Testing the role of heart mitochondrial stability and function in heart failure of ectotherms exposed to heat stress

Abstract

Hearts appear to be the first organs to fail in heat stressed animals. 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) temperatures (THF) are only a few degrees from current environmental temperatures. While the ectothermic cardiovascular system may act as an ‘ecological thermometer’, the exact mechanism mediating HF remains unresolved. This thesis hypothesised that heat-stressed cardiac mitochondria drives HF in ectotherms and first investigated this in a common New Zealand fish Notolabrus celidotos. This thesis further tested cardiac mitochondria in wrasses from cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats to explore the effects of temperature across species from differing thermal habitats. Then, utilising N. celidotus, a species’ capacity to acclimate cardiac mitochondria was assessed following acclimation to mean seasonal temperatures. Finally, this thesis compared the thermal tolerance of heart and cardiac mitochondrial function in native (Ovalipes catharus) and invasive (Charybdis japonica) paddle crabs to test the role of mitochondria in providing a species an ecological advantage. High resolution respirometry coupled to fluorimeters were used to assess the temperature-mediated changes in cardiac mitochondrial respiration, ROS and ATP production, and these changes were overlaid with the THF (27.8 ± 0.4 oC) of N. celidotus. Even at saturating oxygen levels, several mitochondrial components were compromised before the onset of THF suggesting that impairment of oxygen consumption by heart mitochondria preceded oxygen limitation. Importantly, the capacity to efficiently produce ATP in the heart was limited at 25 oC, and this was prior to the acute THF for N. celidotus. Membrane leakiness increased significantly at 25 oC, as did cytochrome c release and permeability to NADH. Maximal flux rates and the capacity for the electron transport system (ETS) to uncouple were also altered at 25 oC. These data indicate that mitochondrial membrane integrity is lost, depressing ATP synthesis capacity and promoting cytochrome c release, prior to THF. It was concluded that mitochondria can mediate HF in heat stressed hearts in fish and plays a significant role in thermal stress tolerance. The contribution of cardiac mitochondrial dysfunction to heat stress induced HF was compared in cold temperate (N. fucicola), temperate (N. celidotus) and tropical (T. lunare) wrasse species. 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 ETS flux. In situ assays conducted across a range of temperatures using naïve 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. These data indicated that species distribution may be related to the thermal limits of mitochondrial stability and function. While acute thermal stress may drive HF, ectotherms have a capacity to acclimate. The contributing role of compromised cardiac mitochondrial function to HF in N. celidotus acclimated to mean winter (cold acclimated CA, 15 oC) and summer (warm acclimated WA, 21 oC) temperatures was assessed. Heat stress mediated HF occurred at a THF of 26.7 ± 0.4 oC in CA fish, and at 28.2 ± 0.6 oC WA fish. Biochemical analyses revealed that WA N. celidotus had elevated plasma lactate indicating increased dependence on anaerobic pathways. When cardiac mitochondria were tested with increasing temperatures, relative to WA fish, CA fish maintained higher RCR values at higher temperatures. However, apparent breakpoints in the RCR with substrates supporting Complex I (CI) oxygen flux occurred below THF for both acclimated groups. WA cardiac mitochondria were less sensitive to increasing temperature for respirational flux supported by CI, CII, and chemically uncoupled flux through the ETS. These findings concluded that while acclimation to summer temperatures alters cardiac mitochondrial function in N. celidotus, these may come at an energetic cost, thereby increasing susceptibility of this species to further habitat warming. To understand if more thermo-stable mitochondria provide an advantage to invasive species in warming oceans, the influence of temperature on heart function and cardiac mitochondria were compared between the native New Zealand paddle crab Ovalipes catharus and the invasive paddle crab Charybdis japonica. Doppler ultrasound showed that with increasing temperature O. catharus and C. japonica elevated their heart rates (p≤0.05). However, C. japonica showed greater plasticity in heartbeat duration, and contraction rate with increasing temperature, while O. catharus was more inclined to increase heart rate, and already had a shorter more rapid contraction at 19 oC. In situ testing of mitochondrial function showed that Leak-I was highest for O. catharus at all temperatures. C. japonica showed a greater inner mitochondrial membrane integrity which suggested tighter coupling of OXP. Although CI lost function on exposure to 30 oC in both species, this occurred more rapidly in O. catharus. Additionally, O. catharus had higher CCO rates at all temperatures compared to C. japonica indicating elevated concentrations of CCO in O. catharus. The scope to increase CCO flux was greater in the more stenothermal O. catharus than in C. japonica. Overall, the substantial differences in heart function and cardiac mitochondria between crab species indicated that mitochondrial integrity may limit survival and future distributions of the native species. Based on the above investigations, it can be concluded that heart mitochondrial dysfunction contributes significantly to HF in marine ectotherms exposed to increasing temperatures, thereby defining the thermal limits of the ectothermic heart. All mitochondrial components were studied at maximum oxygen saturation and therefore, the changes observed were a direct impact of elevated temperature stress. This finding is contrary to the previously held notion that whole-animal tolerance at upper thermal limits resulted from a mismatch between oxygen demand and oxygen supply to the tissues of ectotherms (Pörtner and Farrell, 2008; Pörtner and Knust, 2007; Pörtner et al., 2004). Currently, emerging studies indicate that at temperatures where aerobic scope and cardiac function are maximised, ectotherms face restricted growth (Healy and Schulte, 2012), a decrease in population abundance (Clark et al., 2013; Gräns et al., 2014) and based on evidence from this thesis, cardiac mitochondria of the ectotherm heart is also compromised (Iftikar and Hickey, 2013; Iftikar et al., 2010; Iftikar et al., 2014).