RESUMO
BACKGROUND: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. METHODS: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. RESULTS: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. CONCLUSIONS: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.
Assuntos
Cardiomegalia/metabolismo , Proteínas Relacionadas à Folistatina/metabolismo , Proteína Forkhead Box O1/metabolismo , Uridina Quinase/metabolismo , Animais , Cardiomegalia/genética , Proteínas Relacionadas à Folistatina/genética , Proteína Forkhead Box O1/genética , Camundongos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Uridina Quinase/genéticaRESUMO
BACKGROUND: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. METHODS: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. RESULTS: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa2+ uptake impair mitochondrial energetics and contractile function. CONCLUSIONS: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Sistemas CRISPR-Cas , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Técnicas de Inativação de Genes , Células HeLa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Função Ventricular EsquerdaRESUMO
RATIONALE: Sorafenib is an effective treatment for renal cell carcinoma, but recent clinical reports have documented its cardiotoxicity through an unknown mechanism. OBJECTIVE: Determining the mechanism of sorafenib-mediated cardiotoxicity. METHODS AND RESULTS: Mice treated with sorafenib or vehicle for 3 weeks underwent induced myocardial infarction (MI) after 1 week of treatment. Sorafenib markedly decreased 2-week survival relative to vehicle-treated controls, but echocardiography at 1 and 2 weeks post MI detected no differences in cardiac function. Sorafenib-treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. High doses of sorafenib induced necrotic death of isolated myocytes in vitro, but lower doses did not induce myocyte death or affect inotropy. Histological analysis documented increased myocyte cross-sectional area despite smaller heart sizes after sorafenib treatment, further suggesting myocyte loss. Sorafenib caused apoptotic cell death of cardiac- and bone-derived c-kit+ stem cells in vitro and decreased the number of BrdU+ (5-bromo-2'-deoxyuridine+) myocytes detected at the infarct border zone in fixed tissues. Sorafenib had no effect on infarct size, fibrosis, or post-MI neovascularization. When sorafenib-treated animals received metoprolol treatment post MI, the sorafenib-induced increase in post-MI mortality was eliminated, cardiac function was improved, and myocyte loss was ameliorated. CONCLUSIONS: Sorafenib cardiotoxicity results from myocyte necrosis rather than from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cell proliferation by inducing apoptosis exacerbates damage by decreasing endogenous cardiac repair. In the setting of MI, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.
Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/mortalidade , Niacinamida/análogos & derivados , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/farmacologia , Animais , Apoptose/efeitos dos fármacos , Gatos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Metoprolol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Niacinamida/efeitos adversos , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , SorafenibeRESUMO
RATIONALE: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. OBJECTIVE: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin's vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. METHODS AND RESULTS: Adiponectin significantly reduced tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression and attenuated TNFα-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin-induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNFα-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNFα-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. CONCLUSIONS: These results demonstrate for the first time that adiponectin inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.