Fathima I. Iftikar

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.

Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus): new angles to the osmorespiratory compromise

SUMMARY Earlier studies demonstrated that oscars, endemic to ion-poor Amazonian waters, are extremely hypoxia tolerant, and exhibit a marked reduction in active unidirectional Na+ uptake rate (measured directly) but unchanged net Na+ balance during acute exposure to low PO2, indicating a comparable reduction in whole body Na+ efflux rate. However, branchial O2 transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O2 permeability. Direct measurements of 22Na appearance in the water from bladder-catheterized fish confirmed a rapid 55% fall in unidirectional Na+ efflux rate across the gills upon acute exposure to hypoxia (PO2=10–20 torr; 1 torr=133.3 Pa), which was quickly reversed upon return to normoxia. An exchange diffusion mechanism for Na+ is not present, so the reduction in efflux was not directly linked to the reduction in Na+ influx. A quickly developing bradycardia occurred during hypoxia. Transepithelial potential, which was sensitive to water [Ca2+], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K+ loss rates, and 3H2O exchange rates (diffusive water efflux rates) across the gills fell by 55–75% during hypoxia, with recovery during normoxia. Osmotic permeability to water also declined, but the fall (30%) was less than that in diffusive water permeability (70%). In total, these observations indicate a reduction in gill transcellular permeability during hypoxia, a conclusion supported by unchanged branchial efflux rates of the paracellular marker [3H]PEG-4000 during hypoxia and normoxic recovery. At the kidney, glomerular filtration rate, urine flow rate, and tubular Na+ reabsorption rate fell in parallel by 70% during hypoxia, facilitating additional reductions in costs and in urinary Na+, K+ and ammonia excretion rates. Scanning electron microscopy of the gill epithelium revealed no remodelling at a macro-level, but pronounced changes in surface morphology. Under normoxia, mitochondria-rich cells were exposed only through small apical crypts, and these decreased in number by 47% and in individual area by 65% during 3 h hypoxia. We suggest that a rapid closure of transcellular channels, perhaps effected by pavement cell coverage of the crypts, allows conservation of ions and reduction of ionoregulatory costs without compromise of O2 exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug-drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and β-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

Respiratory responses to progressive hypoxia in the Amazonian oscar, Astronotus ocellatus.

This study determined the respiratory responses to progressive hypoxia in oscar, an extremely hypoxia-tolerant Amazonian cichlid. Oscar depressed oxygen consumption rates (MO2), beginning at a critical O2 tension (Pcrit) of 46Torr, to only 14% of normoxic rates at 10Torr. Total ventilation (Vw) increased up to 4-fold, entirely due to a rise in ventilatory stroke volume (no change in ventilatory frequency), and water convection requirement (Vw/MO2) increased substantially (up to 15-fold). Gill O2 extraction fell steadily, from 60% down to 40%. Although O2 transfer factor (an index of gill O2 diffusion capacity) increased transiently in moderate hypoxia, it decreased at 10Torr, which may have caused the increased expired-arterial PO2 difference. Venous PO2 was always very low (< or =7Torr). Anaerobic metabolism made a significant contribution to ATP supply, indicated by a 3-fold increase in plasma lactate that resulted in an uncompensated metabolic acidosis. Respiration of isolated gill cells was not inhibited until below 5Torr; because gill water PO2 always exceeded this value, hypoxic ion flux arrest in oscars [Wood et al., Am. J. Physiol. Reg. Integr. Comp. Physiol. 292, R2048-R2058, 2007] is probably not caused by O2 limitation in ionocytes. We conclude that metabolic depression and tolerance of anaerobic bi-products, rather than a superior capacity for O2 supply, allow oscar to thrive in extreme hypoxia in the Amazon.

The Ionoregulatory Responses to Hypoxia in the Freshwater Rainbow Trout Oncorhynchus mykiss

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Low O2 acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function

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Temperature acclimation of mitochondria function from the hearts of a temperate wrasse (Notolabrus celidotus)

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