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Characterizing the metabolic capacity of the anoxic hagfish heart.

Abstract

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes–processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid-base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfish’s exceptional anoxia tolerance.

Publication
Journal of Experimental Biology 218: 3754-3761
Date
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Hagfish are benthic fishes that spend considerable periods of time buried in the sediment and the putrefying carcasses of aquatic animals where they feed (Lesser et al., 1997; Martini, 1998). These environments are routinely hypoxic and even anoxic, so it is unsurprising that hagfish display an exceptional tolerance to hypoxia and anoxia. Hansen and Sidell (1983) demonstrated that anoxia and cyanide poisoning did not have an effect on the in situ function of the heart from the Atlantic hagfish Myxine glutinosa. Similarly, the Pacific hagfish Eptatretus stoutii has been shown to tolerate 36 h of anoxia at 10°C with a regular heartbeat and full recovery (Cox et al., 2010, 2011). Long-term anoxic survival like this demands the maintenance of certain organ functions within the animal, and one such critical organ is the heart. The maintenance of cardiac function under anoxic conditions allows for the mobilization of metabolic fuel stores (e.g. glycogen) from storage tissues (e.g. liver) and the removal of waste from metabolically active tissues (Stecyk et al., 2008). These two functions are critical to the anoxic survival of the animal and indeed, Cox et al. (2010) found that these are well maintained in anoxic hagfish: cardiac power output, a proxy for energy usage, decreased by only 25% in hagfish exposed to 36 h of anoxia. The maintenance of anoxic cardiac function in hagfish is thought to be associated with a relatively low cardiac metabolic requirement (Forster, 1991), such that the hagfish heart’s routine power output – the lowest ever measured in a fish – is within the heart’s maximum glycolytic potential (Farrell and Stecyk, 2007). The importance of glycolytic pathways to cardiac function in hagfish was demonstrated by Hansen and Sidell (1983). Using an in situ preparation on Atlantic hagfish, they found that despite inhibition of cardiac mitochondrial function with cyanide or sodium azide, cardiac output was maintained for at least 3 h. However, inhibiting glycolytic function with iodoacetate resulted in a significant decrease in cardiac output. The authors also found that the activity ratio of the enzymes pyruvate kinase (PK) to cytochrome c oxidase (CO) were 5.6-fold higher in the hagfish ventricle compared with that of cod. This was interpreted as the hagfish ventricle having a ‘more anaerobically geared’ metabolism (Hansen and Sidell, 1983). Together, these results suggest that anaerobic glycolysis plays an important role in maintaining cardiac function, and thus survival, in the anoxic hagfish.

Because the glycolytic capacity of the hagfish heart is thought to be capable of supporting routine cardiac function, and because this cardiac function is well maintained in anoxic environments, we hypothesized that the metabolic rate of the hagfish heart would be similarly maintained in anoxic environments. We tested this hypothesis by performing direct calorimetry on the excised, cannulated hearts of Pacific hagfish, E. stoutii, which are capable of beating for at least 24 h post-excision under anoxic conditions (Wilson, 2014). Direct calorimetry – the measurement of heat produced by, in this case, an excised heart – was used because it is the only technique that enables the continuous measurement of metabolic rate under anoxic conditions. In addition to the calorimetry measurements, we ran additional parallel anoxic exposures on excised, cannulated hearts and sampled them at three time points throughout the exposures to measure a variety of cardiac metabolites: lactate and glycogen levels were measured to estimate reliance on anaerobic ATP-production pathways, and intracellular pH (pHi) was measured to determine how the heart’s ability to maintain acid–base balance was influenced by anoxic exposure.

In addition to the excised heart experiments, we exposed two groups of hagfish to 36 h of normoxia and anoxia. Hagfish heart tissue was analysed for the same metabolites described above and blood was analysed for a number of haematological parameters. This allowed us to compare the response of the in vitro heart with that of the in vivo heart, which was important because the in vitro heart would experience an almost immediate lack of O2 upon induction of environmental anoxia, whereas in vivo, the heart becomes progressively hypoxic until true anoxia is reached when the fish’s large O2 stores are depleted; this could take a considerable period of time given the hagfish’s low haemoglobin P50 value [∼7–12 mmHg PO2 (Wells et al., 1986; Forster et al., 1992)], large blood volume and low metabolic rate (Cox et al., 2010, 2011). This fact made the in vivo exposures especially important to testing our hypothesis that metabolic activity is maintained in the hagfish heart during prolonged periods of anoxia.