The fish heart as a model system for the study of myoglobin

Camp. Biocham. Phvsrol. Vol. 76A, No. 3, pp. 487-493. Printed in Great B&in

THE

1983

c

FISH HEART AS A MODEL SYSTEM THE STUDY OF MYOGLOBIN WILLIAM

Biology Department. Mount Island Biological Laboratory,

R.

0300-9629!83 $3.00 + 0.00 1983 Pergamon Press Ltd

FOR

DRIEDZIC

Allison University, Sackville, N.B. Canada EOA 3C0. and Mount Desert Salsbury Cove, ME 04672, USA, and Huntsman Marine Laboratory, St. Andrews, N.B. Canada EOG 30 (Receiced

I8 February 1983)

A model is presented for myoglobin study based upon naturally occurring differences in myocardial myoglobin content in fish. 2. The sea raven (Hemitripterus americanus) and the ocean pout (Macrozoarces americanus) have heart myoglobin contents of approx. 65 and 5 nmol/g wet wt. respectively. 3. The maximal activities of enzymes associated with energy metabolism are similar in the two hearts. 4. Isolated perfused hearts performed with similar efficiencies based upon similar rates of work, oxygen consumption and lactate production. 5. Under normoxic perfusion conditions both hearts met 98”~” of the ATP demand by oxidative mechanisms. 6. Myoglobin-rich sea raven hearts performed significantly better than myoglobin-poor ocean pout hearts under conditions of hypoxia and glycolytic blockage. 7. The performance of sea raven hearts was impaired during hypoxia by decreasing the content of functional myoglobin with hydroxylamine. 8. No effect upon performance was observed with the ocean pout heart. 9. The data provide the first evidence that myoglobin plays a role in the maintenance of contractility in heart under hypoxic conditions. Abstract-l.

INTRODUCTION

mechanical performance (Cole et (11.. 1978). Finally it is important to note that not all vertebrate hearts are myoglobin rich. Millikan (1939) in his classical review points out that hearts which contract at very high frequencies such as those in small birds and mice may have little or no myoglobin (precise data were not provided) but more recently it has been observed that some fish hearts which are slow beating may be essentially devoid of myoglobin while other species with similar cardiac demands may have hearts high in myoglobin content (Driedzic and Stewart, 1982). It is obvious that the role of myoglobin in heart warrants further careful study. The naturally occurring variation in fish myocardial myoglobin content is being exploited to gain insight into the role of this protein. The studies to be reported here will focus upon two benthic species found in the North Atlantic, sea raven (Hemitripterus americanus) and ocean pout (Mucrozoarces americanus). The hearts of both species have in common the essential lack of coronary arteries and hence must receive oxygen and nutrients directly from the blood in the ventricular lumen. The two hearts differ in the content of myoglobin, the magnitude of which is qualitatively apparent in Fig. I. The heart of the sea raven is darkly coloured even after extensive washout of blood, whereas the ocean pout heart is very pale in colour.

The intracellular protein myoglobin occurs in high concentrations in red skeletal muscle and in the heart of many animals. Myoglobin binds reversibility with oxygen and can facilitate oxygen diffusion through membranes in vitro (see Wittenberg, 1970 for an extended review). In skeletal muscle myoglobin content correlates well with activities of enzymes which are associated with aerobically based mechanisms of ATP production (Holloszy and Booth, 1976). Direct evidence for an involvement of myoglobin in oxygen flux has been attained from studies in which the content of functional myoglobin was modified by agents which convert the molecule into a ferric state incapable of binding oxygen. A decrease in the content of ferrous myoglobin resulted in a decrease in the steady-state oxygen consumption rate of pigeon breast muscle fibers under hypoxic conditions (Wittenberg et al.. 1975) and decreases in both oxygen consumption and tension development by the dog gastrocenimius-plantaris muscle (Cole, 1982). In light of the above information there seems little doubt that at least under some conditions myoglobin plays an important role in oxygen flux in red skeletal muscle. The role of myoglobin in heart tissue is still open to question and a number of findings suggest that the situation is much more complex than with skeletal muscle. Foremost, the activities of oxidative enzymes need not correlate with myoglobin content (Burleigh and Schimke, 1969; Meszaros et al., 1980). Secondly, perfused dog hearts treated with nitrite to decrease the content of functional myoglobin were able to maintain control levels of oxygen consumption and

BIOCHEMICAL PROFILE OF RED AND WHITE FISH HEARTS

The myoglobin content of the sea raven heart is about 65 nmol/g wet wt and that of the ocean pout 487

488

WILLIAMR. DRIEDZK

Fig. I. Perfused isolated sea raven (A) and ocean pout (B) hearts.

about 5 nmol/g wet wt (Table 1). The organization of energy metabolism in these hearts as well as in ocean pout white skeletal muscle, a tissue of known high anaerobic metabolism (Driedzic et cd., 1981) was assessed by determining the concentration of various components and the maximal activity of selected enzymes (Tables 1 and 2). The content of cytochrome c was much higher and that of high energy phosphates much lower in heart than in skeletal muscle. The activities of enzymes obligatory related to aerobic energy metabolism such as citrate synthase, 2-oxoglutarate dehydrogenase and cytochrome oxidase, were far higher in heart than in skeletal muscle. In contrast the activity of enzymes associated with anaerobic carbohydrate metabolism notably phosphofructokinase and pyruvatc kinase were higher in

white skeletal muscle than in heart. All of the proteins, creatine phosphate and ATP displayed similar levels in the sea raven and ocean pout hearts. The results show that the red-white dichotomy which exists for skeletal muscle in terms of energy metabolism does not apply to red and white fish hearts. In fact, with the exception of the difference in myoglobin content the metabolic organization in fish hearts appears remarkably similar actoss teleost species (see Driedzic and Stewart, 1982). The important feature of this finding in terms of utilizing the fish heart as a model for myoglobin study is that one need not be concerned that the tissues differ greatly in their maximum potential contribution to ADP rephosphorylation by aerobic and anaerobic mechanisms.

Myoglobin

function

Table I. Content of proteins and high-energy phosphates associated with energy metabolism in sea raven heart, ocean pout heart and ocean pout white skeletal muscle

Myoglobin Cytochrome Creatine phosphate ATP

c

Sea raven heart

Ocean pout heart

63.8 + 2. I 2.78 + 0.01

1.70k 0. I8

0.54f0.19 1.30+0.24

1.04+0.09 1.74+0.17

5.1 + I.8

in fish heart

489

Afterload Reservoir

Ocean pout white skeletal muscle 0.9 f 0.2 0.49 k 0.08 20.41 k 1.58 3.83 _+ 0.73

Values represent the means + SEM of a minimum of four individuals. Myoglobin and cytochrome c are expressed as nmol/g wet wt; creatine phosphate and ATP as pmol/g wet wt. Heart myoglobin, creatine phosphate and ATP values were determined after tissue perfusion; all other values based upon samples taken from resting animals. Sources: Turner and Driedzic (1980); Driedzic er ul. (19R 1); Driedzic and Stewart (1982).

ISOLATED PERFUSED PREPARATION

HEART

The biochemical data presented above provide information only into the extremes of energy demand. Activity within these boundaries has been obtained with perfused isolated hearts. The single flow through system and in particular the absence of coronary arteries render the sea raven and ocean pout hearts ideal for perfusion experiments. The preparation involves excising the heart, cannulating the atrium and filling the heart with Ringers from a reservoir at physiological filling pressures. The ventra aorta is cannulated and connected to a second reservoir which is situated higher than the filling level. The ventricle fills by gravity and then pumps its contents into the second reservoir (Fig. 2). The hearts are electrically paced and may be driven to contract over a range of fequencies. The preparation is responsive to alterations in filling and afterload pressures such that an increase in filling pressure results in increases in mean pressure development (Fig. 3a, area under curve) and cardiac output, and an increase in afterload results in an increase in mean pressure development (Fig. 3b). Furthermore, the preparation is

Table 2. Maximal heart,

Fig. 2. Isolated

perfused

heart

apparatus.

stable for extended periods of time (Fig. 3c) with no appreciable decrease in mechanical function. The perfused isolated heart is therefore amenable to the quantitation of power output, oxygen consumption and lactate production and as such is extremely suitable for mechanical/metabolic studies.

activities of enzymes associated with energy metabolism in sea raven ocean pout heart and ocean pout white skeletal muscle

_______ Citrate synthase 2-Oxoglutarate dehydrogenase Cytochrome oxidase Hexokinase Phosphofructokinase Pyruvate kinase Lactate dehydrogenase

Sea raven heart ___ 12.05 * 1.13 1.02* 0.09 35.8 k 8.0 2.52 + 0.70 1.31 kO.11 37.04 + 2.88 154.67 _+ 18.46

Ocean pout heart 12.78 + 1.17 1.80+0.13 29.8 + 4.8 2.45 +- 0.35 1.17+0.15 36.34 + 0.42 127.79 k 4.16

Ocean pout skeletal muscle 0.23 k ND 0.80 (n ND 2.51 + 67.55 k 177.40 f

0.01 = 2)

0.23 5.90 5.53

Enzymes were assayed at 10°C and are expressed as (nmol of substrate converted to product/min) per g wet wt of tissue. All values represent the mean + SEM of 3-6 individuals. Source: Driedzic and Stewart (I 982). ND = not detectable.

WILLIAM R. DR~EOZK

490

k-1

-1

16 ---17

32~

min.41

33

49 -50

Fig. 3. Representative pressure development records of perfused isolated fish hearts. (A) Single contractions at different tilling pressures. (B) Response of a heart to increased afterload at a fixed filling pressure. (C) Performance of a heart under fixed conditions for 50 min. (A) and (B) are traces from sea raven hearts. (C) is a trace from an ocean pout heart. All hearts were perfused at 10 C.

CONTRIEUTIONS OF AEROBIC AND ANAEROBIC ENERGY METABOLISM IN PERFUSED ISOLATED HEARTS

Rates of oxygen consumption and lactate production have been assessed for sea raven and ocean pout hearts perfused at a filling pressure of 1.3 cm H,O and an afterload resistance of I5 cm H20 (Driedzic and Scott, in preparation). Under these conditions the sea raven heart has a slightly greater cardiac output which when heart size is taken into consideration results in a significantly greater power output per g of tissue for sea raven than ocean pout hearts (Table 3). A single passage of perfusate through the heart reduces the oxygen tension from 50 to 65mm Hg O? dependent upon species. In this experiment the perfusate was gassed with 21:;; oxygen to eIiminate any direct passage of oxygen through the myocardium to the atmosphere. The oxygen consumption was higher for the sea raven heart than the ocean pout heart but the important feature is that the aerobic efficiencies of energy conversion were similar. The anerobic contribution to energy metabolism was calculated by assessing the rate of lactate production (Table 4). The lactate content of hearts perfused with the ventral aorta open to the atmosphere for 5 min to establish zero time conditions was determined. Then, the lactate content of hearts perfused for 50 min under working conditions, as well as the amount of lactate which was washed out of the heart

into the perfusate, was measured. From these data it was possible to calculate the net lactate production during the perfusion period. This value was not significantly different between sea raven and ocean pout hearts. On the basis of the oxygen consumption and lactate production rates it is possible to estimate the

Tabie

3. ~ydrodyllamic and metabolic responses of perfused isolated sea raven and ocean pout hearts Sea raven

M&n pressure (cm F_l?o) Flow (ml/min) Power (kerg/g dry wt set) APO? (mm Hg) O? Consumption (nmol/g dry wt set) (Y,,) Aerobic efficiency

Ocean

pout

20.6 i: 0.5

19.0 f 0.6

5.9 * 0.6 17.3 i: 2.1 51.6+ 13.1

4.6 _+ 0.9 11.7 i_ 3.3 62.61 Il.9

91.7 i: 23.1 4.2

76.5 i_ 7.6 3.4

All values represent the means i SEM of a minimum of five individuals. Hearts were perfused at a fixed filling pressure of I .3 cm H,O and required to work against an afterload of 15cm H,O. The perfusate was maintained at IO ‘C, gassed with 1:; CO, in air and adjusted to pH 7.8 with HCO;. All hearts were paced at 20 beats per min. Power output was calculated as a product of pressure times flow. Oxygen consumption was calculated from the decrease in oxygen tension after passage through the heart times cardiac output.

Myoglobin function in fish heart

491

Table 4. Rate of lactate production by perfused isolated sea

raven and ocean pout hearts Sea raven

Ocean pout

13.7k2.1 22.1 F3.4

9.1 * 1.1 15.5k4.9

17.9 * 7.9

34.3 * 17.5

8.8 i 3.7

14.1 + 6.7

Lactate in heart (~mol/g dry wt) 0 min 50 min Lactate released from heart (pmotig dry wt) Net lactate production (nmolig dry wt set)

See Table 3 for perfusion conditions. Values represent means + SEM of five or six individuals. Table 5. Contributions regeneration by perfused

the

, 0.15%510

to aerobic and anaerobic ATP isolated sea raven and ocean pout hearts Sea raven

Aerobic ATP equivalents (nmoljg dry wt set) Anaerobic ATP equivalents (nmol/g dry wt set) (“G) Oxidative ATP production

Ocean

Fig. 4. Absorption spectra before and after addition

9 98

14 98

Data were transformed from Tables 3 and 4 on the basis of 6mol of ATP generated per mole 0, consumed and I mol ATP generated per mol of lactate produced.

aerobic and anaerobic contributions to ADP rephosphorylation (Table 5). For both hearts the contribution of anaerobic metaboism was minimal with oxidative mechanisms accounting for approx 98% of the ATP regenerated. The importance of these findings with respect to utilizing this system as a model for myoglobin function analysis is that the perfused hearts are not being oxygen limited and that both hearts operate at similar energy conversion efficiencies. This work sets the stage for investigations into the role of myoglobin in the oxygen deficient state. IMPORTANCE

OF MYOGLOBIN

HYPOXIC

UNDER

CONDITIONS

The influence of functional myoglobin during hypoxia was assessed by monitoring performance under various conditions of oxygen availability and metabolic intervention (Driedzic et al., 1982). Hearts were perfused with media equilibrated with either 21 or 5% oxygen. On the basis of an extraction of 50-65 mm Hg 0, on a single passage through the heart, an oxygen tension of 5% would be inadequate to support

Table 6. Effectiveness

Additions

Lactate (pmol/g

to Ringer’s

See text for the perfusion individuals,

600

(nm)

of sea raven heart homogenate of hydroxylamine (0.03 mM).

aerobic needs even if all of the oxygen were to be extracted. The perfusate in some cases contained iodoacetate (0.5 mM) and/or hydroxylamine (1.0 mM); the former to block glycolysis and the latter to decrease the content of ferrous myoglobin. These perturbations warrant further comment. The effectiveness of iodoacetate as a glycolytic block was determined. Sea raven hearts were perfused for 15 min at a filling pressure of 5 cm H,O and allowed to pump against atmospheric pressure. The perfusate contained either NaCN (1 .O mM) to inhibit oxidative metabolism or NaCN plus iodoacetate (0.5 mM). Following the perfusion period the content of lactate in the hearts was not significantly different between the two groups (Table 6). The amount of lactate released into the perfusate though was significantly higher in the absence of iodoacetate: it appears that lactate production is about 85% inhibited under these conditions. Although the blockage of glycolysis was not absolute the level of inhibition was considered adequate for the series of experiments to be described below. The compound hydroxylamine has been shown to convert ferrous myoglobin to a ferric state incapable of binding oxygen (Wittenberg et al., 1975). The in vitro potency of this agent is presented in Fig. 4 which shows absorption spectra for a sea raven heart homogenate in the presence and absence of hydroxylamine. Hydroxylamine was added to the same cuvette at a hydroxylamine/myoglobin molar ratio calculated to occur if the intracellular hydroxylamine fully equilibrated with the perfusate compartment (1 mM). Note that this concentration results in complete ablation of the characteristic absorption peaks

of iodoacetate as a glycolytic sea raven hearts

Glucose (10 mM) + NaCN Glucose (10 mM) + NaCN + iodoacetate (0.5 mM)

570

pout

459

551

540 Wavelength

(1 mM)

(I mM) conditions.

inhibitor

in heart dry wt)

13,47 + 1,51 18.32 + 3.57 Values represent

in perfused

isolated

Lactate released from heart (Pmol/g dry wt) 130.29 + 46.79 17.29 k 4.39

the means 2 SEM of four

492

WILLIAM R. DRIEDZIC SEA RAVEN __..__._~

__~ 10

21%O,.IA,Pyr,HA

0

5

10

15

20

OCEAN

-. .

25

1 lo/---

POUT

21%O,,IA, Pyr,HA

1

30 Time (mini

Fig. 5. Power output of perfused isolated sea raven and ocean pout hearts. All values represent the means + SEM of a minimum of six individuals. Hearts were perfused at a fixed filling pressure of 3 cm H,O and required to work against an afterload of 20 cm H,O. The perfusate was equilibrated with 5 and 2pA oxygen and contained various combinations of pyruvate (2 mM), iodoacetate (0.5 mM) and/or hydroxylamine (1 .OmM). For further perfusion conditions see Table 3. The data are expressed as erg/g of fish sec. To convert to units of g dry wt of heart the following relationship may be used. Sea raven. 0.097 g dry heart/kg fish; Ocean pout, 0.132 g dry heart/kg fish; Source: Dried& et al., 1982.

of ferrous

myoglobin.

In the experiments

presented

below the inclusion of hydroxylamine (1 .OmM) in the perfusate decreased the content of functional myoglobin in the sea raven hearts from approx. 70 to 25 nmol/g wet wt. ‘In all cases the content of myoglobin in the ocean pout hearts was below 3 nmoI/g wet wt. Both sea raven and ocean pout hearts perfused with media gassed with 21% oxygen and containing no additional compounds maintained initial power outputs for at least 30min. The inclusion of iodoacetate in the perfusate resulted in contractile failure in 15-20min for the hearts of both species; the supplementation of pyruvate (2.0 mM) to circumvent the glycolytic block restored performance to the control level. That is, hearts perfused with Ringers gassed with 21% oxygen and containing iodoacetate and pyruvate performed as well as those hearts perfused with no additional compounds in the media. Hearts perfused with Ringers containing both iodoacetate and pyruvate and equilibrated with 5% oxygen rapidly failed, (Fig. 5). Sea raven hearts declined to 504 of the mrttal power output m 12.5 mm, whereas ocean pout hearts reached this Level of deterioration within 5 min. These data alone suggest that myoglobin confers a protective effect during hypoxia. This hypothesis was tested by including hydroxylamine in the perfusate. Hearts perfused with media equilibrated with 21% oxygen and containing iodoacetate, pyruvate and hydroxyiamine performed as well as hearts perfused under the same conditions but with the excmsion of hydroxyIamine. That is, hydroxylamine per se does not have a general deleterious effect upon contractile performance. Under hypoxic conditions hearts perfused with media containing iodoacetate, pyruvate and hydroxylamine rapidly failed. Sea raven hearts displayed 50% of the initial power output after 7 min, whereas ocean pout hearts were reduced to this Ievel of activity after 4min of perfusion. Figure 5 shows the difference in performance in hypoxic, glycolytic inhibited hearts with and without the myoglobin effector. The myoglobin-rich sea raven hearts entered the initial stages of contractile failure at a much faster

rate when the content of functional myoglobin was decreased. Although the myoglobin inhibitor had a marked effect upon sea raven heart performance there was no further impai~ent of contractile function of the myog~obin-poor ocean pout heart. The

simplest interpretation of these data is that myoglobin confers an enhancement of performance during hypoxia. Collectively the data presented in this paper provide the first evidence that myogIobin plays a role in the maintenance of contractility in heart during an hypoxic challenge. It is probable that this is due to the facilitation of oxygen diffusion through the cytoplasm to the mitochondria; however, this has yet to be proven. CONCLUDING

COMMENTS

Evidence is presented in this review that the fish heart is a suitable model for the study of myoglobin function. The system is potentially useful in assessing the quantitative contribution of myoglobin facilitated oxygen flux to the overall oxygen consumption rate and the relationship of myoglobin to cellular functional integrity. As a first step in this direction it has been established that myoglobin plays a role under hypoxic conditions. It should be emphasized though that the work reported lends no insight into why species have variable myocardial myoglobin content. There may be a number of conditions other than hypoxia under which myoglobin could play an important role. For instance, myog~obin content may be associated in some fashion with maximum cardiac performance as suggested by Giovane et ul. (1980). However, this is clearly a complex problem which probably involves other aspects of oxygen delivery such as mitochondria placement and blood residence time. The importance of myoglobin may vary with temperature due to the relationship amongst oxygen soiubility, oxygen demand and rates of diffusion (Stevens, 1982). The power of the present model is that it may be utilized in a broad sense to establish what conditions influence the importance of myoglobin.

Myoglobin

function

Acknowledgements-Work conducted in the author’s laboratory was supported in part by operating grants from the New Brunswick Heart Foundation and N.S.E.R.C. of Canada. In addition, support was received from N.S.E.R.C. awarded directly to the H.M.L., N.I.H. Biomedical Support Grant No. SO7 RR05764-07 and N.S.F. Grant No. DEB8100823 to M.D.I.B.L.

REFERENCES

Burleigh I.G. and Schmike R. T. (1969) The activities of some enzymes concerned with energy metabolism in mammalian muscle of differing pigmentation. Biochem. J. 113, 157-165. Cole R. P. (1982) Myoglobin function in exercising skeletal muscle. Science, N. Y. 216, 523-525. Cole R. P., Wittenberg B. A. and Caldwell P. R. B. (1978) Myoglobin function in the isolated flurocarbon-perfused dog heart. Am. J. Physiol. 234, H567-H572. Driedzic W. R. and Stewart J. M. (I 982) Myoglobin content and the activities of enzymes of energy metabolism in red and white fish hearts. J. camp. Physiol. (in press). Driedzic W. R., McGuire G. and Hatheway M. (1981) Metabolic alterations associated with increased energy

in fish heart

493

demand in fish white muscle. J. camp. Physiol. 141, 425432. Driedzic W. R., Stewart J. M. and Scott D. L. (1982) The protective effect of myoglobin during hypoxic perfusion of isolated fish hearts. J. molec. CeN. Curdiol. (in press). Giovane A., Greco G., Maresca A. and Tota B. (1980) Myoglobin in the heart ventricle of tuna and other fishes. Experientia 36, 2 19-220. Holloszy J. 0. and Booth F. W. (1976) Biochemical adaptations to endurance training exercise in muscle. Ann. Revs. Physiol. 38, 273-29 1. Meszaros K.. Chance B. and Holtzer H. (1980) Myoglobin and cytochrome oxidase in the myocardium of the developing chick. J. Molec. Cell. Cardiol. 12, 965-975. Millikan G. A. (1939) Muscle hemoglobin. Physiol. Reo. 19, 503-523. Stevens E. D. (1982) The effect of temperature on facilitated oxygen diffusion and its relation to warm tuna muscle. Can. J. Zoo/. 60, 1148-1152. Wittenberg J. B. (1970) Myoglobin-facilitated oxygen diffusion: Role of myoglobin in oxygen entry into muscle. Physiol. Rea. 50, 559-636. Wittenberg B. A., Wittenberg J. B. and Caldwell P. R. B. (1975) Role of myoglobin in the oxygen supply to red skeletal muscle. J. hiol. Chem. 250, 9038-9043.