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The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PGC-1α is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through coactivation of coupled transcription factors, such as ERR, Nrf1, Gabpa, and YY1. We hypothesized that PGC-1α plays an essential role in transcription of TCA cycle genes. Thus, by utilizing localization peaks of PGC-1α to TCA cycle gene promoters, it would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful to understand how those genes are dysregulated in pathological conditions.
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BACKGROUND: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are. METHODS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice. RESULTS: Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in Drp1 MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption. CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.
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Cardiomiopatías , Mitofagia , Animales , Ratones , Autofagia/fisiología , Cardiomiopatías/genética , Dinaminas/genética , Dinaminas/metabolismo , Corazón , Dinámicas Mitocondriales , Mitofagia/fisiología , Obesidad/genéticaRESUMEN
Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.
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Isquemia Miocárdica , Tiorredoxinas , Animales , Ratones , Autofagia , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Cisteína/metabolismo , Disulfuros , Isquemia Miocárdica/genética , Oxidación-Reducción , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Nicotinamide adenine dinucleotide (NAD+) kinase (NADK) phosphorylates NAD+, thereby producing nicotinamide adenine dinucleotide phosphate (NADP). Both NADK genes and the NADP(H)-producing mechanism are evolutionarily conserved among archaea, bacteria, plants and mammals. In mammals, NADK is activated by phosphorylation and protein-protein interaction. Recent studies conducted using genetically altered models validate the essential role of NADK in cellular redox homeostasis and metabolism in multicellular organisms. Here, we describe the evolutionary conservation, molecular properties, and signaling mechanisms and discuss the pathophysiological significance of NADK.
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NAD , Plantas , Animales , NAD/metabolismo , NADP/metabolismo , Plantas/metabolismo , Transducción de Señal , Mamíferos/metabolismoRESUMEN
Aims: PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1 -/-) mice and to investigate the molecular mechanisms of its transcriptional control. Methods and results: Echocardiography showed that ejection fraction and fractional shortening were lower in Perm1 -/- mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1 -/- hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1 -/- hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1's binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2. Conclusion: This study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.
RESUMEN
PERM1 (PGC-1/ERR-induced regulator in muscle 1) is a muscle-specific protein induced by PGC-1 and ERRs. Previous studies have shown that PERM1 promotes mitochondrial biogenesis and metabolism in cardiomyocytes in vitro. However, the role of endogenous PERM1 in the heart remains to be investigated with loss-of-function studies in vivo. We report the generation and characterization of systemic Perm1 knockout (KO) mice. The baseline cardiac phenotype of the homozygous Perm1 KO mice appeared normal. However, RNA-sequencing and unbiased pathway analyses showed that homozygous downregulation of PERM1 leads to downregulation of genes involved in fatty acid and carbohydrate metabolism in the heart. Transcription factor binding site analyses suggested that PPARα and PGC-1α are involved in changes in the gene expression profile. Chromatin immunoprecipitation assays showed that PERM1 interacts with the proximal regions of PPAR response elements (PPREs) in endogenous promoters of genes involved in fatty acid oxidation. Co-immunoprecipitation and reporter gene assays showed that PERM1 promoted transcription via the PPRE, partly in a PPARα and PGC-1α dependent manner. These results suggest that endogenous PERM1 is involved in the transcription of genes involved in fatty acid oxidation through physical interaction with PPARα and PGC-1α in the heart in vivo.
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Metabolismo de los Lípidos , Proteínas Musculares , PPAR alfa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Animales , Ácidos Grasos , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Miocitos Cardíacos , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.
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Cardiomegalia , Miocitos Cardíacos , Proteínas Señalizadoras YAP/metabolismo , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Ciclo del Ácido Cítrico , Transportador de Glucosa de Tipo 1/genética , Glucólisis , Ratones , Miocitos Cardíacos/metabolismoRESUMEN
While mRNA vaccines against SARS-CoV-2 are exceedingly effective in preventing symptomatic infection, their immune response features remain to be clarified. In the present prospective study, 225 healthy individuals in Japan, who received two BNT162b2 doses, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions) with various determinants were examined and the potency of sera against variants of concerns was determined. Significant rise in NT50s was seen in sera on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation seen between NT50s and adverse effects. NT50s and SARS-CoV-2-S1-binding-IgG levels on day 28 post-1st dose and pain scores following the 2nd dose were greater in women than in men. The average half-life of NT50s was ~ 68 days, and 23.6% (49 out of 208 individuals) failed to show detectable neutralizing activity on day 150. While sera from elite-responders (NT50s > 1,500: the top 4% among the participants) potently to moderately blocked all variants of concerns examined, some sera with low NT50s failed to block the B.1.351-beta strain. Since BNT162b2-elicited immunity against SARS-CoV-2 is short, an additional vaccine or other protective measures are needed.
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Vacuna BNT162/efectos adversos , COVID-19/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacuna BNT162/farmacocinética , COVID-19/sangre , Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/farmacocinética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Humanos , Inmunogenicidad Vacunal/inmunología , Pruebas Inmunológicas , Japón , Cinética , Masculino , Persona de Mediana Edad , Estudios Prospectivos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidadRESUMEN
RATIONALE: Obesity-associated cardiomyopathy characterized by hypertrophy and mitochondrial dysfunction. Mitochondrial quality control mechanisms, including mitophagy, are essential for the maintenance of cardiac function in obesity-associated cardiomyopathy. However, autophagic flux peaks at around 6 weeks of high-fat diet (HFD) consumption and declines thereafter. OBJECTIVE: We investigated whether mitophagy is activated during the chronic phase of cardiomyopathy associated with obesity (obesity cardiomyopathy) after general autophagy is downregulated and, if so, what the underlying mechanism and the functional significance are. METHODS AND RESULTS: Mice were fed either a normal diet or a HFD (60 kcal% fat). Mitophagy, evaluated using Mito-Keima, was increased after 3 weeks of HFD consumption and continued to increase after conventional mechanisms of autophagy were inactivated, at least until 24 weeks. HFD consumption time-dependently upregulated both Ser555-phosphorylated Ulk1 (unc-51 like kinase 1) and Rab9 (Ras-related protein Rab-9) in the mitochondrial fraction. Mitochondria were sequestrated by Rab9-positive ring-like structures in cardiomyocytes isolated from mice after 20 weeks of HFD consumption, consistent with the activation of alternative mitophagy. Increases in mitophagy induced by HFD consumption for 20 weeks were abolished in cardiac-specific ulk1 knockout mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. Rab9 S179A knock-in mice, in which alternative mitophagy is selectively suppressed, exhibited impaired mitophagy and more severe cardiac dysfunction than control mice following HFD consumption for 20 weeks. Overexpression of Rab9 in the heart increased mitophagy and protected against cardiac dysfunction during HFD consumption. HFD-induced activation of Rab9-dependent mitophagy was accompanied by upregulation of TFE3 (transcription factor binding to IGHM enhancer 3), which plays an essential role in transcriptional activation of mitophagy. CONCLUSIONS: Ulk1-Rab9-dependent alternative mitophagy is activated during the chronic phase of HFD consumption and serves as an essential mitochondrial quality control mechanism, thereby protecting the heart against obesity cardiomyopathy.
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Cardiomiopatías/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitofagia , Obesidad/complicaciones , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/patología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Thioredoxin 1 (Trx1) is a major antioxidant that acts adaptively to protect the heart during the development of diabetic cardiomyopathy. The molecular mechanism(s) responsible for regulating the Trx1 level and/or activity during diabetic cardiomyopathy is unknown. ß-hydroxybutyrate (ßHB), a major ketone body in mammals, acts as an alternative energy source in cardiomyocytes under stress, but it also appears to be involved in additional mechanisms that protect the heart against stress. ßHB upregulated Trx1 in primary cultured cardiomyocytes in a dose- and a time-dependent manner and a ketogenic diet upregulated Trx1 in the heart. ßHB protected cardiomyocytes against H2O2-induced death, an effect that was abolished in the presence of Trx1 knockdown. ßHB also alleviated the H2O2-induced inhibition of mTOR and AMPK, known targets of Trx1, in a Trx1-dependent manner, suggesting that ßHB potentiates Trx1 function. It has been shown that ßHB is a natural inhibitor of HDAC1 and knockdown of HDAC1 upregulated Trx1 in cardiomyocytes, suggesting that ßHB may upregulate Trx1 through HDAC inhibition. ßHB induced Trx1 acetylation and inhibited Trx1 degradation, suggesting that ßHB-induced inhibition of HDAC1 may stabilize Trx1 through protein acetylation. These results suggest that ßHB potentiates the antioxidant defense in cardiomyocytes through the inhibition of HDAC1 and the increased acetylation and consequent stabilization of Trx1. Thus, modest upregulation of ketone bodies in diabetic hearts may protect the heart through the upregulation of Trx1.
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BACKGROUND: While mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the features of immune response remain to be clarified. METHODS: In the present prospective observational study, 225 healthy individuals in Kumamoto General Hospital, Japan, who received two BNT162b2 doses in February 2021, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT 50 ; assessed using infectious virions and live target cells) with SARS-CoV-2-S1-binding-IgG and -IgM levels, adverse effects (AEs), ages, and genders were examined. The average half-life of neutralizing activity and the average time length for the loss of detectable neutralizing activity were determined and the potency of serums against variants of concerns was also determined. FINDINGS: Significant rise in NT 50 s was seen in serums on day 28 post-1st dose. A moderate inverse correlation was seen between NT 50 s and ages, but no correlation was seen between NT 50 s and AEs. NT 50 s and IgG levels on day 28 post-1st dose and pain scores following the 2nd shot were greater in women than in men. The average half-life of neutralizing activity in the vaccinees was approximately 67.8 days and the average time length for their serums to lose the detectable neutralizing activity was 198.3 days. While serums from elite-responders (NT 50 s>1,500-fold: the top 4% among all participants' NT 50 s) potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT 50 s failed to block the infectivity of a beta strain. INTERPRETATION: BNT162b2-elicited immune response has no significant association with AEs. BNT162b2-efficacy is likely diminished to under detection limit by 6-7 months post-1st shot. High-level neutralizing antibody-containing serums potently to moderately block the infection of SARS-CoV-2 variants; however, a few moderate-level neutralizing antibody-containing serums failed to do so. If BNT162b2-elicited immunity memory is short, an additional vaccine or other protective measures would be needed. RESEARCH IN CONTEXT: Evidence before this study: While mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the salient features of immune response including the persistence of protection remain to be clarified. There is a report that anti-SARS-CoV-2 antibodies persist through 6 months after the second dose of mRNA-1273 vaccine (Doria-Rose et al. N Engl J Med . 2021;384:2259-2261); however, more definite immune kinetics following mRNA-vaccine-elicited protection have to be clarified. The mRNA-vaccine-elicited protection against SARS-CoV-2 variants are also to be determined. Added value of this study: In the present prospective study, 225 twice-BNT162b2-dose-receiving individuals in Japan were enrolled. No significant correlation was seen between 50% neutralizing titers (NT 50 s), determined by using infectious SARS-CoV-2 virions and live target cells, and adverse effects. Largely, NT 50 s and IgG levels were greater in women than in men. Following 28 days post-2 nd shot, significant reduction was seen in NT 50 s, IgG, and IgM levels. The average half-life of NT 50 s was â¼68 days and the average time-length for participants' serums to lose the detectable activity was â¼198 days. Although serums from elite-responders potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT 50 s failed to block the infectivity of a beta strain. Implications of all the available evidence: BNT162b2 efficacy is likely to be diminished to under detection limit by 6-7 months post-1 st shot on average. Individuals with moderate NT 50 s may fail to block beta variants. If BNT162b2-elicited immune memory is lost soon, additional vaccine(s) or other protective means would be needed.
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[Figure: see text].
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Antioxidantes/metabolismo , Citocinas/metabolismo , Cardiomiopatías Diabéticas/enzimología , Miocitos Cardíacos/enzimología , Nicotinamida Fosforribosiltransferasa/metabolismo , Estrés Oxidativo , Animales , Apoptosis , Autofagia , Células Cultivadas , Citocinas/genética , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Fibrosis , Glutatión/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Mitofagia , Miocitos Cardíacos/patología , NAD/metabolismo , NADP/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Ratas Wistar , Sirtuinas/genética , Sirtuinas/metabolismo , Tiorredoxinas/metabolismoRESUMEN
RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.
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Cardiomiopatías Diabéticas/metabolismo , Proteína Forkhead Box O1/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , PPAR alfa/metabolismo , Anciano , Animales , Línea Celular , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Femenino , Proteína Forkhead Box O1/genética , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miocitos Cardíacos/patología , PPAR alfa/genética , Transcripción GenéticaRESUMEN
Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Biogénesis de Organelos , Proteoma , Proteómica , Animales , Línea Celular , Linaje de la Célula , Metabolismo Energético , Humanos , Ratones , Mitocondrias Cardíacas/genética , Proteínas Mitocondriales/genética , Ratas , Factores de TiempoRESUMEN
BACKGROUND: Our work aimed to investigate the association between vigorous physical activity and visit-to-visit systolic blood pressure variability (BPV). METHODS: We conducted a post hoc analysis of SPRINT (Systolic Blood Pressure Intervention Trial), a well-characterized cohort of participants randomized to intensive (<120 mm Hg) or standard (<140 mm Hg) systolic blood pressure targets. We assessed whether patients with hypertension who habitually engage in vigorous physical activity would have lower visit-to-visit systolic BPV compared with those who do not engage in vigorous physical activity. Visit-to-visit systolic BPV was calculated by SD, average real variability (ARV), and SD independent of the mean (SDIM) using measurements taken during the 1-, 2-, 3-, 6-, 9-, and 12-month study visits. A medical history questionnaire assessed vigorous physical activity, which was divided into 3 categories according to the frequency of vigorous physical activity. RESULTS: A total of 7,571 participants were eligible for analysis (34.8% female, mean age 67.9 ± 9.3 years). During a follow-up of 1-year, vigorous physical activity could significantly reduce SD, ARV, and SDIM across increasing frequency of vigorous physical activity. There were negative linear trends between frequency of vigorous physical activity and visit-to-visit systolic BPV. CONCLUSIONS: Long-term engagement in vigorous physical activity was associated with lower visit-to-visit systolic BPV. CLINICAL TRIALS REGISTRATION: SPRINT (Systolic Blood Pressure Intervention Trial); Trial Number: NCT01206062, https://clinicaltrials.gov/ct2/show/NCT01206062.
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Presión Sanguínea , Ejercicio Físico , Anciano , Presión Sanguínea/fisiología , Ejercicio Físico/fisiología , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Visita a Consultorio MédicoRESUMEN
Lysosomal dysfunction caused by mutations in lysosomal genes results in lysosomal storage disorder (LSD), characterized by accumulation of damaged proteins and organelles in cells and functional abnormalities in major organs, including the heart, skeletal muscle, and liver. In LSD, autophagy is inhibited at the lysosomal degradation step and accumulation of autophagosomes is observed. Enlargement of the left ventricle (LV) and contractile dysfunction were observed in RagA/B cardiac-specific KO (cKO) mice, a mouse model of LSD in which lysosomal acidification is impaired irreversibly. YAP, a downstream effector of the Hippo pathway, was accumulated in RagA/B cKO mouse hearts. Inhibition of YAP ameliorated cardiac hypertrophy and contractile dysfunction and attenuated accumulation of autophagosomes without affecting lysosomal function, suggesting that YAP plays an important role in mediating cardiomyopathy in RagA/B cKO mice. Cardiomyopathy was also alleviated by downregulation of Atg7, an intervention to inhibit autophagy, whereas it was exacerbated by stimulation of autophagy. YAP physically interacted with transcription factor EB (TFEB), a master transcription factor that controls autophagic and lysosomal gene expression, thereby facilitating accumulation of autophagosomes without degradation. These results indicate that accumulation of YAP in the presence of LSD promotes cardiomyopathy by stimulating accumulation of autophagosomes through activation of TFEB.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cardiomiopatías/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/patología , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/genética , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Factores de Transcripción/genética , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patología , Proteínas Señalizadoras YAPRESUMEN
The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Animales , Metilación de ADN , Modelos Animales de Enfermedad , Regulación hacia Abajo , Complejo I de Transporte de Electrón/genética , Metabolismo Energético/genética , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Histonas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Musculares/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación Oxidativa , Fenilefrina/farmacología , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismo , RNA-Seq , Ratas , Receptores de Estrógenos/genética , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Metabolic adaption is crucial for the heart to sustain its contractile activity under various physiological and pathological conditions. At the molecular level, the changes in energy demand impinge on the expression of genes encoding for metabolic enzymes. Among the major components of an intricate transcriptional circuitry, peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) plays a critical role as a metabolic sensor, which is responsible for the fine-tuning of transcriptional responses to a plethora of stimuli. Cumulative evidence suggests that energetic impairment in heart failure is largely attributed to the dysregulation of PGC-1α. In this review, we summarize recent studies revealing how PGC-1α is regulated by a multitude of mechanisms, operating at different regulatory levels, which include epigenetic regulation, the expression of variants, post-transcriptional inhibition, and post-translational modifications. We further discuss how the PGC-1α regulatory cascade can be impaired in the failing heart.
RESUMEN
AIMS: Thioredoxin 1 (Trx1) is an evolutionarily conserved oxidoreductase that cleaves disulphide bonds in oxidized substrate proteins such as mechanistic target of rapamycin (mTOR) and maintains nuclear-encoded mitochondrial gene expression. The cardioprotective effect of Trx1 has been demonstrated via cardiac-specific overexpression of Trx1 and dominant negative Trx1. However, the pathophysiological role of endogenous Trx1 has not been defined with a loss-of-function model. To address this, we have generated cardiac-specific Trx1 knockout (Trx1cKO) mice. METHODS AND RESULTS: Trx1cKO mice were viable but died with a median survival age of 25.5 days. They developed heart failure, evidenced by contractile dysfunction, hypertrophy, and increased fibrosis and apoptotic cell death. Multiple markers consistently indicated increased oxidative stress and RNA-sequencing revealed downregulation of genes involved in energy production in Trx1cKO mice. Mitochondrial morphological abnormality was evident in these mice. Although heterozygous Trx1cKO mice did not show any significant baseline phenotype, pressure-overload-induced cardiac dysfunction, and downregulation of metabolic genes were exacerbated in these mice. mTOR was more oxidized and phosphorylation of mTOR substrates such as S6K and 4EBP1 was impaired in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mitochondrial respiration and metabolic gene promoter activity, suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F, an oxidation-resistant mutation, prevented Trx1 knockdown-induced mTOR oxidation and inhibition and attenuated suppression of metabolic gene promoter activity. CONCLUSION: Endogenous Trx1 is essential for maintaining cardiac function and metabolism, partly through mTOR regulation via Cys1483.