Inhibition of apoptosis by progesterone in cardiomyocytes

Abstract

While gender-based differences in heart disease have raised the possibility that estrogen (ES) or progesterone (PG) may have cardioprotective effects, recent controversy regarding hormone replacement therapy has questioned the cardiac effects of these steroids. Using cardiomyocytes, we tested whether ES or PG has protective effects at the cellular level. We found that PG but not ES protects cardiomyocytes from apoptotic cell death induced by doxorubicin (Dox). PG inhibited apoptosis in a dose-dependent manner, by 12 ± 4.0% at 1 μm and 60 ± 1.0% at 10 μm. The anti-apoptotic effect of PG was also time dependent, causing 18 ± 5% or 62 + 2% decrease in caspase-3 activity within 1 h or 72 h of pretreatment. While PG causes nuclear translocation of its receptor within 20 min, the cytoprotective effect of PG was canceled by mifepristone (MF), a PG receptor antagonist. Analyses using Affymetrix high-density oligonucleotide array and RT-PCR found that PG induced Bcl-xL, metallothionine, NADPH quinone oxidoreductase 1, glutathione peroxidase-3, and four isoforms of glutathione S-transferase. Western blot analyses revealed that PG indeed induced an elevation of Bcl-xL protein in a dose- and time-dependent manner. Nuclear run-on assay indicated that PG induced Bcl-xL gene transcription. Inhibiting the expression of Bcl-xL using siRNA reduced the cytoprotective effect of PG. Our data suggests that PG induces a cytoprotective effect in cardiomyocytes in association with induction of Bcl-xL gene.

Figure 1. Morphological Evidence of PG Inhibiting Dox Induced Apoptosis

Primary cultured cardiomyocytes were pretreated with 10 μM PG for 24 hrs and then treated with 0.8 μM Dox in the absence or presence of 10 μM PG. At 24 hrs after Dox treatment, the morphology was recorded under a phase contrast microscope. Detached cells were combined with adherent cells for Annexin V-Fluo staining for recording under a fluorescent microscope.

Figure 2. PG Inhibits Dox Induced Caspases

Primary cultured cardiomyocytes were treated with 0.8 μM Dox for 24 hrs following pretreatment of PG at indicated dose for 24 hrs (A) or at 10 μM over indicated time (B). For Dox dose response, cardiomyocytes were treated with 10 μM PG and Dox at indicated doses for 16 hrs (C). Following pretreatment with 10 μM PG in the absence or presence of 10 μM 17β-estradiol (ES) or 10 μM testosterone (TS), cells were treated with 0.8 μM Dox for 24 hrs before harvesting for measurements of caspase activity (D, E). The data are from one experiment representative of three with means ± standard deviations from three samples. A superscript letter indicates significant difference (P<0.05) from means labeled with a different letter as determined by ANOVA analysis.

Figure 3. Expression of PR in Cardiac Tissue or Cardiomyocytes

Cardiac tissue was collected from 8 weeks old male or female C57BL6 mice or Sprague Dawley rats by quick frozen in liquid nitrogen. The uterus tissue from a pregnant rat was collected immediately after birth to serve as a positive control. Animal tissues were grinded in liquid nitrogen bath for dissolving in lysis buffer for Western blot analyses (A). Primary cultured cardiomyocytes were treated with 10 μM PG and harvested at indicated time for isolation of cytoplasmic or nuclear fractions (B). An equal amount of proteins (20 μg/lane) was loaded for Western blot analyses.

Figure 4. PR Receptor Antagonist MF Blocks Cytoprotective Effect of PG

Primary cultured cardiomyocytes were treated with 10 μM PG in the absence or presence of 1 μM MF for 24 hrs before addition of 0.8 μM Dox. The cells were harvested at 24 hrs later for measurements of caspase 3 using DEVD-AMC as a substrate (A), or for Western blot to measure caspase-3 cleavage or mitochondrial release of cytochrome c (B, C). A superscript letter indicates significant difference (P<0.05) from means labeled with a different letter as determined by ANOVA analysis.

Figure 5. RT-PCR Verification of Microarray Detected Genes Induced by PG

Total RNA was harvested from primary cultured cardiomyocytes 24 hrs after 10 μM PG treatment for RT-PCR analyses as described in the Method. GAPDH was used as a loading control.

Figure 6. PG Induces Bcl-xL protein

Primary cultured cardiomyocytes were treated with 10 μM PG (A, C) or indicated dose (B) for the time points shown or 24 hrs (B, C). MF (1 μM) was added to cells 1 hr before PG (C). Cell lysates were used for Western blots to measure Bcl-xL protein (20 μg protein/lane, upper panels) with vinculin being used as a loading control (bottom panels). The Data shown is representative of 3 experiments.

Figure 7. PG Causes An Increase in the Rate of Bcl-xL Transcription

Primary cultured cardiomyocytes were treated with 10 μM PG for 24 hrs before being harvested for nuclear run-on assay (A) or measurements of Bcl-xL mRNA stability by addition of 5 μg/ml actinomycin D (ActD) over indicated time (B). The relative Ct method was used to calculate mRNA levels with normalization to β-Actin (A). The level of Bcl-xL was set to 100 at 0 time point for determining Bcl-xL mRNA stability (B). The data represent average ± standard deviations of three independent experiments. An asterisk (*) indicates p<0.05 when the treated group was compared to control using Student’s t test.

Figure 8. bcl-x Gene Promoter Activity by PG Treatment

Primary cultured cardiomyocytes were transfected with luciferase constructs under the control of −905 bp human bcl-x promoter (A) or −3.2 kb mouse bcl-x promoter (B) using Fugene6 liposomes. At 48 hrs after transfection, cells were placed in 0.5% FBS for 24 hrs before treatment with 1 μM corticosterone (CT, A), 10 μM PG (A) or PG at indicated doses (B) for 24 hrs before harvesting for measurement of luciferases. A superscript letter indicates significant difference (P<0.05) from means labeled with a different letter as determined by ANOVA analysis.

Figure 9. Cardioprotective Effect of PG Is Dependent on Bcl-xL

Primary cultured cardiomyocytes were transfected with Bcl-xL siRNA or negative control siRNA as described in the Methods. At 48 hrs after siRNA transfection, cells were collected to verify Bcl-xL down regulation (upper panel) by Western blot with vinculin (bottom panel) as a loading control (A) or were pretreated with 10 μM PG for 24 hrs prior to 16 hrs treatment with 1 μM Dox for measurement of caspase-3 activity (B). The data is from one experiment representative of three. A superscript letter indicates significant difference (P<0.05) from means labeled with a different letter as determined by ANOVA analysis.

Figure 10. PG Inhibits Caspase-3 Activation by A Variety of Toxicants

Primary cultured cardiomyocytes were pretreated with 10 μM PG 24 hrs prior to treatment with 100 μM H₂O₂, 1 μM rotenone (Rot), 0.5 μM palmitate (Palm), 5 μM 2-deoxyglucose (2-DG) or 25 μM glutamate (Glu) in the absence or presence of 10 μM PG. Dox treatment (0.8 μM) was included for comparison. Cells were harvested at 24 hrs after for measurements of caspase-3 activity. The data represents mean ± SEM from triplicate samples of one experiment representative of three. A superscript letter indicates significant difference (P<0.05) from means labeled with a different letter as determined by ANOVA analysis.