genética , Actinas/genética , Animais , Animais Geneticamente Modificados/genética , Ventrículos do Coração/metabolismo , Camundongos , Miocárdio/metabolismo , Coelhos
10.
J Am Heart Assoc ; 7(14)2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29987122

RESUMO

BACKGROUND: Desmin filament proteins interlink the contractile myofibrillar apparatus with mitochondria, nuclei and the sarcolemma. Mutations in the human desmin gene cause cardiac disease, remodeling, and heart failure but the pathophysiological mechanisms remain unknown. METHODS AND RESULTS: Cardiomyocyte-specific overexpression of mutated desmin (a 7 amino acid deletion R172-E178, D7-Des Tg) causes accumulations of electron-dense aggregates and myofibrillar degeneration associated with cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause impairment of cardiac contractility, the molecular mechanism of cardiomyocyte death remains elusive. In the present study, we report that the D7-Des Tg mouse hearts undergo aberrant mitochondrial fission associated with increased expression of mitochondrial fission regulatory proteins. Mitochondria isolated from D7-Des Tg hearts showed decreased mitochondrial respiration and increased apoptotic cell death. Overexpression of mutant desmin by adenoviral infection in cultured cardiomyocytes led to increased mitochondrial fission, inhibition of mitochondrial respiration, and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des overexpression in cardiomyocytes. CONCLUSIONS: Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Taken together, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.


Assuntos
Cardiomiopatias/genética , DNA/genética , Desmina/genética , Mitocôndrias Cardíacas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Células Cultivadas , Análise Mutacional de DNA , Desmina/metabolismo , Modelos Animais de Doenças , Imuno-Histoquímica , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley
13.
Circ Heart Fail ; 10(10)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29021349

RESUMO

BACKGROUND: Alterations in autophagy have been reported in hypertrophic cardiomyopathy (HCM) caused by Danon disease, Vici syndrome, or LEOPARD syndrome, but not in HCM caused by mutations in genes encoding sarcomeric proteins, which account for most of HCM cases. MYBPC3, encoding cMyBP-C (cardiac myosin-binding protein C), is the most frequently mutated HCM gene. METHODS AND RESULTS: We evaluated autophagy in patients with HCM carrying MYBPC3 mutations and in a Mybpc3-targeted knockin HCM mouse model, as well as the effect of autophagy modulators on the development of cardiomyopathy in knockin mice. Microtubule-associated protein 1 light chain 3 (LC3)-II protein levels were higher in HCM septal myectomies than in nonfailing control hearts and in 60-week-old knockin than in wild-type mouse hearts. In contrast to wild-type, autophagic flux was blunted and associated with accumulation of residual bodies and glycogen in hearts of 60-week-old knockin mice. We found that Akt-mTORC1 (mammalian target of rapamycin complex 1) signaling was increased, and treatment with 2.24 mg/kg·d rapamycin or 40% caloric restriction for 9 weeks partially rescued cardiomyopathy or heart failure and restored autophagic flux in knockin mice. CONCLUSIONS: Altogether, we found that (1) autophagy is altered in patients with HCM carrying MYBPC3 mutations, (2) autophagy is impaired in Mybpc3-targeted knockin mice, and (3) activation of autophagy ameliorated the cardiac disease phenotype in this mouse model. We propose that activation of autophagy might be an attractive option alone or in combination with another therapy to rescue HCM caused by MYBPC3 mutations.


Assuntos
Autofagia/fisiologia , Cardiomiopatias/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Mutação/genética , Miocárdio/metabolismo , Animais , Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos Transgênicos , Fenótipo
14.
J Am Heart Assoc ; 6(8)2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28862927

RESUMO

BACKGROUND: Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid ß-protein, tau, and α-synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation-prone proteins and thereby disrupting aggregate formation. However, the effects of CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated αB-crystallin (CryABR120G). METHODS AND RESULTS: A proteotoxic model of desmin-related cardiomyopathy caused by cardiomyocyte-specific expression of CryABR120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild-type CryAB or CryABR120G. Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryABR120G expression in a dose-dependent manner, whereas CLR03 had no effect. Ubiquitin-proteasome system function was analyzed using a ubiquitin-proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein. CLR01 improved proteasomal function in CryABR120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryABR120G transgenic mice. CONCLUSIONS: CLR01 can inhibit CryABR120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress, presumably through clearance of the misfolded protein via increased proteasomal function. CLR01 or related compounds may be therapeutically useful in treating the pathogenic sequelae resulting from proteotoxic heart disease.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Organofosfatos/farmacologia , Agregação Patológica de Proteínas , Cadeia B de alfa-Cristalina/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Vetores Genéticos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Compostos Organofosforados/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteólise , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdução Genética , Transfecção , Ubiquitinação , Cadeia B de alfa-Cristalina/genética
15.
J Am Heart Assoc ; 6(9)2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28871043

RESUMO

BACKGROUND: Cardiac stress can trigger production of a 40-kDa peptide fragment derived from the amino terminus of the cardiac myosin-binding protein C. Cardiac stress, as well as cMyBP-C mutations, can trigger production of 1 such truncated protein fragment, a 40-kDa peptide fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40-kDa fragment in mouse cardiomyocytes (cMyBP-C40k) leads to cardiac disease, fibrosis, and death within the first year. Fibrosis can occur in many cardiovascular diseases, and mitogen-activated protein kinase--activated protein kinase-2 signaling has been implicated in a variety of fibrotic processes. Recent studies demonstrated that mitogen-activated protein kinase--activated protein kinase-2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C40k cardiomyocyte expression. METHODS AND RESULTS: Nontransgenic and cMyBP-C40k inducible transgenic mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long-term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy, or probability of survival. In the inducible transgenic group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy, and prolonged survival. CONCLUSIONS: Pharmaceutical inhibition of mitogen-activated protein kinase--activated protein kinase-2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic cMyBPC40k disease.


Assuntos
Cardiomiopatias/prevenção & controle , Proteínas de Transporte/metabolismo , Hipertrofia Ventricular Esquerda/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Remodelação Ventricular/efeitos dos fármacos , Actinas/metabolismo , Animais , Cardiomiopatias/enzimologia , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima
16.
Am J Physiol Renal Physiol ; 313(3): F699-F705, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28679593

RESUMO

The early events that signal renal dysfunction in presymptomatic heart failure are unclear. We tested the hypothesis that functional and mechanistic changes occur in the kidney that precede the development of symptomatic heart failure. We employed a transgenic mouse model with cardiomyocyte-specific overexpression of mutant α-B-crystallin that develops slowly progressive cardiomyopathy. Presymptomatic transgenic mice displayed an increase in serum creatinine (1.17 ± 0.34 vs. wild type 0.65 ± 0.16 mg/dl, P < 0.05) and in urinary neutrophil gelatinase-associated lipocalin (NGAL; 278.92 ± 176.24 vs. wild type 49.11 ± 22.79 ng/ml, P < 0.05) but no renal fibrosis. Presymptomatic transgenic mouse kidneys exhibited a twofold upregulation of the Ren1 gene, marked overexpression of renin protein in the tubules, and a worsened response to ischemia-reperfusion injury based on serum creatinine (2.77 ± 0.66 in transgenic mice vs. 2.01 ± 0.58 mg/dl in wild type, P < 0.05), urine NGAL (9,198.79 ± 3,799.52 in transgenic mice vs. 3,252.94 ± 2,420.36 ng/ml in wild type, P < 0.05), tubule dilation score (3.4 ± 0.5 in transgenic mice vs. 2.6 ± 0.5 in wild type, P < 0.05), tubule cast score (3.2 ± 0.4 in transgenic mice vs. 2.5 ± 0.5 in wild type, P < 0.05), and TdT-mediated dUTP nick-end labeling (TUNEL)-positive nuclei (10.1 ± 2.1 in the transgenic group vs. 5.7 ± 1.6 per 100 cells counted in wild type, P < 0.01). Our findings indicate functional renal impairment, urinary biomarker elevations, and induction of renin gene and protein expression in the kidney that occur in early presymptomatic heart failure, which increase the susceptibility to subsequent acute kidney injury.


Assuntos
Lesão Renal Aguda/etiologia , Síndrome Cardiorrenal/etiologia , Cardiomiopatias/etiologia , Insuficiência Cardíaca/etiologia , Rim/patologia , Traumatismo por Reperfusão/etiologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Lesão Renal Aguda/fisiopatologia , Animais , Doenças Assintomáticas , Biomarcadores/urina , Síndrome Cardiorrenal/genética , Síndrome Cardiorrenal/patologia , Síndrome Cardiorrenal/fisiopatologia , Cardiomiopatias/genética , Creatinina/urina , Modelos Animais de Doenças , Progressão da Doença , Predisposição Genética para Doença , Insuficiência Cardíaca/genética , Rim/metabolismo , Rim/fisiopatologia , Lipocalina-2/urina , Camundongos Transgênicos , Mutação , Fenótipo , Renina/genética , Renina/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de Tempo , Regulação para Cima , Cadeia B de alfa-Cristalina/genética
17.
Circ Res ; 121(6): 604-616, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28655832

RESUMO

RATIONALE: Postmitotic cells, such as cardiomyocytes, seem to be particularly susceptible to proteotoxic stimuli, and large, proteinaceous deposits are characteristic of the desmin-related cardiomyopathies and crystallin cardiomyopathic diseases. Increased activity of protein clearance pathways in the cardiomyocyte, such as proteasomal degradation and autophagy, has proven to be beneficial in maintaining cellular and cardiac function in the face of multiple proteotoxic insults, holding open the possibility of targeting these processes for the development of effective therapeutics. OBJECTIVE: Here, we undertake an unbiased, total genome screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. METHODS AND RESULTS: Primary mouse cardiomyocytes that accumulate aggregates as a result of a mutant CryAB (αB-crystallin) causative for human desmin-related cardiomyopathy were used for a total genome-wide screen to identify gene products that affected aggregate formation. We infected cardiomyocytes using a short hairpin RNA lentivirus library in which the mouse genome was represented. The screen identified multiple candidates in many cell signaling pathways that were able to mediate significant decreases in aggregate levels. CONCLUSIONS: Subsequent validation of one of these candidates, Jak1 (Janus kinase 1), a tyrosine kinase of the nonreceptor type, confirmed the usefulness of this approach in identifying previously unsuspected players in proteotoxic processes.


Assuntos
Cardiomiopatias/genética , Clonagem Molecular/métodos , Cristalinas/genética , Desmina/genética , Ensaios de Triagem em Larga Escala/métodos , Miócitos Cardíacos/metabolismo , Transcriptoma , Animais , Agregação Celular/genética , Células Cultivadas , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Camundongos , Miócitos Cardíacos/fisiologia , Ratos
18.
Pflugers Arch ; 468(10): 1685-95, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27568194

RESUMO

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.


Assuntos
Proteínas de Transporte/metabolismo , Miocárdio/metabolismo , Miosinas/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Camundongos , Contração Muscular , Mutação , Ligação Proteica , Sarcômeros/metabolismo
19.
Autophagy ; 12(11): 2252-2253, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27573291

RESUMO

Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC. Our data focus on using gain- and loss-of-function approaches to modify UBE2I levels and measure the effects on cardiomyocyte autophagic flux. UBE2I expression does have an impact on macroautophagy/autophagy as increased SUMOylation results in increased autophagy. We show that increased SUMOylation is cardioprotective and can decrease morbidity in proteotoxic cardiac disease.


Assuntos
Autofagia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Humanos , Modelos Biológicos , Ratos , Sumoilação
20.
Circ Res ; 118(12): 1894-905, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27142163

RESUMO

RATIONALE: SUMOylation plays an important role in cardiac function and can be protective against cardiac stress. Recent studies show that SUMOylation is an integral part of the ubiquitin proteasome system, and expression of the small ubiquitin-like modifier (SUMO) E2 enzyme UBC9 improves cardiac protein quality control. However, the precise role of SUMOylation on other protein degradation pathways, particularly autophagy, remains undefined in the heart. OBJECTIVE: To determine whether SUMOylation affects cardiac autophagy and whether this effect is protective in a mouse model of proteotoxic cardiac stress. METHODS AND RESULTS: We modulated expression of UBC9, a SUMO E2 ligase, using gain- and loss-of-function in neonatal rat ventricular cardiomyocytes. UBC9 expression seemed to directly alter autophagic flux. To confirm this effect in vivo, we generated transgenic mice overexpressing UBC9 in cardiomyocytes. These mice have an increased level of SUMOylation at baseline and, in confirmation of the data obtained from neonatal rat ventricular cardiomyocytes, demonstrated increased autophagy, suggesting that increased UBC9-mediated SUMOylation is sufficient to upregulate cardiac autophagy. Finally, we tested the protective role of SUMOylation-mediated autophagy by expressing UBC9 in a model of cardiac proteotoxicity, induced by cardiomyocyte-specific expression of a mutant α-B-crystallin, mutant CryAB (CryAB(R120G)), which shows impaired autophagy. UBC9 overexpression reduced aggregate formation, decreased fibrosis, reduced hypertrophy, and improved cardiac function and survival. CONCLUSIONS: The data showed that increased UBC9-mediated SUMOylation is sufficient to induce relatively high levels of autophagy and may represent a novel strategy for increasing autophagic flux and ameliorating morbidity in proteotoxic cardiac disease.


Assuntos
Cardiomiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Autofagia , Cardiomiopatias/genética , Células Cultivadas , Camundongos , Ratos , Ratos Sprague-Dawley , Enzimas de Conjugação de Ubiquitina/genética , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo
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