L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.

To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+ transient compared to the GFP control. The Ca2+ transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with ß-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.

Binge alcohol exposure modulates rodent expression of biomarkers of the immunoinflammatory response to orthopaedic trauma.

BACKGROUND: Alcohol is a known modulator of the immune system and host-defense response. Alcohol abuse is common in trauma patients, although the influence of alcohol intoxication on the inflammatory response following major orthopaedic injury remains unknown. The aim of this investigation was to examine the influence of binge alcohol exposure on biomarkers of the systemic inflammatory response following bilateral traumatic femoral fracture in a rodent model. METHODS: Ninety-two Sprague-Dawley rats were administered intraperitoneal injections of either saline solution or alcohol for three days. These animals then underwent a sham procedure or bilateral femoral intramedullary pinning and mid-diaphyseal closed fracture via blunt guillotine. The animals were killed at specific time points after the injury. Serum and lung tissue were collected, and twenty-five inflammatory markers were analyzed by immunoassay. Histological sections of lung tissue were evaluated by a board-certified pathologist. RESULTS: Bilateral femoral fracture significantly (p < 0.05) increased multiple serum biomarkers of inflammation. Binge alcohol treatment prior to injury significantly suppressed the increase in serum levels of interleukin (IL)-6, white blood cells, IL-2, IL-10, and C-reactive protein after the fracture. However, alcohol-treated animals were found to have increased pulmonary levels of IL-6, IL-1ß, IL-2, and macrophage inflammatory protein-1α following bilateral femoral fracture. In addition, lung tissue harvested following alcohol treatment and injury demonstrated increased pathologic changes, including parenchymal, alveolar, and peribronchial leukocyte infiltration and significantly elevated pulmonary wet-to-dry ratio, indicative of pulmonary edema. CONCLUSIONS: Our results indicate that acute alcohol intake prior to bilateral femoral fracture with fixation in rats modulates the inflammatory response after injury in a tissue-dependent manner. Although serum biomarkers of inflammation were suppressed in alcohol-treated animals following injury, several measures of pulmonary inflammation including cytokine levels, histological changes, and findings of pulmonary edema were significantly increased following fracture with the presence of alcohol.

Correlation of measurable serum markers of inflammation with lung levels following bilateral femur fracture in a rat model.

INTRODUCTION: Evaluation of the systemic inflammatory status following major orthopedic trauma has become an important adjunct in basing post-injury clinical decisions. In the present study, we examined the correlation of serum and lung inflammatory marker levels following bilateral femur fracture. MATERIALS AND METHODS: 45 Sprague Dawley rats underwent sham operation or bilateral femoral intramedullary pinning and mid-diaphyseal closed fracture via blunt guillotine. Animals were euthanized at specific time points after injury. Serum and lung tissue were collected, and 24 inflammatory markers were analyzed by immunoassay. Lung histology was evaluated by a blinded pathologist. RESULTS: Bilateral femur fracture significantly increased serum markers of inflammation including interleukin (IL)-2, IL-6, IL-10, GM-CSF, KC/GRO, MCP-1, and WBC. Femur fracture significantly increased serum and lung levels of IL-1a and KC/GRO at 6 hours. Lung levels of IL-6 demonstrated a trend towards significance. Histologic changes in pulmonary tissue after fracture included pulmonary edema and bone elements including cellular hematopoietic cells, bone fragments and marrow emboli. DISCUSSION AND CONCLUSION: Our results indicate that bilateral femur fracture with fixation in rats results in increases in serum markers of inflammation. Among the inflammatory markers measured, rise in the serum KC/GRO (CINC-1), a homolog to human IL-8, correlated with elevated levels of lung KC/GRO. Ultimately, analysis of serum levels of KC/GRO (CINC-1), or human IL-8, may be a useful adjunct to guide clinical decisions regarding surgical timing.

Heterogeneous translational dynamics of rhodamine B in polyelectrolyte multilayer thin films observed by single molecule microscopy.

The lateral diffusion dynamics of rhodamine B (RB) in polyelectrolyte multilayer (PEM) thin films has been studied with single-molecule confocal fluorescence microscopy. The films were made with sodium poly(sodium 4-styrenesulfonate) (PSS) and poly(diallydimethlyammonium chloride) (PDDA). Analysis of the real-time emission intensity traces reveals three diverse components of translational motion: (1) fast diffusion of RB through the confocal detection volume; (2) reversible tracer adsorption processes; and (3) nanoconfined diffusion. These processes cover a wide range of time scales. Analysis via fluorescence correlation spectroscopy (FCS) involves multicomponent fitting of the autocorrelated emission data. The model includes a free Brownian diffusion parameter, D, and two rate constants of desorption, k(-1) and k(-2). For RB in a PSS/PDDA thin film made with 0.01 M NaCl in the polyelectrolyte buildup solutions, D = 1.7 x 10(-7) cm(2)/s, k(-1) = 30 s(-1), and k(-2) = 0.1 s(-1). FCS was also performed on RB/PEM samples made with NaCl concentrations of the buildup solutions ranging from 0.01 to 0.7 M. A weak dependence of D and k(-1) on NaCl concentration was observed while k(-2) increased linearly with [NaCl].

Effect of corticosteroids on the biomechanical strength of rat rotator cuff tendon.

BACKGROUND: The effect of corticosteroids on tendon properties is poorly understood, and current data are contradictory and diverse. The biomechanical effect of steroids on rotator cuff tendon has not been studied, to our knowledge. The current study was undertaken to characterize the biomechanical effects of corticosteroid exposure on both uninjured and injured rat rotator cuff tendon. METHODS: One hundred and twenty-three male Sprague-Dawley rats were randomly assigned to four groups: control (C), tendon injury (I), steroid exposure (S), and tendon injury plus steroid exposure (I+S). Unilateral tendon injuries consisting of a full-thickness defect across 50% of the total width of the infraspinatus tendon were created. Steroid treatment consisted of a single dose of methylprednisolone placed into the subacromial space. At one, three, and five weeks postoperatively, the shoulders were harvested and the infraspinatus tendon was subjected to biomechanical testing. Two specimens from each group were used for histological analysis. RESULTS: At one week, maximum load, maximum stress, and stiffness were all significantly decreased in Group S compared with the values in Group C. Mean maximum load decreased from 37.9 N in Group C to 27.5 N in Group S (p < 0.0005). Mean maximum stress decreased from 18.1 MPa in Group C to 13.6 MPa in Group S (p < 0.0005). Mean stiffness decreased from 26.3 N/mm in Group C to 17.8 N/mm in Group S (p < 0.0005). At one week, mean maximum stress in Group I+S (17.0 MPa) was significantly decreased compared with the value in Group I (19.5 MPa) (p < 0.0005). At both the three-week and the five-week time point, there were no significant differences between Group C and Group S or between Group I and Group I+S with regard to mean maximum load, maximum stress, or stiffness. Histological analysis showed fat cells and collagen attenuation in Groups S and I+S. These changes appeared to be transient. CONCLUSIONS: A single dose of corticosteroids significantly weakens both intact and injured rat rotator cuff tendons at one week. This effect is transient as the biomechanical properties of the steroid-exposed groups returned to control levels by three weeks.

Ethanol potentiates the acute fatty infiltration of liver caused by burn injury: prevention by insulin treatment.

Burn injury is a significant and severe representation of critical illness. Nearly, 50% of patients admitted to hospitals for burn injuries have detectable levels of ethanol in their circulations and these patients have poorer clinical outcomes than burned individuals without measurable circulating ethanol. We report here data on a clinically relevant form of hepatic injury, the development of microvesicular steatosis, in a murine model wherein animals were either given ethanol or saline, and were subjected to burn or sham injury. Because better glycemic control with insulin has been shown in clinical studies to impart major clinical benefit, an additional group of burn ethanol animals were treated with insulin. Insulin significantly reduced blood glucose in injured animals to levels no different from those seen in animals that were neither ethanol exposed nor burned. A single intraperitoneal injection of ethanol was insufficient to raise blood alanine aminotransferase (ALT), measured as an index of liver injury. However, burn injury led to significant increases in ALT at 24 and 48 hours, which had returned to preinjury levels by 7 days. This ALT rise was completely prevented with insulin treatment. A single injection of ethanol did not evoke increased microvesicular steatosis but did potentiate the ability of burn to do so at 24 hours after injury. The burn induced increase in microvesicular steatosis was also seen at 48 hours, but had subsided by 7 days. The increased microvesicular steatosis was prevented by insulin therapy. Thus, ethanol potentiates the ability of burn to cause acute liver injury, which is completely preventable by insulin therapy. These findings may have substantial clinical significance and suggest this model may be useful for the study of the mechanisms of hepatic injury as well as the mechanisms, probably multiple, of insulin action in this setting.