cytochrome-c-t and Heart-Defects--Congenital

The regulation of phosphate (Pi) handling is crucial during calcification of the aortic valve. Gene profiling of Pi transporters revealed that VIC culture expresses SLC201A1/Pit1 and SLC20A2/Pit2. On exposure to a mineralizing medium (2 mM Pi), the expression of Pi transporters in VIC culture is increased several folds, with the highest magnitude for SLC20A1. By using siRNAs, we established that silencing SLC20A1 significantly reduced Pi-induced mineralization of VICs. In human pathological specimens, we found that the expression of SCL20A1 was increased in CAVD tissues compared to control non-mineralized aortic valves. Treatment of VIC culture with Pi promoted the loss of mitochondrial membrane potential (ΔΨm) and cytochrome c release within the cytosol, leading to apoptosis. Inhibition of Pi transporters with phosphonoformic acid (PFA) prevented Pi-mediated apoptosis of VICs. Moreover, we discovered that the level of the Akt-1 transcript is diminished in CAVD tissues compared with control valves. Accordingly, treatment with Pi caused a reduction of the Akt-1 transcript in VIC culture, and treatment with PFA or siRNA against SLC20A1 restored the level of Akt-1. Overexpression of Akt-1 (pCMVAkt-1) prevented both Pi-induced apoptosis and mineralization of VIC culture. These results strongly suggest that overexpression of SLC20A1 promotes apoptosis and mineralization by altering the level of Akt-1.

Topics: Aged; Aged, 80 and over; Aortic Valve; Apoptosis; Bicuspid Aortic Valve Disease; Calcinosis; Cells, Cultured; Cytochromes c; Endothelial Cells; Female; Gene Expression; Heart Defects, Congenital; Heart Valve Diseases; Humans; Male; Membrane Potential, Mitochondrial; Middle Aged; Mitochondria; Phosphates; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Sodium-Phosphate Cotransporter Proteins, Type III

The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspase 3; Caspase 7; Caspases; Cell Nucleus; Cell Shape; Cell Survival; Cells, Cultured; Cytochromes c; DNA Fragmentation; Female; Fibroblasts; Heart; Heart Defects, Congenital; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Membranes; Permeability; T-Lymphocytes