Evidence that myoglobin does not support heart performance at maximal levels of oxygen demand

Sculpin (Myoxocephalus octodecimspinosus Mitchell) weighing 186 ± 14g were caught by otter trawl in waters off St Andrews, NB, Canada. Fish were transported to Mount Allison University and held in filtered, recirculated sea water at 10-13°C. Ventricle strips were bathed in medium containing (inmmol 1⁻¹): NaCl, 150; MgSO₄, 2; KC1, 5; CaC1₂, 3; NaH₂PO₄, 0·17; Na₂HPO₄, 2·33; glucose, 10; and gassed with 0·5% CO₂:21% O₂ (balance N₂). After equilibration with the gas mixture, 11·2mmoll⁻¹ NaHCO₃ was added to bring the final pH to 7·8 at 10°C. Experiments were conducted in a 30-ml bath which was maintained at 10°C by water-jacketing. In some studies the gas mixture was changed to 0·5 % CO₂:99·5 % N₂ and various combinations of 0·5mmoll⁻¹ DNP and/or lmmoll⁻¹ hydroxyl-amine (final concentration) were added to the medium as the experiment proceeded. Addition of these compounds did not alter pH and hydroxylamine (1·0 mmol 1⁻¹) does not impede heart performance under normoxic conditions (Driedzic et al. 1982). The change in gas mixture resulted in a decrease in oxygen level to 0·9 ± 0·1 % within 3 min, as determined by oxygen electrodes. The new level was maintained for the balance of the experiment. Isolated sculpin hearts were perfused with media containing hydroxylamine to assess its efficacy in converting functional (iron II) myoglobin to the non-functional (iron III) state. Following perfusion hearts were blotted and subsequently homogenized ( 10 % w/v) in 75 mmol 1⁻¹ Tris, 5 mmol 1⁻¹ EDTA, 3mmoll⁻¹ MgCb, pH7·2 at 20°C. Homogenates were centrifuged at 10000 g for 10 min and myoglobin content of the supernatant was quantified by a direct spectrophotometric method (Sidell, 1980). The absorption spectrum of the supernatant was recorded between 490 and 610 nm and the myoglobin concentration was calculated from the net absorbance at 581 nm utilizing an empirically determined millimolar extinction coefficient (peak minus baseline; see Sidell, 1980) of 12-8 for oxymyoglobin. Typical absorption spectra for treated and control hearts are presented in Driedzic (1983). Iron II myoglobin content in control hearts was 78·7 ± 10·1 nmol g⁻¹ wet mass (N = 6). Hydroxylamine treatment decreased the level to 7·5 ± 1·2nmolg⁻¹ (N =6). Sculpin hearts lack coronary arteries and exchange gases and nutrients directly with the fluid in the ventricular lumen. It was assumed that myoglobin in ventricle strips would be altered in a similar fashion to that in perfused hearts. Spectral scans of tissue homogenates showed that DNP does not directly alter the oxidation state of myoglobin.

Hearts were exposed, excised and then rinsed in cold bathing medium. One or two strips were prepared from each heart. A myocardial strip (diameter approx. 1 mm), taken from a similar site on each ventricle, was mounted for isometric force recording as described by Gesser (1977). One end of the strip was fixed in a plastic clamp while the other end was tied with surgical silk to an isometric force transducer (Harvard363) connected to a recorder (BiotronixBL-882). After a 15-min recovery period, the ventricle strip was electrically paced at 30 beats min⁻¹via two parallel platinum electrodes, one on each side of the strip. Voltage was about 50 % above the minimum necessary to give a maximal mechanical response. Preparations were allowed to stabilize for 10—15 min and at time zero the gas mixture was altered to 0·5 % CO₂:99·5 % N2 and/or DNP was added. Force development was monitored for the next 10 min. In some experiments hydroxylamine was added 5 min prior to time zero. Relative force was calculated as a percentage of that developed at time zero. All data are expressed as mean ± S.E.M. Response curves were fitted to either linear or exponential equations. Differences between regression coefficients (following linear transformations if necessary) were compared using a t-test and a probability of less than 0·05 was considered to be significant.

Sculpin ventricle strips maintained stable force development under normoxic conditions (Fig. 1A). The inclusion of hydroxylamine in the bathing media had no, effect upon performance. Under hypoxic conditions hearts lost about 30% of initial force development after 10 min. The addition of hydroxylamine to the medium, under hypoxia, led to a significantly (P< 0·025) greater rate of failure (Fig. IB). The hydroxylamine-induced failure implies that myoglobin is essential to the maintenance of performance by ventricle strips under hypoxia. This observation is important to the present investigation since it shows the preparation is a suitable model to elucidate potential roles of myoglobin function.

Inclusion of the metabolic uncoupler, DNP, in the bathing medium resulted in contractile failure (Fig. 1C). After 10min of treatment, under normoxia (middle curve), preparations maintained only 15-20% of the initial force level. Ventricle strips bathed in medium gassed with 0·5% CO₂:99·5% O₂ and containing DNP performed significantly (P< 0·005) better than strips equilibrated in 0·5% CO₂:21% O₂. This confirms that the DNP effect was due to an oxygen-related problem. There was no discernible difference between control heart strips and those receiving hydroxylamine under conditions of normoxia and DNP treatment. Therefore the induced decrease in functional myoglobin content did not lead to an exacerbation of the decline in performance at high levels of oxygen demand. This interpretation requires that the maximal rate of failure of the preparations is not being reached. This potential limitation was assessed by subjecting heart strips to hypoxia and metabolic uncoupling. Sculpin heart muscle deteriorated rapidly under conditions of hypoxia and DNP treatment (Fig. ID). The inclusion of hydroxylamine in the medium led to a significantly (P = 0·005) more rapid failure with all hearts reaching zero contractility in 8 min. The extremely rapid contractile failure is faster than the response under the condition of normoxia, DNP and hydroxylamine treatment.

The presence of functional myoglobin has a protective effect upon performance of ventricle strips under conditions of severe hypoxia. This has been observed for perfused fish hearts and in that preparation is directly attributable to a myoglobin-related enhancement of oxygen consumption (Bailey & Driedzic, 1986; Driedzic et al. 1982). In perfused rat hearts, levels of high-energy phosphates are depleted more rapidly, during hypoxia, in preparations with decreased concentrations of functional myoglobin than in controls (Taylor et al. 1986). Although at variance with earlier findings for heart (Cole, Wittenberg & Caldwell, 1978; Jones & Kennedy, 1982), it is now apparent that myoglobin plays a critical role in cardiac metabolism during hypoxia, at least under in vitro conditions. This conclusion is consistent with theoretical predictions (Meyer et al. 1984).

Myoglobin, however, does not appear to be necessary in supporting performance and, by inference, oxygen consumption under conditions of extreme energy demand and normoxia. In this case normoxia is defined as a constant extracellular oxygen level of 21 %. If myoglobin function were critical under these conditions the experimental design would have revealed major differences in response to DNP treatment versus DNP plus hydroxylamine treatment. Essential to this conclusion is the observation that the rate of ventricle strip failure was not limited by the resolution of the technique. It is possible that the oxygen gradient from extracellular space to the mitochondrial inner membrane, which is established by respiration itself (Kennedy & Jones, 1986), is adequate to support oxygen diffusion under conditions of normoxia and maximal oxygen demand. Regardless, in this situation myoglobin-supported oxygen consumption is non-essential. This conclusion is contrary to predictions from simple mathematical models which imply that maximum oxygen consumption can be higher in the presence of myoglobin than in its absence (Meyer et al. 1984).

In summary, the present experiments confirm the role of myoglobin in protecting heart performance during hypoxia. The experiments are the first designed to test the contention that myoglobin-enhanced oxygen consumption plays a critical role under conditions of extreme energy demand. The results suggest that myoglobin may not be essential in meeting the demands of maximal respiration, at least under normoxia. The role of myoglobin appears to be confined to situations of low extracellular oxygen availability.

Work was supported by operating grants from the New Brunswick Heart Foundation, NSERC of Canada and the Canadian Donner Foundation. AAC was the recipient of an NSERC Postgraduate Scholarship. We would like to thank Dr J. Bailey for his critical input.