Anthony J. R. Hickey

A systematic review of experimental treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome

Sepsis and multiple organ dysfunction syndrome (MODS) are major causes of morbidity and mortality in the intensive care unit. Recently mitochondrial dysfunction has been proposed as a key early cellular event in critical illness. A growing body of experimental evidence suggests that mitochondrial therapies are effective in sepsis and MODS. The aim of this article is to undertake a systematic review of the current experimental evidence for the use of therapies for mitochondrial dysfunction during sepsis and MODS and to classify these mitochondrial therapies. A search of the MEDLINE and PubMed databases (1950 to July 2009) and a manual review of reference lists were conducted to find experimental studies containing data on the efficacy of mitochondrial therapies in sepsis and sepsis-related MODS. Fifty-one studies were included in this review. Five categories of mitochondrial therapies were defined-substrate provision, cofactor provision, mitochondrial antioxidants, mitochondrial reactive oxygen species scavengers, and membrane stabilizers. Administration of mitochondrial therapies during sepsis was associated with improvements in mitochondrial electron transport system function, oxidative phosphorylation, and ATP production and a reduction in cellular markers of oxidative stress. Amelioration of proinflammatory cytokines, caspase activation, and prevention of the membrane permeability transition were reported. Restoration of mitochondrial bioenergetics was associated with improvements in hemodynamic parameters, organ function, and overall survival. A substantial body of evidence from experimental studies at both the cellular and the organ level suggests a beneficial role for the administration of mitochondrial therapies in sepsis and MODS. We expect that mitochondrial therapies will have an increasingly important role in the management of sepsis and MODS. Clinical trials are now required.

Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats.

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause‐and‐effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20‐month‐old SHR and Wistar‐Kyoto controls by iTRAQ™‐labelling combined with multidimensional LC/MS/MS. Of 137 high‐scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.

Do Mitochondria Limit Hot Fish Hearts? Understanding the Role of Mitochondrial Function with Heat Stress in Notolabrus celidotus

Hearts are the first organs to fail in animals exposed to heat stress. Predictions of climate change mediated increases in ocean temperatures suggest that the ectothermic heart may place tight constraints on the diversity and distribution of marine species with cardiovascular systems. For many such species, their upper temperature limits (Tmax) and respective heart failure (HF) temperature (THF) are only a few degrees from current environmental temperatures. While the ectothermic cardiovascular system acts as an “ecological thermometer,” the exact mechanism that mediates HF remains unresolved. We propose that heat-stressed cardiac mitochondria drive HF. Using a common New Zealand fish, Notolabrus celidotus, we determined the THF (27.5°C). Haemoglobin oxygen saturation appeared to be unaltered in the blood surrounding and within heat stressed hearts. Using high resolution respirometry coupled to fluorimeters, we explored temperature-mediated changes in respiration, ROS and ATP production, and overlaid these changes with THF. Even at saturating oxygen levels several mitochondrial components were compromised before THF. Importantly, the capacity to efficiently produce ATP in the heart is limited at 25°C, and this is prior to the acute THF for N. celidotus. Membrane leakiness increased significantly at 25°C, as did cytochrome c release and permeability to NADH. Maximal flux rates and the capacity for the electron transport system to uncouple were also altered at 25°C. These data indicate that mitochondrial membrane integrity is lost, depressing ATP synthesis capacity and promoting cytochrome c release, prior to THF. Mitochondria can mediate HF in heat stressed hearts in fish and play a significant role in thermal stress tolerance, and perhaps limit species distributions by contributing to HF.

Temperature sensitivity of cardiac mitochondria in intertidal and subtidal triplefin fishes

The heart is acutely sensitive to temperature in aquatic ectotherms and appears to fail before any other organ as the thermal maximum is reached, although the exact cause of this failure remains unknown. The heart is highly aerobic and therefore dependent on mitochondrial oxidative phosphorylation (OXPHOS) to meet energy requirements, but the role of cardiac mitochondria in limiting heart function at high temperatures remains unclear. We used permeabilised ventricle fibres to explore heart mitochondrial function in situ in three closely related species of small New Zealand triplefin fishes in response to temperature. We compared this to measures of whole animal respiration rates and critical oxygen tensions in these fishes. Bellapiscis medius, an intertidal species, had the greatest tolerance to hypoxia at higher temperatures and had more efficient OXPHOS at 30°C than the two subtidal species Forsterygion varium and F. malcolmi. B. medius also displayed the highest cytochrome c oxidase flux, which may in part explain how B. medius tolerates higher temperatures and hypoxia. Triplefin heart mitochondria exhibit decreased coupling to phosphorylation with increasing temperature. This most likely impairs ATP supply to the heart at elevated temperatures, potentially contributing to heart failure at ecologically relevant temperatures.

New Zealand triplefin fishes (family Tripterygiidae): contrasting population structure and mtDNA diversity within a marine species flock

Triplefin fishes (Family Tripterygiidae) dominate the New Zealand temperate coastal fish fauna in diversity (26 endemic species, 14 genera). Most species appear to have evolved as a local radiation and mostly occupy sympatric distributions throughout New Zealand. To investigate the forces driving current gene‐flow patterns and past evolutionary histories, we searched for common patterns of population genetic subdivision within eight species sampled throughout their distributions [mitchochondrial DNA (mtDNA) control region, n = 1086]. We hypothesised that common phylogeographical and population differentiation patterns would reveal past or ongoing physical processes, with differences reflecting stochastic or species‐specific processes. Striking differences between species were apparent, with strong phylogeographical structure detected in Grahamina capito and the estuarine species G. nigripenne. G. capito fell into three distinct geographically restricted lineages. G. nigripenne largely separated into northern and southern lineages (ΦST 0.834). Strong population structuring and isolation by distance was evident in Bellapiscis medius, B. lesleyae and Forsterygion lapillum (ΦST 0.686, 0.436 and 0.115, respectively). High gene flow was apparent in G. gymnota and Ruanoho whero, and F. varium. However, for the latter species, isolation was apparent with samples collected from the offshore Three Kings Islands. Overall, a strong relationship was found between habitat depth and population structure among species, and species inhabiting shallower water habitats showed lower genetic diversity with higher levels of population subdivision. High‐latitude populations generally showed low haplotype and nucleotide diversity. These data suggest that processes resulting from intraspecific differences in habitat preference, climatic histories and/or larval ecologies have subdivided populations of shallow water triplefin species.

Mice Lacking the Neuropeptide α-Calcitonin Gene-Related Peptide Are Protected Against Diet-Induced Obesity

Alpha-calcitonin gene-related peptide (alphaCGRP) is a neuropeptide that is expressed in motor and sensory neurons. It is a powerful vasodilator and has been implicated in diverse metabolic roles. However, its precise physiological function remains unclear. In this study, we investigated the role of alphaCGRP in lipid metabolism by chronically challenging alphaCGRP-specific knockout (alphaCGRP(-/-)) and control mice with high-fat diet regimens. At the start of the study, both animal groups displayed similar body weights, serum lipid markers, and insulin sensitivity. However, alphaCGRP(-/-) mice displayed higher core temperatures, increased energy expenditures, and a relative daytime (nonactive) depression in respiratory quotients, which indicated increased beta-oxidation. In response to fat feeding, alphaCGRP(-/-) mice were comparatively protected against diet-induced obesity with an attenuated body weight gain and an overall reduction in adiposity across all the three diets examined. AlphaCGRP(-/-) mice also displayed improved glucose handling and insulin sensitivity, lower im and hepatic lipid accumulation, and improved overall metabolic health. These findings define a new role for alphaCGRP as a mediator of energy metabolism and opens up therapeutic opportunities to target CGRP action in obesity.

Characterization of proteomic changes in cardiac mitochondria in streptozotocin-diabetic rats using iTRAQ™ isobaric tags.

Diabetes now affects more than 5% of the worlds population and heart failure is the most common cause of death amongst diabetic patients. Accumulating evidence supports a view that myocardial mitochondrial structural and functional changes are central to the onset of diabetic heart failure, but the exact nature of these changes at the proteomic level remains unclear.Here we report on proteomic changes in diabetic rat heart mitochondria following 120 days of streptozotocin‐diabetes using the recently developed iTRAQ™ labeling method, which permits quantification of proteins directly from complex mixtures, bypassing the limitations associated with gel‐based methods such as 2‐DE. Of 252 unique proteins identified, 144 were represented in at least three of six individual paired experiments. Relative amounts of 65 proteins differed significantly between the groups, confirming that the cardiac mitochondrial proteome is indeed impacted by diabetes. The most significant changes were increased protein levels of enzymes involved in mitochondrial oxidation of long‐chain fatty acids, which was also confirmed by enzyme assays, and decreased levels of multiple enzymes involved in oxidative phosphorylation and catabolism of short‐chain fatty acids and branched‐chain amino acids. We also found significant changes in levels of several enzymes linked to oxidative stress.

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of β-sheet-containing aggregates by human amylin: A potential role for defective chaperone biology in Type 2 diabetes

Misfolding of the islet β-cell peptide hA (human amylin) into β-sheet-containing oligomers is linked to β-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent β-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective β-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

Antiphospholipid antibodies internalised by human syncytiotrophoblast cause aberrant cell death and the release of necrotic trophoblast debris.

Antiphospholipid antibodies (aPL) are the strongest maternal risk factor for pre-eclampsia, a hypertensive disease of human pregnancy. Pre-eclampsia is triggered by a toxic factor released from the placenta that activates the maternal endothelium. Antiphospholipid antibodies cause the release of necrotic trophoblast debris from the placental syncytiotrophoblast and this debris can activate endothelial cells. In this study, we investigated how aPL affects syncytiotrophoblast death and production of necrotic trophoblast debris by examining the interaction between aPL and human first trimester placental explants. Human polyclonal and murine monoclonal aPL, but not control antibodies, were rapidly internalised by the syncytiotrophoblast. Inhibitors of endocytosis or the low-density lipoprotein receptor (LDLR) family, but not toll-like receptors, decreased the internalisation of aPL and prevented the release of necrotic trophoblast debris from the syncytiotrophoblast. Once internalised, aPL increased inner mitochondrial membrane leak and Cytochrome c release while depressing oxidative flux through Complex IV of the electron transport system in syncytiotrophoblast mitochondria. These data suggest that the human syncytiotrophoblast internalises aPL by antigen-dependent endocytosis involving LDLR family members. Once internalised by the syncytiotrophoblast, aPL affects the death-regulating mitochondria, causing extrusion of necrotic trophoblast debris which can activate maternal endothelial cells thereby contributing to the pathogenesis of pre-eclampsia.

Proteins Associated with Immunopurified Granules from a Model Pancreatic Islet β-Cell System: Proteomic Snapshot of an Endocrine Secretory Granule

beta-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E beta-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and beta-cell death in type-2 diabetes.