Figure 3.

Mevalonate cascade stimulates autophagy in primary human myocardial atrial fibroblast. (a) Myocardial atrial fibroblast cells were either left untreated (top left) or they were treated with 10 μM simvastatin (top right, and lower panels) for 72 h. Cells were then imaged by TEM. Magnification: 3.4 × 10³. Structures identified as autophagosomes can be seen as double-membrane vacuoles in magnified part of the figures. (b) Western blot analysis of cell lysates from myocardial atrial fibroblast. Cells were treated with 10 μM simvastatin for the indicated time periods, and then immunoblotted using the indicated specific antibodies. GAPDH was used as loading control. (c) Myocardial atrial fibroblast treated with simvastatin (+Simva, 10 μM, 72 h) showed increased Lysotracker Red staining, a marker of lysosomal activation. (d) Myocardial atrial fibroblast cells treated with simvastatin showed an increase in LC3-β (green) punctuate indicating autophagosome formation. (e) Western blot analysis of cell lysates from myocardial atrial fibroblast cells. Cells were treated with 10 μM simvastatin for the indicated time points and then immunoblotted using the indicated specific antibodies. Simvastatin induced late cathepsin L and early cathepsin-B activation. (f and g) Myocardial atrial fibroblast were pretreated with specific cathepsin-B inhibitor (CA-074, 4 h, f) or cathepsin-B and -L inhibitor (ZFF-fmk, 4 h, g) and then co-treated with simvastatin (10 μM) for indicated time points. Cell viability was measured using MTT assay. Cathepsin inhibitors significantly inhibited simvastatin-induced cell death in myocardial atrial fibroblast (***P<0.001)