Early structural changes of the heart after experimental polytrauma and hemorrhagic shock

Abstract

Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction.

Fig 1. Plasma levels of inflammatory mediators and hemoglobin.

Levels of (A) haemoglobin (Hb) in blood and (B) macrophage chemotactic protein 1 (MCP-1), (C) keratinocyte chemoattractant (KC) and (D) complement component C3a in plasma of animals 4 h after infliction of polytrauma and hemorrhagic shock (PTHS; n = 7 for Hb, n = 8 for MCP-1, KC and C3a) and native control animals (CTRL; n = 8 for Hb, n = 4 for MCP-1, n = 5 for C3a and KC). Results (B, C, D) are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. For statistical comparison of experimental means, unpaired t-tests (A, C, D) and Mann-Whitney rank sum test (B) were performed. *: p<0.05.

Fig 2. Histological and humoral markers of cardiac damage.

(A) Plasma levels of cardiac troponin I (cTnI) as a marker of cardiac damage from animals 4 h after the insult of polytrauma and hemorrhagic shock (PTHS; n = 8) and native controls (CTRL, n = 4). Results are presented as amount of protein per total protein in plasma to unmask diluting effects from volume resuscitation. (B) Representative images of immunohistochemical (IHC) assessment of tissue cleaved caspase 3. Magnification: 100x (bar: 50 μm). (C) Representative hematoxylin and eosin stained sections of cardiac tissue, showing no signs of marked histomorphological changes. Magnifications: 100x (upper images, bar: 50 μm) and 200x (lower images, bar: 100 μm) for each group. (D) Representative images and densitometric analysis of IHC preparations of tissue high-mobility group box 1 (HMGB1) showing increased signal in samples from PTHS animals. Magnification: 100x (bar: 50 μm). For histological evaluation: n = 5 (PTHS); n = 5 (CTRL). DSR: density sum red. Experimental means were compared for statistical significance using the unpaired t-test (D) and Mann-Whitney rank sum test (A). *: p<0.05.

Fig 3. Morphological alterations of cardiac gap junction proteins.

(A) Representative images of immunohistochemical (IHC) and immunofluorescence (IF) assessment of connexin 43 (Cx43) in cardiomyocytes show marked lateralization and disruption of gap junctions 4 h after experimental polytrauma and hemorrhagic shock (PTHS, n = 6) compared to native controls (CTRL, n = 5). Magnification (IHC): 100x (bar: 50 μm). Magnification (IF): 200x. (B) Densitometric analysis of IHC red signal and (C) evaluation of total amount of Cx43 in tissue homogenates by western blotting show slightly increased mean values for PTHS. Insets show representative bands from the same blot for CTRL and PTHS. n = 6 (PTHS); n = 4 (CTRL). DSR: density sum red. Experimental means were compared by unpaired t-test. *: p<0.05.