RESUMO
Diabetic cardiomyopathy (DCM) is a major cause of mortality/morbidity in diabetes mellitus patients. Although tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous cardiovascular target, its effect on myocardial cells and mitochondria in DCM and the underlying mechanisms remain unknown. Here, we determined the involvement of BH4 deficiency in DCM and the therapeutic potential of BH4 supplementation in a rodent DCM model. We observed a decreased BH4:total biopterin ratio in heart and mitochondria accompanied by cardiac remodeling, lower cardiac contractility, and mitochondrial dysfunction. Prolonged BH4 supplementation improved cardiac function, corrected morphological abnormalities in cardiac muscle, and increased mitochondrial activity. Proteomics analysis revealed oxidative phosphorylation (OXPHOS) as the BH4-targeted biological pathway in diabetic hearts as well as BH4-mediated rescue of down-regulated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) signaling as a key modulator of OXPHOS and mitochondrial biogenesis. Mechanistically, BH4 bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and activated downstream AMP-activated protein kinase/cAMP response element binding protein/PGC-1α signaling to rescue mitochondrial and cardiac dysfunction in DCM. These results suggest BH4 as a novel endogenous activator of CaMKK2.
Assuntos
Biopterinas/análogos & derivados , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias Diabéticas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/genética , Animais , Biopterinas/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Diabetes Mellitus/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Coração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Biogênese de Organelas , Fosforilação Oxidativa , Ratos , Ratos Long-Evans , Transdução de Sinais/fisiologiaRESUMO
Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3UTR to stabilize TNF-alpha mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-alpha mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC(50)) below 10 microM. The IC(50) of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 microM, respectively, for binding of HuR protein to TNF-alpha mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-alpha mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-alpha mRNA and decreased levels of secreted TNF-alpha. From these results, we could find inhibitors for the TNF-alpha mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.