Figure 4
Mevalonate cascade inhibition induces endoplasmic reticulum stress and activates UPR in primary human myocardial atrial fibroblast. (a) Western blot analysis of cell lysates from myocardial atrial fibroblasts. Cells were treated with 10 μM simvastatin before sample preparation at the indicated times. Immunoblotting was performed using the indicated specific antibodies. Simvastatin increased the expression of IREα1, CHOP and ATF4 and enhanced ATF6 cleavage, XBP1 splicing, eif2α and PERK phosphorylation. Equal protein loading was confirmed by detecting GAPDH protein. (b) Simvastatin increased nuclear accumulation of endoplasmic reticulum-related proteins. Cells were treated with simvastatin (10 μM) for indicated time point and cytosolic and nuclear fractions were prepared. Simvastatin increased ATF4, cleaved ATF6 and spliced XBP1 nuclear content. HDAC1 was used to assess fraction purity and as an equal loading control. (c) Simvastatin activated endoplasmic reticulum-related caspase. Cells were treated with simvastatin then cell lysates were probed for caspase-4 cleavage by immunoblot. Simvastatin induced caspase-4 activation. Equivalent loading was confirmed using GPADH. (d and e) Myocardial atrial fibroblasts were pretreated with caspase-4 inhibitor (Z-LEVD-FMK, 10 μM) for 4 h and then co-treated with simvastatin (10 μM, 96 h). Cell viability was measured using MTT assay. Caspase-4 inhibitor significantly decreased simvastatin-induced cell death and caspase-3/-7 activation (f) (***P<0.001)
