RESUMO
RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.
Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Miocárdio/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Dependovirus/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Genes Sintéticos , Vetores Genéticos/genética , Coração/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição MEF2/biossíntese , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Polimerase III/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-ß-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-ß-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of ß-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2-5 and GATA4 in stabilized-ß-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/ß-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing ß-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/ß-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.
Assuntos
Cromatina/genética , Fator de Transcrição GATA4/genética , Insuficiência Cardíaca/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , beta Catenina/genética , Adulto , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Progressão da Doença , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Ligação Proteica , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
The role of erythropoietin (Epo) in myocardial repair after infarction remains inconclusive. We observed high Epo receptor (EPOR) expression in cardiac progenitor cells (CPCs). Therefore, we aimed to characterize these cells and elucidate their contribution to myocardial regeneration on Epo stimulation. High EPOR expression was detected during murine embryonic heart development followed by a marked decrease until adulthood. EPOR-positive cells in the adult heart were identified in a CPC-enriched cell population and showed coexpression of stem, mesenchymal, endothelial, and cardiomyogenic cell markers. We focused on the population coexpressing early (TBX5, NKX2.5) and definitive (myosin heavy chain [MHC], cardiac Troponin T [cTNT]) cardiomyocyte markers. Epo increased their proliferation and thus were designated as Epo-responsive MHC expressing cells (EMCs). In vitro, EMCs proliferated and partially differentiated toward cardiomyocyte-like cells. Repetitive Epo administration in mice with myocardial infarction (cumulative dose 4 IU/g) resulted in an increase in cardiac EMCs and cTNT-positive cells in the infarcted area. This was further accompanied by a significant preservation of cardiac function when compared with control mice. Our study characterized an EPO-responsive MHC-expressing cell population in the adult heart. Repetitive, moderate-dose Epo treatment enhanced the proliferation of EMCs resulting in preservation of post-ischemic cardiac function.
Assuntos
Eritropoetina/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Receptores da Eritropoetina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Transdução de SinaisRESUMO
The multiphasic regulation of the Wnt/ß-catenin canonical pathway is essential for cardiogenesis in vivo and in vitro. To achieve tight regulation of the Wnt/ß-catenin signaling, tissue- and cell-specific coactivators and repressors need to be recruited. The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. Using a yeast-two-hybrid screen, we identified four-and-a-half-LIM-domain 2 (FHL2) as a cardiac-specific ß-catenin interaction partner and activator of Wnt/ß-catenin-dependent transcription. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells (ESCs). In this model, stable FHL2 gain-of-function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state. Mechanistically, FHL2 overexpression enhanced nuclear accumulation of ß-catenin and activated Wnt/ß-catenin-dependent transcription leading to sustained upregulation of the early cardiogenic gene Igfbp5. In an alternative P19 cell model, transient FHL2 overexpression led to early activation of Wnt/ß-catenin-dependent transcription, but not sustained high-level of Igfbp5 expression. This resulted in enhanced cardiogenesis. We propose that early Wnt/ß-catenin-dependent transcriptional activation mediated by FHL2 is important for the transition to and expansion of early cardiogenic mesodermal cells. Collectively, our findings offer mechanistic insight into the early cardiogenic code and may be further exploited to enhance cardiac progenitor cell activity in vitro and in vivo.
Assuntos
Células-Tronco Embrionárias/fisiologia , Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Musculares/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , beta Catenina/biossíntese , beta Catenina/metabolismoRESUMO
BACKGROUND: The term Gliomatosis cerebri (GC), a radiology-defined highly infiltrating diffuse glioma, has been abandoned since molecular GC-associated features have not been established yet. METHODS: We conducted a multinational retrospective study of 104 children and adolescents with GC providing comprehensive clinical and (epi-)genetic characterization. RESULTS: Median overall survival (OS) was 15.5 months (interquartile range, 10.9-27.7) with a 2-years survival rate of 28%. Histopathological grading correlated significantly with median OS: CNS WHO grade II: 47.8 months (25.2-55.7); grade III: 15.9 months (11.4-26.3); grade IV: 10.4 months (8.8-14.4). By DNA methylation profiling (n=49), most tumors were classified as pediatric-type diffuse high-grade glioma (pedHGG), H3-/IDH-wildtype (n=31/49, 63.3%) with enriched subclasses pedHGG_RTK2 (n=19), pedHGG_A/B (n=6), and pedHGG_MYCN (n=5), but only one pedHGG_RTK1 case. Within the pedHGG, H3-/IDH-wildtype subgroup, recurrent alterations in EGFR (n=10) and BCOR (n=9) were identified. Additionally, we observed structural aberrations in chromosome 6 in 16/49 tumors (32.7%) across tumor types. In the pedHGG, H3-/IDH-wildtype subgroup TP53 alterations had a significant negative effect on OS. CONCLUSION: Contrary to previous studies, our representative pediatric GC study provides evidence that GC has a strong predilection to arise on the background of specific molecular features (especially pedHGG_RTK2, pedHGG_A/B, EGFR and BCOR mutations, chromosome 6 rearrangements).
RESUMO
A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sistemas do Segundo Mensageiro/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Doença Crônica , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
Pancreatic ß-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Immunoblotting , Células Secretoras de Insulina/metabolismo , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Conventional totally implantable venous access ports (TIVAPs) are not approved for power contrast injections but often remain the only venous access site in oncologic patients. Therefore, these devices can play an important role if patients with a TIVAP are scheduled for a contrast-enhanced computed tomography (ceCT) as vascular access may become more difficult during the course of chemotherapy. PURPOSE: To evaluate the feasibility and safety of power injections in conventional TIVAPs in the forearm and to analyze the feasibility of bolus triggering during CT scans. MATERIAL AND METHODS: In this retrospective study we analyzed 177 power injections in 141 patients with TIVAPs in the forearm. Between October 2008 and March 2010 all patients underwent power injections (1.5 mL/s, 150 psi) via the TIVAP for ceCT because conventional vascular access via a peripheral vein had failed. Adequate functioning and catheter's tip location after injection were evaluated. Peak injection pressure and attenuation levels of aorta, liver and spleen were analyzed and compared with results of 50 patients who were injected via classical peripheral cannulas (3 mL/s, 300 psi). Feasibility of automatic scan initiation was evaluated. In vitro the port was stressed with 5 mL/s (300 psi). RESULTS: One TIVAP showed tip dislocation with catheter rupture. Three (2.1%) devices were explanted owing to assumed infection within 4 weeks after the injection. Mean injection pressure was 121.9 +/-24.1 psi. Triggering with automatic scan initiation succeeded in 13/44 (29.6%) scans. Injection via classical cannulas resulted in significantly higher enhancement (p < 0.05). In vitro the port system tolerated flow rates of up to 5 mL/s, injection pressures of up to 338 psi. CONCLUSION: Power injection is a safe alternative for patients with TIVAPs in the forearm if classic vascular access ultimately fails. Triggering was successful in one-third of the attempts. Image quality in the arterial phase scan may be hampered. In vitro results suggest that the device tolerates even higher flow rates.
Assuntos
Cateterismo Venoso Central , Cateteres de Demora , Meios de Contraste/administração & dosagem , Aumento da Imagem/métodos , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Viabilidade , Feminino , Antebraço , Humanos , Pessoa de Meia-Idade , Radiologia Intervencionista/métodos , Estudos Retrospectivos , Adulto JovemRESUMO
We analyzed the effect of conditional, alphaMHC-dependent genetic beta-catenin depletion and stabilization on cardiac remodeling following experimental infarct. beta-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CT(Deltaex3-6): 24 +/- 1.9%; beta-cat(Deltaex3-6): 30.2 +/- 1.6%, P < 0.001). beta-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: beta-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnT(pos) cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by alphaMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (alphaMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kit(pos) and stem cell antigen-1 (Sca-1)(pos) precursor cell population but not with the Islet-1(pos) precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1(pos) cardiac precursor cells from beta-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward alpha-actin(pos) and cTnT(pos) cardiomyocytes after 10 days (CT(Deltaex3-6): 38.0 +/- 1.0% alpha-actin(pos); beta-cat(Deltaex3-6): 49.9 +/- 2.4% alpha-actin(pos), P < 0.001). We conclude that beta-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4(pos)/Sca-1(pos) resident cardiac progenitor cells.
Assuntos
Mioblastos Cardíacos/fisiologia , Infarto do Miocárdio/metabolismo , Regeneração , Remodelação Ventricular , beta Catenina/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células , Regulação para Baixo , Genes Reporter , Camundongos , Camundongos Transgênicos , Mioblastos Cardíacos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Regeneração/genética , Remodelação Ventricular/genética , beta Catenina/genética , beta-Galactosidase/genéticaRESUMO
AIMS: Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias. METHODS AND RESULTS: We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, vTbx5KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA-sequencing of a ventricular-specific TBX5KO mouse and TBX5 chromatin immunoprecipitation was used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in vTbx5KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1, Kcnj5, Kcng2, Cacna1g, Chrm2), in cytoskeleton organization (Fstl4, Pdlim4, Emilin2, Cmya5), and cardiac protection upon stress (Fhl2, Gpr22, Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5KO mice, TBX5 protein-level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced. CONCLUSIONS: This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia.
Assuntos
Arritmias Cardíacas/prevenção & controle , Morte Súbita Cardíaca/prevenção & controle , Ventrículos do Coração/metabolismo , Hipertrofia Ventricular Esquerda/terapia , Proteínas com Domínio T/metabolismo , Disfunção Ventricular Esquerda/terapia , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Sequenciamento de Cromatina por Imunoprecipitação , Morte Súbita Cardíaca/etiologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Terapia Genética , Frequência Cardíaca , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Preparação de Coração Isolado , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA-Seq , Proteínas com Domínio T/genética , Transcrição Gênica , Transcriptoma , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda , Remodelação VentricularRESUMO
The armadillo-related protein beta-catenin has multiple functions in cardiac tissue homeostasis: stabilization of beta-catenin has been implicated in adult cardiac hypertrophy, and downregulation initiates heart formation in embryogenesis. The protein is also part of the cadherin/catenin complex at the cell membrane, where depletion might result in disturbed cell-cell interaction similar to N-cadherin knockout models. Here, we analyzed the in vivo role of beta-catenin in adult cardiac hypertrophy initiated by angiotensin II (Ang II). The cardiac-specific mifepristone-inducible alphaMHC-CrePR1 transgene was used to induce beta-catenin depletion (loxP-flanked exons 3 to 6, beta-cat(Deltaex3-6) mice) or stabilization (loxP-flanked exon 3, beta-cat(Deltaex3) mice). Levels of beta-catenin were altered both in membrane and nuclear extracts. Analysis of the beta-catenin target genes Axin2 and Tcf-4 confirmed increased beta-catenin-dependent transcription in beta-catenin stabilized mice. In both models, transgenic mice were viable and healthy at age 6 months. beta-Catenin appeared dispensable for cell membrane function. Ang II infusion induced cardiac hypertrophy both in wild-type mice and in mice with beta-catenin depletion. In contrast, mice with stabilized beta-catenin had decreased cross-sectional area at baseline and an abrogated hypertrophic response to Ang II infusion. Stabilizing beta-catenin led to impaired fractional shortening compared with control littermates after Ang II stimulation. This functional deterioration was associated with altered expression of the T-box proteins Tbx5 and Tbx20 at baseline and after Ang II stimulation. In addition, atrophy-related protein IGFBP5 was upregulated in beta-catenin-stabilized mice. These data suggest that beta-catenin downregulation is required for adaptive cardiac hypertrophy.
Assuntos
Remodelação Ventricular , beta Catenina/fisiologia , Angiotensina II/farmacologia , Animais , Cardiomegalia/etiologia , Regulação da Expressão Gênica , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas com Domínio T/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
BACKGROUND: The combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. OBJECTIVES: This study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. METHODS: Transcriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells and engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. RESULTS: The authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. CONCLUSIONS: The authors unraveled a network interplay defined by KLF15-Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF.
Assuntos
Insuficiência Cardíaca/metabolismo , Fatores de Transcrição Kruppel-Like/deficiência , Proteínas de Membrana/biossíntese , Regulação para Cima/fisiologia , Remodelação Ventricular/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Krueppel-like factor 15 (KLF15) is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO) line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/métodos , Técnicas de Inativação de Genes , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Miócitos Cardíacos/citologia , Proteínas Nucleares/genética , Linhagem Celular , Homozigoto , HumanosRESUMO
Wnt/ß-catenin signalling controls adult heart remodelling in part via regulation of cardiac progenitor cell (CPC) differentiation. An enhanced understanding of mechanisms controlling CPC biology might facilitate the development of new therapeutic strategies in heart failure. We identified and characterized a novel cardiac interaction between Krueppel-like factor 15 and components of the Wnt/ß-catenin pathway leading to inhibition of transcription. In vitro mutation, reporter assays and co-localization analyses revealed that KLF15 requires both the C-terminus, necessary for nuclear localization, and a minimal N-terminal regulatory region to inhibit transcription. In line with this, functional Klf15 knock-out mice exhibited cardiac ß-catenin transcriptional activation along with functional cardiac deterioration in normal homeostasis and upon hypertrophy. We further provide in vivo and in vitro evidences for preferential endothelial lineage differentiation of CPCs upon KLF15 deletion. Via inhibition of ß-catenin transcription, KLF15 controls CPC homeostasis in the adult heart similar to embryonic cardiogenesis. This knowledge may provide a tool for reactivation of this apparently dormant CPC population in the adult heart and thus be an attractive approach to enhance endogenous cardiac repair.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Coração/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/biossíntese , beta Catenina/biossíntese , Animais , Regulação para Baixo , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Transcrição Gênica , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt , beta Catenina/antagonistas & inibidoresRESUMO
AIMS: We have previously shown that cardiac-specific inhibition of NF-kappaB attenuates angiotensin II (AngII)-induced left ventricular (LV) hypertrophy in vivo. We now tested whether NF-kappaB inhibition is able to block LV remodelling upon chronic pressure overload and chronic AngII stimulation. METHODS AND RESULTS: Cardiac-restricted NF-kappaB inhibition was achieved by expression of a stabilized IkappaBalpha mutant (IkappaBalphaDeltaN) in cells with an active alpha-myosin heavy chain (alphaMHC) promoter employing the Cre/lox technique. Upon low-gradient trans-aortic constriction (TAC, gradient 21 +/- 3 mmHg), hypertrophy was induced in both male and female control mice after 4 weeks. At this time, LV hypertrophy was blocked in transgenic (TG) male but not female mice with NF-kappaB inhibition. Amelioration of LV hypertrophy was associated with activation of NF-kappaB by dihydrotestosterone in isolated neonatal cardiomyocytes. LV remodelling was not attenuated by NF-kappaB inhibition after 8 weeks TAC, demonstrated by decreased fractional shortening (FS) in both control and TG mice irrespective of gender. Similar results were obtained when TAC was performed with higher gradients (48 +/- 4 mmHg). In TG mice, FS dropped to similar low levels over the same time course [FS sham, 29 +/- 1% (mean +/- SEM); FS control + 14 days TAC, 13 +/- 3%; FS TG + 14 days TAC, 9 +/- 5%]. Similarly, LV remodelling was accelerated by NF-kappaB inhibition in an AngII-dependent genetic heart failure model (AT1-R(alphaMHC)) associated with significantly increased cardiac fibrosis in double AT1-R(alphaMHC)/TG mice. CONCLUSION: NF-kappaB inhibition attenuates cardiac hypertrophy in a gender-specific manner but does not alter the course of stress-induced LV remodelling, indicating NF-kappaB to be required for adaptive cardiac hypertrophy.