Myoglobin and cytochrome oxidase in the myocardium of the developing chick

Journal of Molecular

and Cellular Cardiology (1980) 12, 965-975

Myoglobin

KAROLY

and Cytochrome Oxidase in the Myocardium of the Developing Chick

Ml%ZAROS,*

BRITTON

CHANCE

AND HOWARD

HOLTZER

Johnson Research Foundation and ~e~~art~~ent of Anatomy, University of Pennsylvania, School of Medicine, PhiladeQhia, Pennsylvania, (Received 12 February 1979, accepted in revised form 12 hfarch

U.S.A.

1980)

K. MI'zARos, B. CHANCE AND H. HOLTZER. Myoglobin and Cytochrome Oxidase in the Myocardium of the Developing Chick. 3oumzl of ~lolecula~ cmtad Cellzdnr &~rd~olo~~( 1980) 12, 965-975. Myocardial tissue of chick embryos and developing chickens of 3 to 30 days of age was investigated by sensitive spectrophotometry. Spectral and kinetic evidence showed that no hemoglobin was present in the myocardial tissue preparations. Difference spectra of anoxic vs. oxygenated heart tissue of 3- and 4-day-old embryos demonstrated the oxidation-reduction changes of cytochromcs only. At variance with the results of previous studies. myoglobin was first detected at an age af 5 days. At later developmental stages myoglobin dominated the spectrum. Therefore, in order to demonstrate the presence of cytochromes, myoglobin was transformed into derivatives incapable of oxygen binding by treatment of the tissue with nitrite or ethylhydroperoxide. The molar ratio of myoglobin to cytochrome oxidase increased rapidly from 5 to I4 days of age, thereafter a slow decrease was observed. KEY WORDS: Chick; Cytochrome oxidase; Development; Myoglobin; Tissue spectrophotometry.

Embryo;

Heart;

Myocardium;

1. Introduction Aerobic or red muscles perform mechanical work by using ATP generated principally by oxidative phosphorylation 121. Myoglobin, capable of reversible oxygen binding [22}, is found’in red muscles in high amounts. Respiratory enzymes are present and function in most embryonic and adult cehs of animals; however, myoglobin appears only at a certain stage of differentiation of red muscles. Earlier studies on the onset of myoglobin synthesis in avian embryos by Kagen and co-\vorkers [13-I/5’] have yielded controversial results. The appearance of radioactively labeled myoglobin has been demonstrated in cell-free systems prepared from l-day-old chick embryos [I51 and from various body segments of i-day-old embryos [lG]. However, when the label was applied in the yolk sac in zkw, no Iabeled myoglobin could be detected by immunoprecipitation in chick embryos younger than 6 days, and at this age appears first in the heart [14]. Moreover, * On leave from the 1st Department Hungary. o~22-zazai~~~loo965~ i i ~oz.oo~o

of Biochemistry,

Semmelweis Medical

School, Budapest,

0 1980 Academic Press Inc. (London)

Limited

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by a non-radioactive precipitin technique, myoglobin has been first detected in the heart at an age of 14 days, and in the thigh muscle it has appeared close to hatching [I4]. In another study on the thigh muscle by Low and Rich, chemical isolation of myoglobin has been performed; myoglobin accumulation has been found to begin around the 16th day, in accordance with the appearance of myoglobin synthetic activity in cell-free preparations from the thigh muscle at the same age [N]. The heart muscle is the earliest to perform sustained mechanical work, which creates the need for greater oxygen supply and utilization. The aim of this study was to determine the onset of myoglobin synthesis and the development of respiratory enzymes in the myocardium of the chick embryo. Following the pioneering work of Keilin on insect muscle [17], transmission spectrometry has been applied for the investigation of cytochromes in white muscle [4, 5, 131 and red muscle [13], and of myoglobin in perfused rat heart [28]. A sensitive optical method was used in this study for the functional demonstration of both myoglobin and cytochromes in the hemoglobin-free heart tissue fragments prepared from chick embryos and young chickens. (Millikan pioneered the study of myoglobin in muscle [.?.?I). 2. Materials

and Methods

Fertilized eggs were incubated at 37°C:. Chick embryos were removed and placed into Earle’s minimal essential medium. The heart was removed and dissected under a stereo microscope. A gentle massage of the tissue particles removed the traces of blood; spontaneous contractions (which ceased after 15 to 20 min) had a similar effect. The fragments (less than 0.3 mm in diameter) were washed twice with the medium, then transferred into the sample holder of the spectrophotometer. For myocardial fragments from embryos of 3 to 7 days of age a cylindrical sample holder of 1.5 mm diameter was used, which contained tissue from 4 to 15 embryo hearts (0.5 to 1 mg wet weight). For tissue fragments from later developmental stages a larger sample holder of 5 mm diameter was used, into which tissue from 1 to 8 hearts (15 to 20 mg wet weight) was placed. The (vertical) light path was generally about 0.5 mm. During the experiment the heart pieces were immersed in the medium which was changed several times. The space between the surface of the sample and the cover lid was constantly flushed by a slow stream (4 ml/min) of water-saturated oxygen or argon gas. Both the preparation of the tissue and the experiment were carried out at 20°C. Absorption spectra of tissue fragments were recorded by a computer-assisted dual-wavelength spectrophotometer (B. Chance and J. Sorge, pers. comm. [30]). The instrument was equipped with a memory unit for the storage of spectra. The spectrum of the anoxic tissue was generally stored in the memory. Subsequent perturbation of the experimental system (oxygenation) caused spectral changes which were recorded and compared with the spectrum stored in the memory by the

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computer, and the difference spectrum was displayed. The same instrument was used for the dual-wavelength measurement of the kinetics of hemoglobin and myoglobin oxygenation. A linear correlation was found between the magnitude of the absorbance changes in the difference spectra and the amount of tissue up to 25 mg wet weight; thus the quantitative estimation of tissue pigments could be performed on the basis of the difference spectra. Calibration measurements were carried out with both sample holders using solutions of crystalline horse heart myoglobin (type III ; Sigma). The specific gravity of the heart tissue was found to be 1.09 g/cm3. Extraction and assay of isolated chick heart myoglobin was performed according to the method of Akeson and co-workers [I] as modified by Low and Rich [20].

3. Results Kinetic

and spectral euidence for

the absence of hemoglobin from

myocardial

tissue

preparations

An essential condition of the optical measurement of myoglobin in tissue was the absence of blood, i.e. hemoglobin, because of the similar spectral properties of these hemoproteins. In Figure 1 the solid curve is the difference spectrum of anoxic (sample flushed with argon) vs oxygenated myocardial tissue from a 16-day-old chick embryo; the dashed curve is the difference spectrum of anoxic vs oxygenated erythrocytes obtained from the same embryo. The only significant spectral difference appeared in the Soret region: with the tissue a difference

0.08

FIGURE suspension 630 nm.

1. Difference of erythrocyta

spectrum .Itlmic a. oxygenated [-) prepared from a 16-day-old

state) of heart chick embryo.

tissue (&-) and of a Reference wavelength:

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maximum was found at 440 nm (mostly due to myoglobin), whereas for the erythrocytes the maximum appeared at 436 nm. Further, a well-known difference between these oxygen-binding heme compounds is the greater affinity of myoglobin for oxygen [27]. Based on the above physicochemical properties, erythrocytes could be demonstrated in a reconstituted system (Figure 2). The time course of the changes of the absorption difference between 440 and 436 nm occurring in response to oxygen withdrawal was followed in heart tissue samples with or without added erythrocytes. Curve A in Figure 2 was recorded during the deoxygenation of myocardial tissue from a 16-day-old embryo. Thereafter a suspension (20 ~1) of erythrocytes, obtained from the same embryo was added (which increased the 630 to 581 nm absorption change in response to oxygenation-deoxygenation by 200/,). The altered kinetics of the absorption changes at 440 to 436 nm in response to oxygen withdrawal in the presence of erythrocytes is shown by curve B in Figure 2; after a small initial deflection, the increase of the curve was due to the release of oxygen by hemoglobin, then the decrease indicating myoglobin deoxygenation followed. The kinetics observed before the addition of erythrocytes did not indicate the presence of hemoglobin, therefore it was concluded that the applied method of preparation virtually completely removed the blood from the myocardial tissue pieces.

FIGURE 2. Kinetics of the optical changes during the deoxygenation of heart tissue before (curve A) and after (Curve B) the addition of an erythrocyte suspension prepared from the same embryo. Sample and reference wavelengths were 440 nm and 437 nm, respectively.

Demonstration

of myoglobin amytal,

and cytochrome in the myocardial nitrate and ethylhydroperoxide

tissue. Effects of

The spectral changes occurring when oxygen was given to a sample of anoxic heart tissue prepared from 16-day-old embryos are shown in Figure 3 by the solid curve. This difference spectrum largely corresponded to the difference between the myoglobin) and optical spectra of pure ferromyoglobin (i.e. deoxygenated

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969

oxymyoglobin [II, 281, although the differences in the Soret region were relatively small due to light scattering at shorter wat-clengths. Repeated deoxygenation and oxygenation of the tissue was accompanied by optical changes of the same magnitude for about 5 h. Addition of 10 mM amytal (an inhibitor of the electron transport in the respiratory chain) to the medium in which the fragments were immersed doubled the time necessary for complete deoxygenation. However, amytal did not influence the size of the absorption changes (data not shown), which indicated that myoglobin was completely oxygenated when the sample was flushed with oxygen. In order to demonstrate the absorption changes of oxidized and reduced cytochromes the myocardial tissue was treated either with nitrite or ethylperoxide. Nitrite has been shown to oxidize ferromyoglobin to the nonfunctional ferric form in the heart muscle [3, 7, 191. Medium containing 20 rnM NaNO, was added to the fragments. After 15 min an oxygenation-deoxygenation cycle was performed, then the difference spectrum was recorded in the second cycle (Figure 3, dashed curve). No optical changes attributable to myoglobin occurred after nitrite treatment of the myocardium (note the absence of a peak at 581 nm). This difference spectrum showed the absorption bands of cytochromes (cytochrome n at 605 nm, cytochrome c at 550 nm, cytochrome a3 at 445 nm and cytochrome b as a

I

I

450

500

550

600

X (nm) FIGURE xfore (---)

3. Difference spectrum of heart tissue (anoxic vs. oxygenated) from lli-day-&d embryos and after (- - -) treatment with 20 nxvx NaNo,. Reference wavelength: 630 nm.

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shoulder at 430 nm) and of flavoprotein (around 470 nm). A similar pattern of absorption difference spectrum has been found with white muscle [S, 131. The same result was achieved by treating the tissue with ethylhydroperoxide, which oxidized hemoglobin to ferrylllcmoglob~~ [fB], and myoglobin to ferrylmyogIobin [!?I. Oxygen gas was passed through a chamber containing a filter paper impregnated with 0.1 M EtOOH, and this volatile compound was carried to the tissue by the gas Aushing the sample holder. After 30 min argon gas was passed through the chamber containing EtOOH, and the spectral changes in response to oxygenation-deoxygenation were recorded (Figure 4, heart tissue from 6-day-old embryos). No sign of myoglobin oxygenation was seen in the presence of EtOOH; the difference spectrum demonstrated the absorption bands of cytochromes and flavoprotein only. The magnitude of the cytochrome signals was the same after the elimination of functional myoglobin with either nitrite or ethylperoxide in preparations from embryos of the same age. 0.02

0.01 E E 0 D D 2 -0.01 u -0.02 I

450

500

550

600

X km) FIGURE before (--+

4. Difference spectrum of heart tissue (anoxic vs. oxygenated! from 7-day-old embryos and during {- - -) treatment with EtOOH vapour. Reference wavelength: 630 nm.

The amount of functional myoglobin was proportional to the absorption change at 581 nm in the untreated tissue, since no change occurred here during anoxic cycles after the transformation of myoglobin into inactive derivatives (Figures 3 and 4). A molar coefficient for the absorption difference of ferromyoglobin and oxymyoglobin at the wavelength-pair 630 to 581 nm AE,, = 7.3 was calculated from the spectra of purified oxy- and ferromyoglobin 1111. For cytochrome a appearing at 605 nm in nitrite-treated myocardium a similar coefficient for the wavelength-pair 605 to 630 nm, AE, = 1 I.1 was used [lo, 291.

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Ufference spectra of heart tissue (anoxic vs. oxygenated) from embryos and chickens between 3 and 30 days old were recorded before and after treatment with 20 mM NaNO,. Figure 5 shows the difference spectrum of,myocardial tissue prepa.red from 12 embryos of 4 days of age. As indicated by the absence of change at 581 nm, no functional myoglobin was found at this stage. The pattern obtained was similar to that seen after nitrate (or ethylperoxide) treatment of tissue from later stages, displaying the absorption bands of cytochrome, and this pattern did not change after nitrate treatment. ~yoglobin was first detected in myocardial tissue from 5-day-old embryos.

FXGURE 5. Difference spectrum of heart tissue (anoxic vs. oxygenated) embryos. Reference wavelength: 630 nm.

prepared from 4-day-old

Myoglobin accumulated rapidly in the myocardium between days 5 and 14, reaching a pleateau around the 16th day [Figure 6(a)]. In order to check the results of the direct spectrophotometric measurements, myoglobin was extracted from the hearts of 8, 16 and 24-day-old embryos and chicken, and the isolated myoglobin was assayed [ZO]. This method yielded about 10% higher values [see open symbols in Figure 6(a)]. Thecytochrome oxidase {i.e. cytochrome a) content of the heart tissue increased steadily [Figure 6(b)]. The ratio of cytochrome F to cytochrome a did not change significantly during this period. The molar ratio of myoglobin to cytochrome oxidase [Figure 6(c)] increased rapidly during the initial build-up of myoglobin, then slowly decreased. 4. Discussion Optical techniques are suitable for the measurement of tissue pigments without destruction of the structure and function of the tissue. The methods applied in the present study allowed the direct measurement of both myoglobin and cytochromes in the myocardium. The good agreement between the results of the direct spectrophotometric measurement of myoglobin and the assay of extracted myoglobin demonstrated the reliability of the direct optical approach, which has been found to be sensitive to 40 ng (or 2 nmoljg tissue) of functional myoglobin. M.C.C.

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6-

FIGURE 6. Concentration of (a), myoglobin; (b), cytochrome oxidase and (c), their molar ratio in the myocardium of the chick embryo and the developing chick. (Hatching occurred on the 21st day.) Myoglobin and cytochrome oxidase concentrations were determined directly from the difference spectra of tissue samples (0). Also shown are the results of myoglobin estimation by extraction of myoglobin (0).

Myoglobin was found first in the myocardium of chick embryos at an age of 5 days by our method. Among the variety of earlier results (surveyed in the Introduction), a similar result (6 days) has been obtained by in vivo radioactive labeling and immunoprecipitation of myoglobin [14]. The very early (at 1 and 2 days of age) myoglobin synthetic activity of cell-free preparations detected by the by the absence of some radioimmunological assay [15, 161 has b een explained control factors from the in vitro system [14]. In the case of the thigh muscle, however, a close correlation has been found between the synthetic activity of the cellfree preparation and the onset of myoglobin accumulation around the 16th day by Low and Rich [20], who have performed chemical isolation of myoglobin. By a non-radioactive precipitin technique myoglobin has been detected first at more advanced stages, i.e. at 14 days in the heart and at 21 days in the thigh muscle

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[14]; the relatively low sensitivity of the precipitin method (about 10 pg myoglobin~ml~ may account for the difference. In view of the specificity and sensitivity of our optical method for the estimation of functional myoglobin in situ, it is concluded that myoglobin accumulation begins in the chick myocardium around the 5th day of embryonic development. Cytochrome oxidase activity of the chick embryo heart has been investigated by Davidson [8], who has found a 20% decrease of specific enzyme activity between days 19 and 21. No decrease was found by us in the concentration of cytochrome oxydase. Adaptive changes of the respiratory pigments have been described earlier. Both exercise and low environmental oxygen tension have been shown to increase the amount of myoglobin in red muscles [S, 241. On the other hand, the amount of cytochrome oxidase decreases in the heart of chronically hypoxic animals 121, 251. In view of these opposite changes, the molar ratio of myoglobin to cyotochrome.oxidase might be used as a sensitive indicator of the sufficiency of the oxygen supply in the red muscles. From the point of view of respiration, the embryo may be regarded as a very hypoxic adult, as suggested by Mela and co-workers [,?I]. The decrease of the myoglobin-cytochrome ratio after hatching can be considered to be a sign of improved oxygen supply. As regards the onset of myoglobin synthesis, few leads are available to interpret the timing of this event. Although some movement of the legs in the egg ca.nnot be ruled out, it is difficult to conceive that myoglobin synthesis in the thigh muscle would be triggered by hypoxia around the 16th day. Even more intriguing is the case of the embryonic heart, which undergoes regular contractions from the 33rd hour of development 1231, although we were unable to detect functional myoglobin before the 5th day. However, major developmental changes are occurring in the heart at this age, such as the thickening of the heart musculature and the formation of the primitive ventricles, as well as the appearance of sympathetic nerve elements in the heart [26]. These changes may be related to the onset of myoglobin synthesis, however neither applies to the case of the thigh muscle. Most probably the initiation of myoglobin synthesis is scheduled by the genetic program. Acknowledgements

The research reported Grant HL 18708.

in this paper was supported

by U.S. Public

Health

Service

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Addendum 30.

CHANCE, B. Rapid and sensitive 22 (8), 619-638 (f951).

spectrophotometry.

Review of ~cie~~~~eI~tr~rne~t~~