Local wound p38 MAPK inhibition attenuates burn-induced cardiac dysfunction

Abstract

Background: Topical inhibition of activated p38 MAPK within burn wounds attenuates the local and systemic inflammatory response. In this study, we investigated the effects of local activated p38 MAPK inhibition on burn-induced cardiac dysfunction.

Methods: Using a standardized rat model of scald burn injury, rats were given a 30% total body surface area partial thickness burn or sham injury, and the wounds were treated with an activated p38 MAPK inhibitor (SB) or vehicle. Systemic blood pressure measurements were recorded in vivo followed by in vitro assessment of sarcomere contraction in single-cell suspensions of isolated cardiomyocytes.

Results: Systolic blood pressure or maximum left ventricular pressures in vivo and peak cardiomyocyte sarcomere contractility in vitro were significantly reduced after burn injury. These functional deficits were abolished 24 h after burn injury following local p38 MAPK inhibition. In vitro incubation of normal cardiomyocytes with homogenate from burned skin or burn serum resulted in a similar pattern of impaired cardiomyocyte contractility. These effects were reversed in normal cardiomyocytes exposed to burn skin homogenates treated topically with a p38 MAPK inhibitor. A Western blot analysis showed that cardiac p38 MAPK activation was not affected by dermal blockade of activated p38 MAPK, arguing against systemic absorption of the inhibitor and indicating the involvement of systemic cytokine signaling.

Conclusion: Topical activated p38 MAPK inhibition within burned skin attenuates the release of proinflammatory mediators and prevents burn-induced cardiac dysfunction after thermal injury. These results support the inhibition of burn-wound inflammatory signaling as a new therapeutic approach to prevent potential postthermal injury multiorgan dysfunction syndrome.

Figure 1

In vivo measurement of systemic hemodynamics, mean arterial blood pressure (MAP, A), systolic blood pressure (SBP, B), diastolic blood pressure (DBP, C) and heart rate (D) 24 hours after burn or sham injury. Activated p38 MAPK-inhibitor (SB) or vehicle was applied locally at the time 0, 8, and 16 hours after burn or sham injury. *p<0.05 burn SB vs. burn vehicle (ANOVA). n=5 animals in each group.

Figure 2

In vitro measurement of peak sarcomere shortening values (A), contraction velocity (B) and relaxation velocity (C). Activated p38 MAPK-inhibitor (SB) or vehicle was applied locally at the time 0, 8, and 16 hours after burn or sham injury. Cardiomyocytes were harvested 24 hours after burn or sham injury. Using IonWizard^® Software and the IonOptix^® Acquisition System, 10–15 sarcomeres were included in a rectangle-shaped region of interest (ROI) and contractions were recorded for 75 s, averaged and analyzed. The total number of analyzed cells per group and time point was n=45. *p<0.05 burn SB vs. burn vehicle (ANOVA).

Figure 3

In vitro measurement of peak sarcomere shortening values (A, D), contraction velocity (B, E) and relaxation velocity (C, F) in normal cardiomyocytes. Shown in the left panel are CMs exposed to homogenates from normal skin or burn skin treated with activated p38 MAPK-inhibitor (SB) or vehicle (applied locally at the time 0, 8, and 16 hours after burn or sham injury). Shown in the right panel are CMs exposed to serum isolated from normal or burn injured rats treated topically with vehicle or SB at time 0, 8, and 16 hours after burn or sham injury. The serum was collected 24 hours after burn or sham injury. Cardiomyocytes were harvested from healthy rats and incubated with 200 μL of skin homogenate or 100 μL serum for 18 hours. Using IonWizard^® Software and the IonOptix^® Acquisition System, 10–15 sarcomeres were included in a rectangle-shaped region of interest (ROI) and contractions were recorded for 75 s, averaged and analyzed. The total number of analyzed cells per group and time point was n=30. *p<0.05 burn SB vs. burn vehicle (ANOVA).

Figure 4

Expression of base p38 MAPK, phosphorylated p38 MAPK and phosphorylated MAPKAPK protein expression in cardiomyocytes using Western blotting. Ventricular cardiomyocytes were harvested from sham or burn injury rats 24 hours after injury. Burn injury animals were treated with vehicle or topical SB at time 0, 8, and 16 hours after burn injury Results for cardiomyocytes from sham rats are representative for all time points. Representative gel patterns from Western blots are shown in the lower part of Figure 4B. Results were quantified using digital pixel density and image analysis software, normalized to housekeeping GAPDH and expressed as ratios of protein/GAPDH pixel density.