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.

SUMMARYEarlier 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 P O2 , indicating a comparable reduction in whole body Na + efflux rate. However, branchial O 2 transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O 2 permeability. Direct measurements of 22 Na 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 (P O2 =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 [Ca 2+ ], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K + loss rates, and 3 H 2 O 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 [ 3 H]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 O 2 exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

For many aquatic species, the upper thermal limit (T max ) and the heart failure temperature (T HF ) are only a few degrees away from the species' current environmental temperatures. While the mechanisms mediating temperature-induced heart failure (HF) remain unresolved, energy flow and/or oxygen supply disruptions to cardiac mitochondria may be impacted by heat stress. Recent work using a New Zealand wrasse (Notolabrus celidotus) found that ATP synthesis capacity of cardiac mitochondria collapses prior to T HF . However, whether this effect is limited to one species from one thermal habitat remains unknown. The present study confirmed that cardiac mitochondrial dysfunction contributes to heat stress-induced HF in two additional wrasses that occupy cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats. With exposure to heat stress, T. lunare had the least scope to maintain heart function with increasing temperature. Heat-exposed fish of all species showed elevated plasma succinate, and the heart mitochondria from the cold temperate N. fucicola showed decreased phosphorylation efficiencies (depressed respiratory control ratio, RCR), cytochrome c oxidase (CCO) flux and electron transport system (ETS) flux. In situ assays conducted across a range of temperatures using naive tissues showed depressed complex II (CII) and CCO capacity, limited ETS reserve capacities and lowered efficiencies of pyruvate uptake in T. lunare and N. celidotus. Notably, alterations of mitochondrial function were detectable at saturating oxygen levels, indicating that cardiac mitochondrial insufficiency can occur prior to HF without oxygen limitation. Our data support the view that species distribution may be related to the thermal limits of mitochondrial stability and function, which will be important as oceans continue to warm.

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SUMMARYIt was hypothesised that chronic hypoxia acclimation (preconditioning) would alter the behavioural low-O 2 avoidance strategy of fish as a result of both aerobic and anaerobic physiological adaptations. Avoidance and physiological responses of juvenile snapper (Pagrus auratus) were therefore investigated following a 6week period of moderate hypoxia exposure (10.2-12.1kPa P O2 , 21±1°C) and compared with those of normoxic controls (P O2 =20-21kPa, 21±1°C). The critical oxygen pressure (P crit ) limit of both groups was unchanged at ~7kPa, as were standard, routine and maximum metabolic rates. However, hypoxia-acclimated fish showed increased tolerances to hypoxia in behavioural choice chambers by avoiding lower P O2 levels (3.3±0.7 vs 5.3±1.1kPa) without displaying greater perturbations of lactate or glucose. This behavioural change was associated with unexpected physiological adjustments. For example, a decrease in blood O 2 carrying capacity was observed after hypoxia acclimation. Also unexpected was an increase in whole-blood P 50 following acclimation to low O 2 , perhaps facilitating Hb-O 2 off-loading to tissues. In addition, cardiac mitochondria measured in situ using permeabilised fibres showed improved O 2 uptake efficiencies. The proportion of the anaerobic enzyme lactate dehydrogenase, at least relative to the aerobic marker enzyme citrate synthase, also increased in heart and skeletal red muscle, indicating enhanced anaerobic potential, or in situ lactate metabolism, in these tissues. Overall, these data suggest that a prioritization of O 2 delivery and O 2 utilisation over O 2 uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O 2 tolerance.