MethodsRat neonate cardiomyocytes were cultured and treated with AGEs at different concentration. Two classic autophagy markers, microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1, were detected by western blot assay. The inhibition of RAGE and phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR pathway were applied to cells, respectively.ResultsAGEs administration enhanced the expression of Beclin-1 and LC3 II in cardiomyocytes, increased the number of autophagic vacuoles and impaired the cell viability in dose-dependant manners. Also, AGEs inhibited the PI3K/Akt/mTOR pathway via RAGE. Inhibition of RAGE with RAGE antibody reduced expression of Beclin-1 and LC3 II/I and inhibited the cellular autophagy, accompanied by the reactivation of PI3K/Akt/mTOR pathway in cultured cells. Notably, the presence of inhibition of PI3K/Akt/mTOR pathway abolished the protective effect of RAGE inhibition on cardiomyocytes.ConclusionThis study provides evidence that AGEs induces cardiomyocyte autophagy by, at least in part, inhibiting the PI3K/Akt/mTOR pathway via RAGE.Previous studies showed that the accumulation of advanced glycation end products (AGEs) induce cardiomyocyte apoptoisis, leading to heart dysfunction. However, the effect of AGEs on another cell death pathway, autophagy, in cardiomyocytes remains unknown.

Background Mutations in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM). In particular, m.A3243G is the most common T2DM-related mtDNA mutation in many families worldwide. However, the clinical features and pathophysiology of m.A3243G-induced T2DM are largely undefined. Methods Two pedigrees with maternally inherited T2DM were underwent clinical, molecular and biochemical assessments. The mtDNA genes were PCR amplified and sequenced. Mitochondrial adenosine triphosphate (ATP) and reactive oxygen species (ROS) were measured in polymononuclear leukocytes derived from three patients with both the m.A3243G and m.T14502C mutations, three patients with only the m.A3243G mutation and three controls without these mutations. Moreover, GJB2, GJB3 and GJB6 mutations were screened by PCR-Sanger sequencing. Results Members of the two pedigrees manifestated variable clinical phenotypes including diabetes and hearing and vision impairments. The age at onset of T2DM varied from 31 to 66 years, with an average of 41 years. Mutational analysis of mitochondrial genomes indicated the presence of the m.A3243G mutation in both pedigrees. Matrilineal relatives in one of the pedigrees harbored the coexisting of m.A3243G and m.T14502C mutations. Remarkably, the m.T14502C mutation, which causes the substitution of a conserved isoleucine for valine at position 58 in ND6 mRNA, may affect the mitochondrial respiratory chain functions. Biochemical analysis revealed that cell lines bearing both the m.A3243G and m.T14502C mutations exhibited greater reductions in ATP levels and increased ROS production compared with those carrying only the m.A3243G mutation. However, we did not find any mutations in the GJB2, GJB3 and GJB6 genes. Conclusion Our study indicated that mitochondrial diabetes is associated with the tRNA Leu(UUR) A3243G and ND6 T14502C mutations.

Mitochondrial dynamics plays an important role in maintaining normal endothelial cell function and in the pathogenesis of cardiovascular disease. It is not identified whether high-mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule, could influence mitochondrial dynamics in endothelial cells. The objective of this study is to clarify the effect of HMGB1 on mitochondrial dynamics in endothelial cells and the underlying mechanism. EA.hy926 human endothelial cells were incubated with recombinant HMGB1 (rHMGB1); mitochondrial morphology was observed with a confocal microscope and transmission electron microscope (TEM). The expression of dynamin-related protein 1 (Drp1), Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2), Optic atrophy 1 (Opa1), phosphatase and tensin homolog- (PTEN-) induced kinase 1 (PINK1), NOD-like receptor 3 (NLRP3), caspase 1, cleaved caspase 1, 20S proteasome subunit beta 5 (PSMB5), and antioxidative master nuclear factor E2-related factor 2 (NRF2) and the concentration of interleukin 1β (IL-1β) were determined. Specific inhibitors C29, TAK-242, FPS-ZM1, AMD3100, and epoxomicin were used to block toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), C-X-C-chemokine receptor 4 (CXCR4), and PSMB5, respectively. siRNAs were used to silence the expression of NRF2. rHMGB1 promoted mitochondrial fusion in endothelial cells, while no significant proinflammatory effects were found. The expression of mitochondrial fission protein Drp1 and phosphorylated subtypes p-Drp1-S616 and p-Drp1-S637 were all downregulated; no significant expression changes of PINK1 and Mfn1, Mfn2, and Opa1 were found. Inhibition of CXCR4 but not TLR4, RAGE, or TLR2 reversed rHMGB1-induced Drp1 downregulation and mitochondrial fusion. Interestingly, inhibition of TLR4 with TAK-242 promoted Drp1 downregulation and mitochondrial fusion. rHMGB1 increased the expression of NRF2 and PSMB5; inhibition of PSMB5 but not silencing NRF2 abolished rHMGB1-induced Drp1 downregulation and mitochondrial fusion. These results indicate that rHMGB1 promotes NRF2 independent mitochondrial fusion via CXCR4/PSMB5 pathway-mediated Drp1 proteolysis. rHMGB1 may influence mitochondrial and endothelial function through this effect on mitochondrial dynamics.

Type 2 diabetes mellitus (T2DM) is a common endocrine disorder which remains a large challenge for clinicians. Previous studies have suggested that mitochondrial dysfunction plays an active role in T2DM progression, but a detailed mechanism is still elusive. In the current study, two Han Chinese families with maternally inherited T2DM were evaluated using clinical, genetic, molecular, and biochemical analyses. The mitochondrial genomes were PCR amplified and sequenced. Phylogenetic and bioinformatic analyses were used to assess the potential pathogenicity of mitochondrial DNA (mtDNA) mutations. Interestingly, the matrilineal relatives of these pedigrees exhibited variable severity of T2DM, in particular, the age at onset of T2DM varied from 26 to 65 years, with an average of 49 years. Sequence analysis revealed the presence of ND4 G11696A mutation, which resulted in the substitution of an isoleucine for valine at amino acid (AA) position 312. Indeed, this mutation was present in homoplasmy only in the maternal lineage, not in other members of these families, as well as 200 controls. Furthermore, the m.C5601T in the tRNAAla and novel m.T5813C in the tRNACys, showing high evolutional conservation, may contribute to the phenotypic expression of ND4 G11696A mutation. In addition, biochemical analysis revealed that cells with ND4 G11696A mutation exhibited higher levels of reactive oxygen species (ROS) productions than the controls. In contrast, the levels of mitochondrial membrane potential (MMP), ATP, mtDNA copy number (mtDNA-CN), Complex I activity, and NAD+/NADH ratio significantly decreased in cell lines carrying the m.G11696A and tRNA mutations, suggesting that these mutations affected the respiratory chain function and led to mitochondrial dysfunction that was involved in T2DM. Thus, our study broadened the clinical phenotypes of m.G11696A mutation.