Acute Endothelial Cell Dysfunction Caused by a Protein Extract from Damaged Skeletal Muscle

Abstract

INTRODUCTION: Trauma is the fourth leading cause of death. “DAMPs” initiate a delayed SIRS. However, the blockade of inflammatory responses only minimally alters mortality. Our aim was to investigate the acute effects of a select protein extract from damaged skeletal muscle on endothelial cell function. METHODS: A murine trauma model was constructed consisting of the dorsal subcutaneous implantation of a syngeneic donor skeletal muscle matrix. A specific skeletal muscle matrix protein extract was isolated by centrifugation and size exclusion. Animals were studied immediately or 24 hours after resuscitation. Intestinal microvascular leak was assessed by vessel fluorophore illuminated confocal microscopy. Endothelial cell dysfunction was studied in human umbilical vein endothelial cells, fura-2–labeled human intestinal microvascular endothelial cells, and GCamp8 jejunal vessel endothelial cells. Gastrointestinal transit assays generated distribution histograms and calculated geometric centers (n = 3 to 4). RESULTS: Muscle matrix 15% body weight and protein extract IV injection (100 µL) caused a perfuse microvascular leak of the 70-kDa fluorophore and additionally caused a significant delay in gastrointestinal transit (GC = 4.8 ± 0.89 and 4.3 ± 1.34). Extract IV injection greater than 100 µL caused death without recovering mobility. Human umbilical vein endothelial cells acutely contracted by 20% to the trauma extract. Human intestinal microvascular endothelial cells incubated 6 hours in 25% normal human serum developed robust rapid calcium transients to platelet activating factor (50 nM). Human intestinal microvascular endothelial cells incubated in human trauma serum had slowly developing, muted transients to platelet aggregating factor. GCamp8 endothelial cell calcium sparks acutely increased in frequency on trauma extract exposure. CONCLUSION: Damaged skeletal muscle releases a protein that acutely causes endothelial cell dysfunction leading to microvascular leak, ileus, and a precipitous death.