Objective Cardiomyocyte (CM) apoptosis is elevated in end-stage heart failure. Cardiac fibrosis is a common phenotype of heart failure. In previous studies we have shown that in SR-uPA transgenic (SR-uPA+/o) mice there is an increase in plasmin-dependent cardiac fibrosis. We hypothesize that in these mice, plasmin causes the pericellular proteolysis of laminin, an extracellular adhesive glycoprotein that acts as a bridge between proteins attached to the cell membrane and the basal lamina. Consequently, due to the loss of cell-matrix contact, apoptosis is induced. Apoptosis of CM then leads to replacement fibrosis. To test this hypothesis we measured apoptosis in the hearts of SR-uPA+/o and nontransgenic (SR-uPAo/o) littermates.
Methods We measured apoptosis in the hearts of SR-uPA+/o and SR-uPAo/o mice using TUNEL (Roche) staining of apoptotic nuclei in situ with a hematoxylin counterstain. In addition, since caspases are known mediators in the apoptotic pathway, we performed immunostaining and immunoblotting for cleaved caspase 3 (Cell Signaling Technology), a downstream effector caspase, in SR-uPA+/o and SR-uPAo/o heart sections. Mouse thymus served as a positive control for both experiments. Additionally, lysates of camptothecin-treated and nontreated 293 cells were positive and negative controls, respectively, for the immunoblotting.
Results Increased numbers of TUNEL-positive cells were seen in 6-week-old SR-uPA+/o hearts compared with SR-uPAo/o littermates (51.5 [33-67] vs 17.5 [12-28] TUNEL positive cells per 400× field, n = 8, p = .003). Preliminary analysis of counterstained sections reveals TUNEL-positive cells are not CM. In our cleaved caspase-3 studies two SR-uPA+/o hearts had increased numbers of staining cells compared with SR-uPAo/o littermate controls. Furthermore, the immunoblotting was suggestive of greater cleaved caspase-3 activity in 6-week-old SR-uPA+/o versus SR-uPAo/o mice.
Conclusions In SR-uPA+/o mice we found an increase in TUNEL-positive cells and increased cleaved caspase-3 activity. These data indicate that apoptosis may be an important mediator of cardiac fibrosis. However, contrary to our hypothesis, the cells undergoing apoptosis do not appear to be CM and, instead, could be macrophages. Further immunostaining will be needed to conclusively identify the cell type. Additionally, we will increase our sample size in our cleaved caspase-3 immunostaining experiment.
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