Abstract 19761: P27 Inhibits IkBa-Degradation, Attenuates Inflammation, Improves Cardiac Function and Increases Survival Following LPS
Background: A previous cardiac protein interaction screen suggested that cell cycle inhibitor p27 interacts with IKKα, a kinase that regulates phosphorylation-dependent degradation of IκBα, the inhibitor of NF-κB. Bacterial toxin LPS promotes NF-κB-induced myocarditis. Here we test if p27 can interfere with LPS-induced cardiac dysfunction in vivo.
Methods: At 1 and 6 h post-LPS (30 mg/kg i.p.), an HA-tagged TAT-conjugated p27 fusion protein (TAT.p27), a TAT.lacZ control or an equal volume of saline was delivered (20 mg/kg i.p.) to 8-wk old C57BL6 mice. This dose of TAT.p27 was shown capable of entering the heart and increasing total cardiac p27 levels as determined by immunofluorescence for HA-tag and Western blot (WB) for p27. Cardiac structure and function were assessed by 2D-echo and pressure-volume catheters. WBs of heart homogenates for cleaved caspase-3 and Bcl-2, as well as IκBα, were used to assess apoptosis and IκBα turnover. Expression of inflammatory cytokines IL-1β and IL-6 was measured by RT-PCR. Survival was analyzed by Kaplan-Meier plots.
Results: Delivery of TAT.p27 at 1 h post-LPS preserved myocardial levels of IκBα at 1.5, 2 and 4 h post-LPS. Co-immunoprecipitation experiments in intact heart showed an interaction between p27 and IκBα. Delivery of TAT.p27 at 6 h post-LPS resulted in improvements vs. saline and TAT.lacZ controls in fractional shortening (%: 38.2±1.2 vs. 29.1±1.5, P<0.01; vs. 27.3±1.8, P<0.001), cardiac output (µl/min: 17332±4619 vs. 12279± 5304, p<0.05; vs. 12959±3221; p<0.01), stroke volume (µl: 28±7 vs. 21±8, p<0.01; vs. 20±5 µl, p<0.05) and stroke work (mJ: 2121±909 vs. 1472±721, p<0.05; vs. 1366±522, p<0.05). For each above result, N≥13 per group. Apoptosis was significantly decreased in TAT.p27-treated mice subjected to LPS, as were mRNA levels of IL-1β and IL-6 as compared to controls. Importantly, these improvements were associated with better survival in TAT.p27-treated mice vs. saline- and TAT.lacZ-treated controls.
Conclusion: These data suggest that the p27-IκBα and or IKKα interactions, possibly by preserving cardiac IκBα levels and preventing NFκB activation post-LPS, attenuate inflammation, limit cardiomyocyte apoptosis, and improve survival in a model of endotoxin-induced cardiac dysfunction.
- © 2012 by American Heart Association, Inc.