A Dual Role for Death Receptor 5 in Regulating Cardiac Fibroblast Function

Figure 1. DR5 activation induces proliferation in cardiac fibroblasts. (A) A caspase 3/7 assay was used to measure the induction of apoptosis in NRVFs treated over time with TRAIL, bioymifi, or vehicle control. N = 5. (B) An MTS assay was used to measure proliferation in vehicle, TRAIL, or bioymifi treated NRVFs. n = 6, One-Way ANOVA, *p < 0.05. (C) Representative BrdU and Ki67 staining (green) and quantified BrdU (D) or Ki67 (E) staining for vehicle, TRAIL, or bioymifi treated NRVFs. Cells were counterstained with DAPI (blue) to identify nuclei. White arrows indicate BrdU-positive nuclei. n = 6, One-Way ANOVA, *p < 0.05. Representative (F) and quantified (G) BrdU staining (green) in WT and DR5KO AMCF treated with TRAIL, bioymifi, or vehicle control. DAPI (blue) was used to identify nuclei. White arrows indicate BrdU-positive nuclei. n = 5, Two-Way ANOVA, *p < 0.05.

Figure 2. DR5-mediated changes in proliferation occur through ERK1/2 activation. (A) ERK1/2 activation in response to TRAIL in NRVFs was examined using immunoblot analysis for phospho-ERK1/2 in the presence or absence of PD98059. Total-ERK1/2 is shown as a loading control. n = 8, One-Way ANOVA, *p < 0.05. (B) Phosphorylation of ERK1/2 was examined in bioymifi treated NRVFs in the presence or absence of PD98059. Total-ERK1/2 is shown as a loading control. n = 8, One-Way ANOVA, *p < 0.05. (C) Representative BrdU staining (green) and quantified BrdU staining for TRAIL (D) or bioymifi (E) treated NRVFs with our without PD98059 pretreatment. DAPI staining (blue) is used to show nuclei. White arrows indicate BrdU-positive nuclei. n = 8, One-Way ANOVA, *p < 0.05.

Figure 3. DR5 activation induced death in myofibroblasts. (A) Apoptosis was examined by TUNEL staining (red) in rat neonatal myofibroblasts treated with vehicle, TRAIL, or bioymifi. αSMA staining (green) was used to label myofibroblasts and DAPI staining (blue) shows nuclei. (B) Quantification of the number of TUNEL-positive nuclei in myofibroblasts treated with TRAIL, bioymifi, or vehicle control. n = 8. One-Way ANOVA, *p < 0.05. Representative (C) and quantified (D) TUNEL staining for myofibroblasts from adult WT and DR5KO mouse hearts treated with TRAIL or bioymifi. αSMA staining (green) was used to label myofibroblasts and DAPI staining (blue) shows nuclei. Data presented as fold over vehicle from the same mouse. n = 5, Two-Way ANOVA, *p < 0.05.

Figure 4. (A) DR5 and OPG transcript expression was examined with NRVFs and myofibroblasts by RT-qPCR and expressed relative to NRVF transcript levels. n = 6. T-test, *p < 0.05. (B) DR5 protein expression was detected via immunoblot in NRVFs and myofibroblasts. GAPDH is shown as a loading control. The molecular weight ladder (Ldr) is shown in the right lane. Values are expressed relative to NRVFs. n = 6. T-test, *p < 0.05. ERK1/2 activation was examined in myofibroblasts in response to TRAIL (C) or bioymifi (D) treatment over time by immunoblot for phospho-ERK1/2. Total-ERK1/2 is shown as a loading control. n = 5.

Figure 5. DR5 activation induces apoptosis in myofibroblasts. Bax (A) and Bcl-2 (B) transcript expression and the calculated Bax to Bcl-2 ratio (C) were examined in neonatal rat myofibroblasts treated with vehicle, TRAIL or bioymifi using RT-qPCR and expressed relative to vehicle treated cells. n = 5. (D) A Caspase 3/7 Activity assay was used to measure apoptosis in myofibroblasts treated with TRAIL, bioymifi, or vehicle control. Values are expressed relative to vehicle. n = 6, One-Way ANOVA, *p < 0.05 vs. vehicle. (E) Apoptosis was quantified in WT and DR5KO myofibroblasts from adult mouse heart and treated with vehicle, TRAIL or bioymifi. Values are expressed relative to WT vehicle. n = 5, One-Way ANOVA, *p < 0.05.

Figure 6. DR5KO exacerbates fibrosis in vivo. (A) Top: Representative TUNEL (red) staining in hearts of WT and DR5KO animals administered vehicle or isoproterenol. Cardiomyocytes were identified by troponin I (TnI) staining (green) and nuclei were labeled using DAPI (blue). TUNEL positive nuclei are indicated by white arrows. Bottom: Representative Masson's trichrome staining showing fibrosis (blue) in hearts from vehicle and isoproterenol treated WT and DR5KO mice. (B) Quantification of TUNEL positive cardiomyocytes from WT and DR5KO mice administered vehicle or isoproterenol. n = 10 for WT vehicle, 12 for DR5KO vehicle, WT isoproterenol, and DR5KO isoproterenol, Two-Way ANOVA, *p < 0.05 vs. vehicle. (C) Quantification of fibrotic area from Masson's trichrome stained WT and DR5KO hearts after vehicle or isoproterenol treatment. n = 10 for WT vehicle, 12 for DR5KO vehicle, WT isoproterenol and DR5KO isoproterenol, Two-Way ANOVA, *p < 0.05. (D) Fractional shortening was measured echocardiography in WT and DR5KO mice at baseline and with vehicle or isoproterenol infusion. n = 10 for WT vehicle, 12 for DR5KO vehicle, WT isoproterenol and DR5KO isoproterenol, Two-Way ANOVA, *p < 0.05.

Figure 7. Summary of DR5 in cardiac fibroblasts. In naïve cardiac fibroblasts, DR5 activates ERK1/2 resulting in increased proliferation. Upon differentiation/activation of cardiac fibroblasts to myofibroblasts, DR5 signaling changes to a canonical DR5 signaling paradigm leading to caspase activation and cell death.