Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Rapid cortisol signaling in response to acute stress involves changes in plasma membrane order in rainbow trout liver.

The activation of genomic signaling in response to stressor-mediated cortisol elevation has been studied extensively in teleosts. However, very little is known about the rapid signaling events elicited by this steroid. We tested the hypothesis that cortisol modulates key stress-related signaling pathways in response to an acute stressor in fish liver. To this end, we investigated the effect of an acute stressor on biophysical properties of plasma membrane and on stressor-related protein phosphorylation in rainbow trout (Oncorhynchus mykiss) liver. A role for cortisol in modulating the acute cellular stress response was ascertained by blocking the stressor-induced elevation of this steroid by metyrapone. The acute stressor exposure increased plasma cortisol levels and liver membrane fluidity (measured by anisotropy of 1,6-diphenyl-1,3,5-hexatriene), but these responses were abolished by metyrapone. Atomic force microscopy further confirmed biophysical alterations in liver plasma membrane in response to stress, including changes in membrane domain topography. The changes in membrane order did not correspond to any changes in membrane fatty acid components after stress, suggesting that changes in membrane structure may be associated with cortisol incorporation into the lipid bilayer. Plasma cortisol elevation poststress correlated positively with activation of intracellular stress signaling pathways, including increased phosphorylation of extracellular signal-related kinases as well as several putative PKA and PKC but not Akt substrate proteins. Together, our results indicate that stressor-induced elevation of plasma cortisol level is associated with alterations in plasma membrane fluidity and rapid activation of stress-related signaling pathways in trout liver.

Novel insights into cardiac remodelling revealed by proteomic analysis of the trout heart during exercise training.

The changes in the cardiac proteome of rainbow trout (Oncorhynchus mykiss) were quantified during the early phases (4, 7, and 14d) of a typical exercise-training regime to provide a comprehensive overview of the cellular changes responsible for developing a trained heart phenotype. Enhanced somatic growth during the 14d experiment was paralleled by cardiac growth to maintain relative ventricular mass. This was reflected in the cardiac proteome by the increased abundance of contractile proteins and cellular integrity proteins as early as Day 4, including a pronounced and sustained increase in blood vessel epicardial substance - an intercellular adhesion protein expressed in the vertebrate heart. An unexpected finding was that proteins involved in energy pathways, including glycolysis, ß-oxidation, the TCA cycle, and the electron transport chain, were generally present at lower levels relative to Day 0 levels, suggesting a reduced investment in the maintenance of energy production pathways. However, as the fish demonstrated somatic and cardiac growth during the exercise-training program, this change did not appear to influence cardiac function. The in-depth analysis of temporal changes in the cardiac proteome of trout during the early stages of exercise training reveals novel insights into cardiac remodelling in an important model species. BIOLOGICAL SIGNIFICANCE: Rainbow trout hearts have a remarkable ability for molecular, structural, and functional plasticity, and the inherent athleticism of these fish makes them ideal models for studies in comparative exercise physiology. Indeed, several decades of research using exercise-trained trout has shown both conserved and unique aspects of cardiac plasticity induced by a sustained increase in the workload of the heart. Despite a strong appreciation for the outcome of exercise training, however, the temporal events that generate this phenotype are not known. This study interrogates the early stages of exercise training using in-depth proteomic analysis to understand the molecular pathways of cardiac remodelling. Two major and novel findings emerge: (1) structural remodelling is initiated very early in training, as evidenced by a general increase in proteins associated with muscle contraction and integrity at Day 4, and (2) the abundance of proteins directly involved in energy production are decreased during 14d of exercise training, which contrasts the general acceptance of an exercise-induced increase in aerobic capacity of muscle, and suggests that regulation of energy pathways occurs at a different biological level than protein abundance.

Proteomic analysis of sockeye salmon serum as a tool for biomarker discovery and new insight into the sublethal toxicity of diluted bitumen.

Pipelines carrying diluted bitumen (dilbit) from Canada's oil sands traverse North America, including the freshwater habitat of Pacific salmon, posing a risk of environmental release and aquatic exposure. Swimming performance is impacted in juvenile sockeye (Oncorhynchus nerka) exposed to dilbit; therefore biomarkers of dilbit exposure will be valuable for monitoring at-risk salmon stocks. This study characterized changes in the serum proteome of sockeye exposed to a sub-lethal and environmentally relevant concentration of dilbit using isobaric tags for relative and absolute quantitation (iTRAQ), and included a range of experimental conditions to permit identification of biomarkers that are robust across time (1 and 4wk) and exercise level (at rest and following a swim test). Over 500 proteins were identified and quantified in sockeye serum, with dilbit exposure significantly altering the abundance of 24 proteins irrespective of time and exercise, including proteins associated with immune and inflammatory responses, coagulation, and iron homeostasis. An increase in creatine kinase (CK) activity in serum of dilbit-exposed salmon confirmed the higher CK protein abundance measured using iTRAQ. The combination of 4wk dilbit exposure and a swim test had a greater effect on the serum proteome than either treatment alone, including a marked increase in tissue leakage proteins, suggesting that aerobic exercise exacerbates the serum proteome response to dilbit, and the increased cellular damage could impede exercise recovery. This study provides a foundation for the development of bio-monitoring tools for salmon stock assessments, and offers new insights into the sub-lethal toxicity of crude oil exposure in fish.

Novel nongenomic signaling by glucocorticoid may involve changes to liver membrane order in rainbow trout.

Stress-induced glucocorticoid elevation is a highly conserved response among vertebrates. This facilitates stress adaptation and the mode of action involves activation of the intracellular glucocorticoid receptor leading to the modulation of target gene expression. However, this genomic effect is slow acting and, therefore, a role for glucocorticoid in the rapid response to stress is unclear. Here we show that stress levels of cortisol, the primary glucocorticoid in teleosts, rapidly fluidizes rainbow trout (Oncorhynchus mykiss) liver plasma membranes in vitro. This involved incorporation of the steroid into the lipid domains, as cortisol coupled to a membrane impermeable peptide moiety, did not affect membrane order. Studies confirmed that cortisol, but not sex steroids, increases liver plasma membrane fluidity. Atomic force microscopy revealed cortisol-mediated changes to membrane surface topography and viscoelasticity confirming changes to membrane order. Treating trout hepatocytes with stress levels of cortisol led to the modulation of cell signaling pathways, including the phosphorylation status of putative PKA, PKC and AKT substrate proteins within 10 minutes. The phosphorylation by protein kinases in the presence of cortisol was consistent with that seen with benzyl alcohol, a known membrane fluidizer. Our results suggest that biophysical changes to plasma membrane properties, triggered by stressor-induced glucocorticoid elevation, act as a nonspecific stress response and may rapidly modulate acute stress-signaling pathways.