Ashley N. Guillory

Reversal of Growth Arrest with the Combined Administration of Oxandrolone and Propranolol in Severely Burned Children

Background: The hypercatabolic response in severely burned pediatric patients is associated with increased production of catecholamines and corticosteroids, decreased formation of testosterone, and reduced strength alongside growth arrest for up to 2 years after injury. We have previously shown that, in the pediatric burned population, the administration of the testosterone analog oxandrolone improves lean body mass accretion and bone mineral content and that the administration of the &bgr;1-, &bgr;2-adrenoceptor antagonist propranolol decreases cardiac work and resting energy expenditure while increasing peripheral lean mass. Here, we determined whether the combined administration of oxandrolone and propranolol has added benefit. Methods: In this prospective, randomized study of 612 burned children [52% ± 1% of total body surface area burned, ages 0.5–14 years (boys); ages 0.5–12 years (girls)], we compared controls to the individual administration of these drugs, and the combined administration of oxandrolone and propranolol at the same doses, for 1 year after burn. Data were recorded at discharge, 6 months, and 1 and 2 years after injury. Results: Combined use of oxandrolone and propranolol shortened the period of growth arrest by 84 days (P = 0.0125 vs control) and increased growth rate by 1.7 cm/yr (P = 0.0024 vs control). Conclusions: Combined administration of oxandrolone and propranolol attenuates burn-induced growth arrest in pediatric burn patients. The present study is registered at clinicaltrials.gov: NCT00675714 and NCT00239668.

The pre-synaptic Munc13-1 binds alcohol and modulates alcohol self-administration in Drosophila

Munc13‐1 is a pre‐synaptic active‐zone protein essential for neurotransmitter release and involved in pre‐synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13‐1 with EC50s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13‐1 C1 exclusively at Glu‐582, which was identified by mass spectrometry. Mutation of Glu‐582 to alanine, leucine, and histidine reduced the alcohol binding two‐ to five‐fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild‐type Munc13‐1 compared with the mutants. If Munc13‐1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss‐of‐function mutation in the conserved Dunc‐13 in Drosophila melanogaster. The Dunc‐13P84200/+ heterozygotes have 50% wild‐type levels of Dunc‐13 mRNA and display a very robust increase in ethanol self‐administration. This phenotype is reversed by the expression of the rat Munc13‐1 protein within the Drosophila nervous system. The present studies indicate that Munc13‐1 C1 has binding site(s) for alcohols and Munc13‐1 activity is sufficient to restore normal self‐administration to Drosophila mutants deficient in Dunc‐13 activity.

Enhanced Cardiac Function in Gravin Mutant Mice Involves Alterations in the β-Adrenergic Receptor Signaling Cascade

Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (β2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in β-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute β-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca2+ in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased β2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent β2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin’s scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute β-AR stimulation by decreasing β2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.

Protein Kinase A and Phosphodiesterase-4D3 Binding to Coding Polymorphisms of Cardiac Muscle Anchoring Protein (mAKAP)

Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of this study was to examine the effects of naturally occurring mutations in human mAKAP on PKA and PDE4D3 signaling. We have recently identified potentially important human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to β-adrenergic receptor signaling. Three mutations (P1400S, S2195F, and L717V) were cloned and transfected into a mammalian cell line for the purpose of comparing whether those substitutions disrupt mAKAP binding to PKA or PDE4D3. Immunoprecipitation study of mAKAP-P1400S, a mutation located in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding to PDE4D3, with no significant changes in PKA binding or PKA activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site, showed significant increase in both binding propensity to PKA and PKA activity. Additionally, mAKAP-L717V, a mutation flanking the mAKAP-spectrin repeat domain, exhibited a significant increase in PKA binding compared to wild type, but there was no change in PKA activity. We also demonstrate specific binding of wild-type mAKAP to PDE4D3. Binding results were demonstrated using immunoprecipitation and confirmed with surface plasmon resonance (Biacore-2000); functional results were demonstrated using activity assays, Ca(2+) measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.

Human herpes viruses in burn patients: A systematic review

OBJECTIVE The contribution of human herpes viruses, including herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV) to morbidity and mortality after burns remains controversial. This systematic review was undertaken to assess evidence of herpes virus-related morbidity and mortality in burns. MATERIALS AND METHODS PubMed, Ovid, and Web of Science were searched to identify studies of HSV, CMV, or VZV infections in burn patients. Exclusion criteria included: A level of evidence (LoE) of IV or V; nonhuman in vivo studies; and non-English articles. There was no limitation by publication date. RESULTS Fifty articles were subjected to full-text analysis. Of these, 18 had LoE between I-III and were included in the final review (2 LoE I, 16 LoE II-III). Eight had a prospective study design, 9 had a retrospective study design, and 1 included both. CONCLUSIONS No direct evidence linked CMV and HSV infection with increased morbidity and mortality in burns. Following burn, CMV reactivation was more common than a primary CMV infection. Active HSV infection impaired wound healing but was not directly correlated to mortality. Infections with VZV are rare after burns but when they occur, VZV infections were associated with severe complications including mortality. The therapeutic effect of antiviral agents administered after burns warrants investigation via prospective randomized controlled trials.

Cardiovascular Dysfunction Following Burn Injury: What We Have Learned from Rat and Mouse Models

Severe burn profoundly affects organs both proximal and distal to the actual burn site. Cardiovascular dysfunction is a well-documented phenomenon that increases morbidity and mortality following a massive thermal trauma. Beginning immediately post-burn, during the ebb phase, cardiac function is severely depressed. By 48 h post-injury, cardiac function rebounds and the post-burn myocardium becomes tachycardic and hyperinflammatory. While current clinical trials are investigating a variety of drugs targeted at reducing aspects of the post-burn hypermetabolic response such as heart rate and cardiac work, there is still a paucity of knowledge regarding the underlying mechanisms that induce cardiac dysfunction in the severely burned. There are many animal models of burn injury, from rodents, to sheep or swine, but the majority of burn related cardiovascular investigations have occurred in rat and mouse models. This literature review consolidates the data supporting the prevalent role that β-adrenergic receptors play in mediating post-burn cardiac dysfunction and the idea that pharmacological modulation of this receptor family is a viable therapeutic target for resolving burn-induced cardiac deficits.

Delta-opioid augments cardiac contraction through β-adrenergic and CGRP-receptor co-signaling

Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 μg/kg) with or without 10-min pretreatment with either β-adrenergic receptor (β-AR) blocker propranolol (2mg/kg) or CGRP-receptor (CGRPR) blocker CGRP(8-37) (300 μg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p<0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p<0.0001) by 10 min, and 68 ± 19% (p<0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9±5% (p<0.01) by 5 min and 11 ± 6% (p<0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p<0.05) and 28±5% (p<0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p=0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP(8-37), or combined propranolol+CGRP(8-37). Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol+CGRP(8-37). DOR agonists augment myocardial contraction through enhanced β-AR and CGRPR co-signaling.

Biventricular differences in β-adrenergic receptor signaling following burn injury

Burn injury detrimentally affects the myocardium, primarily due to over-activation of β-adrenergic receptors (β-AR). Autopsy reports from our institution reveal that patients often suffer from right ventricle (RV) failure. Since burn injury affects β-AR signaling in the left ventricle (LV), we proposed that β-AR signaling may also be altered in the RV. A rodent model with a scald burn of 60% of the total body surface area was used to test this hypothesis. Ventricles were isolated 7 days post-burn. We examined the expression of β-ARs via Western blotting and the mRNA expression of downstream signaling proteins via qRT-PCR. Cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activity were measured in membrane and cytosolic fractions, respectively, using enzyme immunoassay kits. β1-AR protein expression was significantly increased in the RV following burn injury compared to non-burned RV but not in the LV (p = 0.0022). In contrast, β2-AR expression was unaltered among the groups while Gαi expression was significantly higher in the LV post-burn (p = 0.023). B-arrestin-1 and G-protein coupled receptor kinase-2 mRNA expression were significantly increased in the left ventricle post-burn (p = 0.001, p<0.0001, respectively). cAMP production and PKA activity were significantly lower in the LV post-burn (p = 0.0063, 0.0042, respectively). These data indicate that burn injury affects the β-AR signaling pathway in the RV independently of the LV. Additionally, non-canonical β-AR signaling may be activated in the RV as cAMP production and PKA activity were unchanged despite changes in β1-AR protein expression.

The P50 Research Center in Perioperative Sciences: How the investment by the National Institute of General Medical Sciences in team science has reduced postburn mortality

ABSTRACT Since the inception of the P50 Research Center in Injury and Peri-operative Sciences (RCIPS) funding mechanism, the National Institute of General Medical Sciences has supported a team approach to science. Many advances in critical care, particularly burns, have been driven by RCIPS teams. In fact, burns that were fatal in the early 1970s, prior to the inception of the P50 RCIPS program, are now routinely survived as a result of the P50-funded research. The advances in clinical care that led to the reduction in postburn death were made by optimizing resuscitation, incorporating early excision and grafting, bolstering acute care including support for inhalation injury, modulating the hypermetabolic response, augmenting the immune response, incorporating aerobic exercise, and developing antiscarring strategies. The work of the Burn RCIPS programs advanced our understanding of the pathophysiologic response to burn injury. As a result, the effects of a large burn on all organ systems have been studied, leading to the discovery of persistent dysfunction, elucidation of the underlying molecular mechanisms, and identification of potential therapeutic targets. Survival and subsequent patient satisfaction with quality of life have increased. In this review article, we describe the contributions of the Galveston P50 RCIPS that have changed postburn care and have considerably reduced postburn mortality.

The Potential Role of Oxidative Stress in Cardiac Hypertrophy: A Narrative Review

Background: A common phenotype associated with heart failure is the development of cardiac hypertrophy. Cardiac hypertrophy occurs in response to stress, such as hypertension, coro-nary vascular disease, or myocardial infarction. The most critical pathophysiological conditions in-volved may include dilated hypertrophy, fibrosis and contractile malfunction. The intricate pathophys-iological mechanisms of cardiac hypertrophy have been the core of several scientific studies, which may help in opening a new avenue in preventive and curative procedures. Objectives: To our knowledge from the literature, the development of cardiac remodeling and hyper-trophy is multifactorial. Thus, in this review, we will focus and summarize the potential role of oxida-tive stress in cardiac hypertrophy development. Conclusion: Oxidative stress is considered a major stimulant for the signal transduction in cardiac cells pathological conditions, including inflammatory cytokines, and MAP kinase. The understanding of the pathophysiological mechanisms which are involved in cardiac hypertrophy and remodeling process is crucial for the development of new therapeutic plans, especially that the mortality rates re-lated to cardiac remodeling/dysfunction remain high