Aims: Apoptosis plays a critical role in cardiomyocyte loss during ischaemic heart injury. A detailed understanding of the mechanism involved has a substantial impact on the optimization and development of treatment strategies. Here, we report that the expression of SIRT4, a mitochondrial sirtuin, is markedly down-regulated in hypoxia-induced apoptosis of H9c2 cardiomyoblast cells. Methods and Results: SIRT4 interference significantly alters H9c2 cell viability, apoptotic cell number and caspase-3/7 activity. Furthermore, SIRT4 expression can affect the ratio of pro-caspase 9/caspase 9 or pro-caspase 3/caspase 3, an affect Bax translocation, which in turn alters the development of H9c2 cell apoptosis. Conclusion: These results suggest that SIRT4 is a key player in hypoxia-induced cardiomyocyte apoptosis, and that strategies based on its enhancement might be of benefit in the treatment of ischaemic heart disease.
Background: SIRT5 is located in the mitochondria, and plays a crucial role in the regulation of metabolic process and cellular apoptosis. Cardiomyocytes are abundant in mitochondria. However, the role of SIRT5 in oxidative stress-induced apoptosis is still unknown in cardiomyocytes. Methods and Results: Western blots analysis revealed that SIRT5 is significantly down-regulated in cardiomyocytes upon oxidative stress. MTT assay, DAPI staining, and caspase 3/7 activity assay were used to estimate apoptosis development. The result suggested that compared with the wild-type group, SIRT5 knockdown results in a marked reduction in cell viability, and a significant increase in the number of apoptotic cells and the caspase 3/7 activity. Protein immunoprecipitation revealed a direct interaction between Bcl-Xl and SIRT5. Apoptosis assay and western blot anaylsis suggested that SIRT5 levels could affect the levels of Bcl-Xl expression, but have no effect on the apoptosis development in Bcl-Xl knockdown cells. Conclusion: This study reveals a novel role of SIRT5 in the regulation of oxidative stress-induced apoptosis in cardiomyocytes. Pharmacological interventions on SIRT5 expression may be useful in the treatment of oxidative stress-related cardiac injury.
Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.
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