journal

of Molecular

and Cellular

Cardiology

Histochemistry NOBLJHISA Department

(1976)

8, 599-617

of Creatine

BABA, SANGSIN KIM?

of Pathology, (Received

AND

Ohio State University,

28 May

1975,

Phosphokinase* ESTON CHRISTIS of Medicine,

College

accepted in revisedfinn

FARRELL:

Colmbus,

15 October

Ohio,

USA

1975)

N. BABA, S. Km AND E. C. FARRELL. Histochemistry of Creatine Phosphokinase. Journal of Molecular and Cellular Cara!iolo~ (1976) 8, 599-617. In a preliminary experiment, brief formaldehyde fixation of rat myocardium permitted preservation of creatine phosphokinase (CPK) activity and tine structure. Electrophoresis of the homogenate of the fresh and fixed myocardium demonstrated mitochondrial and three non-mitochondrial CPK isoenzymes. An osmiophilic tetrazolium salt and ferricyanide were used for the electron microscopic demonstration of CPK. With tetrazolium, the reaction products were localized near the inner mitochondril membrane in the intermembranous space of mitochondria, but the reaction was noted on the matrix side of the membranes with ferricyanide. Reaction was also present in the sarcoplasmic reticulum (SR) of the skeletal muscle. Roth heart and skeletal muscle showed diffuse background cytoplasmic reaction. The heat-sensitive braintype isoenayme of CPK was present in the extramitochondriil sarcoplasm of the heart muscle. CPK activity was demonstrated in isolated heart mitochondria and skeletal muscle SR. Myocardial degeneration was produced in the rat myocardium by injection of isoproterenol. Two to 4 h after the injection there was an extensive histochemical reduction of CPK activity in the rnyocardium. Twelve to 24 h after injection, most of the areas with initially reduced CPK reaction recovered the enzyme activity and only the subendocardial layer underwent necrosis without recovery of the CPK activity. The maximal rate of increase in serum CPK activity coincided with the most extensive depletion of the myocardium CPK. Mitochondrial isoenxyme did contribute to elevation of serum CPK and appeared to remain in the injured myocardial cells. KEY

WORDS: Creatine

kinase;

CPK;

Mitochondria;

Isoproterenol;

Myocardium;

Rat.

1. Introduction Creatine is an

phosphokinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2.) (CPK) important

enzyme

in

muscle

metabolism

[7,

36,

461.

Myocardial

CPK

has

been known to be a sensitive measure of myocardial ischemia, and serum CPK assay has been routinely used in clinical pathology [9]. Furthermore, quantitative CPK isoenzyme determination possesses a possible prognostic value by sizing of acute

myocardial

infarction

[31,

57,

58,

621.

There

are

four

CPK

isoenzymes,

and

* This investigation was supported by grants from the Central Ohio Chapter of the American Heart Association. Dr Kim was a recipient of the Research Fellowship from the Central Ohio Chapter. Dr Farrell was a Clinical Chemistry fellow supported by a USPHS grant GM 01805. t Present address: Christ Hospital, Cincinnati, Ohio, U.S.A. $ Present address: Ohio Valley Hospital, Steubenville, Ohio, U.S.A.

600

N. BABA,

S. KIM

AND

E. C. FARRELL

the association of these isoenzymes with intracellular compartments of the heart muscle, skeletal muscle and brain have been recognized [Z, 4, IS, 20, 21, 32, 34, 39, 44, 58, 611. Although several histochemical studies of CPK have been reported [26, 28, 30, 38, 54, 56, 611, ultrastructural histochemical localization of this enzyme has been little investigated [18]. In the first part of this paper, light and electron microscopic histochemical demonstration of CPK activity will be presented using normal rat heart and skeletal muscle. Isoproterenol administered at a sublethal toxic dosehas long been known to produce myocardial degeneration and necrosis resembling ischemic lesions [S, 10, 48, 491. In the second part of this investigation, development of isoproterenol-induced myocardial injuries will be assessed by histochemical staining of myocardial CPK activity and elevation of serum CPK. Although pathogenesisof isoproterenol-induced myocardial degeneration is not clear, the current experimental approach permits production of myocardial necrosiswithout thoracotomy. It is essentialfor a study of serum CPK to avoid direct injury of chest wall musclesby thoracotomy and to prevent unwanted elevations of serum CPK from damaged skeletal muscle.

2. Materials

and Methods

Anima1.s

Male rats of the Sprague-Dawley strain weighing 200 to 300 g were used. CPK in the normal heart and skeletal muscle was studied with a total of 22 rats. Formaldehyde-fixed muscle tissueswere obtained from 10 rats, and the rest of the animals were used for studies of unfixed tissuesand of fixatives other than formaldehyde. For the control study, each observation listed in Table 2 was made on sections obtained from 5 animals. The evaluations made in the Table 2 were based on the observations of all of these sections. Fifty rats received a single subcutaneous injection of isoproterenol hydrochloride at the dose of 75 mg/kg of body weight. Ten animals were sacrificed, each at 2, 4, 8, 16 and 24 h after they were injected. At each time, two sham-injected control animals were studied. Out of 10 animals in each group, 5 were used for conventional histological and electron microscopic observations without histochemistry. The 5 other hearts were fixed with formaldehyde for CPK histochemistry.

Preparation for serum chemistry

Approximately 1 ml of blood was drawn from the jugular vein for serum CPK determination and electrophoresis.

HISTOCHEMISTRY

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601

Histochemicaljixation

Fresh 2% formaldehyde was prepared by heat depolymerization of paraformaldehyde [45]. The fixative was buffered with 0.05M sodium cacodylate (pH 7.2) containing 6% glucoseand 6% dextran, and 10 mM of cysteine was added to reduce lossof CPK activity [42]. The abdominal aorta was infused retrogradely with the cold (4°C) fixative under pressureof between 80 and 120mmHg for 10 min. Two mm cubesof the ventricular myocardium and the pectoralis muscle were dissectedand rinsed for 2 h in a cold wash solution (sodium cacodylate buffer with dextran and glucose).

of whole tissue homogenate

Preparation

Blocks of fresh and fixed heart and skeletal muscle were washed free of red blood cells in saline. The tissueswere homogenized in 5 to 10 volumes of O.lM phosphate buffer (pH 7.2) which contained 0.01~ reduced glutathione [S]. After centrifugation at 20 000 g, the supernatant was collected for CPK determinations.

Preparation

of mitochondrial

pellets

Blocks of fresh and fixed ventricular myocardium were washed, homogenized in 0.05~ Tris-HCl buffer (pH 7.2) and centrifuged at 800 g for 20 min. The supernatant was centrifuged at 8000 g for 20 min to obtain a mitochondrial pellet [40].

Preparation

of sarcoplasmic reticulum (SR)

The supernatant, obtained similarly from skeletal muscle using 0.05~ Tris-HCl buffer (pH 7.2) after 20 min of centrifugation at 8000 g, was recentrifuged at 100 000 g for 10 min and the sediment was discarded. The supernatant was again centrifuged at 100 000 g for 60 min to sediment SR [23].

Enzyme assay

The mitochondrial and microsomal pellets were resuspended in 0.1~ phosphate buffer (pH 7.2) to make approximately a 10% suspension.CPK activities in the tissue homogenates, serum and cell fractions were assayedby the Rosalki method [SO]. The activity was expressedin international units (iu) per gram of wet tissue or litre of serum. One iu corresponds to one p M of creatine phosphate (CP) hydrolyzed per minute.

602

N. BABA,

S. KIM

AND

E. C. FARRELL

Electrojhoresis

Electrophoresis was performed on agarose gel in barbital buffer (pH 8.6, ionic strength 0.05) One ~1 of the sample was placed into each well and 9OV direct current was applied for 40 min. After incubation in the same medium as used for enzyme assay (U.S.), CPK reaction was visualized in gels by fluorescence of NADPHa under U.V. light (340nm). To assess the heat-sensitivity of the isoenzymes, the tissue extracts were pretreated at 42°C for 20 min prior to electrophoresis [II, 671. Light microscopic histochemical studies

The histochemical methods in this study were modifications of the techniques used by Sjiivall [56] and Kishino et al. [3U]. The principle of the histochemical reaction is asfollows : cp+ADp ATP + glucose Glucose-6-phosphate + NADP NADPHz + PMS ---NADP Reduced PMS + NBT ---+.

CPK e (endogenous) HK + (exogenous)

creatine + ATP glucose-6-phosphate + ADP

G-6-PDH +6-phosphogluconate + NADPHa (exogenous) reduced PMS + blue formazan PMS +

The reaction medium contained 0.05~ Tris-HCl (pH 7.2), CP (6.4 mM), ADP (1 mM), 3000 units/d1 hexokinase (HK) (Grade 1)., 15 units/d1 glucose-6-phosphate dehydrogenase (G-6-PDH) (Type XI), magnesiumacetate (lOmM), NADP (ImM), glucose (0.33~), AMP (lOmM), phenazine methosulfate (PMS) and nitro blue tetrazolium (NBT) (0.6 mM).

After perfusion fixation and wash, the 2 mm tissue cubes were immersed for 30 min in a cold 7% aqueous solution of dimethyl-sulfoxide (DMSO) [66] and rapidly frozen with liquid nitrogen. Seven pm thick frozen sectionswere cut and thawed in DMSO. For uniform infiltration of the ingredients into the tissue, the sections were preincubated in the reaction medium for 20 min without CP and NADP. The preincubated sections were kept in the complete reaction medium for 40 min at room temperature under constant agitation. Stained sections were briefly rinsed in 0.05~ cacodylate buffer (pH 7.2) containing 0.3~ sucroseand mounted on glassslideswith glycerin jelly.

HISTOCHEMISTRY

OF CREATINE

Histochemical

PHOSPHOKINASE

603

controls

At first, to study the specificity of CPK reaction, each of the histochemical ingredients, including CP, was deleted from the reaction medium (Table 2). Secondly, effects of specific CPK inhibitor [7] were investigated by preincubation of the tissue with 0.03mM 2,4-dinitrofluorobenzene (DNFB). Thirdly, to study effects of the endogenous NADPHs dehydrogenase (diaphorase) and G-6-PDH, experiments for these enzymes were performed with 0.02m~ NADPHz and 2.3mM disodium glucose-6-phosphate (G-6-P) as substrates. Since diaphorase activities have been shown to be bypassed with PMS [15], these control experiments were repeated with and without PMS. Amounts of PMS, TC-NBT and NADP in these experiments were the same as in the CPK histochemical medium. Becauseof the soluble nature of cytoplasmic CPK, diffusion of CPK during the experiment is quite possible. In order to seewhether or not soluble CPK can penetrate through the organellar membranes into the subcellular particulate compartments, formaldehyde-fixed liver slices and crude liver mitochondria fraction were preincubated with commercial CPK preparation. The liver has been known to be free of CPK [12, 581. Preincubation was done in saline containing 5mg/ml of rabbit muscle CPK for 30 min, and the tissue was rinsed before fixation with formaldehyde.

Electron microscopic histochemical studies Medium

A

Essentially the same as previously described for light microscopy. NBT was replaced with 0.03nm thiocarbamyl nitro blue tetrazolium (TC-NBT) [53].

Medium

B

This contains potassium ferricyanide as the electron receptor [27, 30, 451. Neither tetrazolium salt nor PMS was included. Additional ingredients were: copper sulfate (60 nm), sodiumpotassiumtartrate (100 mM>and potassium ferricyanide (10 m@. The histochemical reactions after the formation ofNADPH2 in the medium B are : NADPHs + Fes+ (CN)s---(s-)-----------, 2 Cu2+ + Fez+ (CN)e ----(4-j-

NADP + Fe2+ (CN)s----(4-j CusFe (CN)s (insoluble, electron dense)

Difference between the media A and B was investigated with normal tissuesobtained from 5 rats. Equal numbers of experiments were repeated for both media.

604

N.

BABA,

S. KIM

AND

E.

C. FARRELL

ElectronMicroscopy Fixation and handling of tissuesfor electron microscopy were similar to the light microscopic technique. Thickness of frozen sections for electron microscopy was 40 pm. The sectionsstained in a Medium A with TC-NBT were heated in a 10% aqueoussolution of OS04 at 60°C for 40 min. Osmicated sectionswere dehydrated in ethanol and propylene oxide and embedded in an epoxy resin mixture [37]. Ultra-thin sectionswere stained with uranyl acetate and lead citrate [47l. Frozen sections and the cell fractions obtained from the fixed tissue were incubated in medium B for 40 min at room temperature. After brief washing, the pellets were fixed in 1% osmium tetroxide, dehydrated and embedded. Ultrathin sectionswere stained only with uranyl acetate. In an attempt to demonstrate intracellular distribution of heat-resistant CPK isoenzymes [I.!?, 131, small piecesof fixed heart tissuewere preheated at 42°C for 20 min prior to staining. For the investigation of isoproterenol-injured myocardium, each tissue segment under study was stained in both media A and B. As the histochemical control for injured myocardium, medium A without the substrate CP was used.

3. Results

E$ectsoffixation on normalrat muscles Enzyme assay The effect of formaldehyde fixation is shown in Table 1, which caused a strong inhibition of myocardial CPK and a lesserdegree of inhibition was noted in fixed skeletal muscle tissue. The difference between heart and skeletal muscle may be the result of the retrograde aortic perfusion during which more fixative flowed through the coronary circulation than through the pectoralis muscle. CPK activities in mitochondrial and SR fractions are alsoshown in Table 1.

Electrophoresis Electrophoresis demonstrated four CPK isoenzymes in fresh tissue homogenate (Plate 1) : BB-type (brain-type), MB (hybrid-type), MM (muscle-type) CPK, and the mitochondrial isoenzyme (,CPK). BB isoenzyme was strongly heat-inactivated, while other isoenzymes were less inhibited. Tissue fixation resulted in poor separation of isoenzymes. &PK appeared in a poorly defined spot and MB- and BB-type CPK formed one single spot. We concluded that portions of the mito-

+

1

.-...

-

_.--_-_II_ I)/

, I I \

,,,,

/,

HISTOCHEMISTRY

TABLE

OF CRRATINE

605

PHOSPHOKINASE

1. CPK contents of tissue homogenates. Effects of formaldehyde tissue fixation on CPK activities and distribution of CPK in mitochondria and SR are demonstrated. In the formaldehyde-fixed myocardial tissue, 6% of CPK activities remains and in the fixed skeletal muscle, 31% of the CPK activities. Cardiac muscle mitochondria contain 20% of the total myocardial CPK and SR of the skeletal muscle 0.2% Tissue

Heart Muscle

Preparation

Homogenates

Mitochondria Skeletal Muscle

Homogenates

Treatment Fresh n=5 Fixed n=5 Fresh n=5 Fresh n=5

Microsomcs

chondrial and cytoplasmic isoenzymes mitochondria contained only a single tained only MM CPK.

Fixed n=5 Fresh n=5

CPK activities

187 f20 iu/g wet wt ( f 1 s.E.) 1.56hO.14 iu/mg protein 11.3 k4.7 iu/g wet wt (6% of fresh tissue) 39.0 *7.4 iu/g wet wt 2.11 f0.45 iu/mg protein 485f81 iu/g wet wt 2.57 fO.17 iu/mg. protein 150+14 iu/g wet wt (31% of fresh tissue) 1.2 f0.4 iu/g wet wt 0.37kO.01 iu/mg protein

survived formaldehyde fixation. band of ,CPK and the isolated

Isolated SR con-

Light microsco&c histochemical studiesonnormalrat heartmuscle The myocardial fibres were intensely stained (Plate 2) and at high magnifications there was a distinct pattern of striation. Stains were also localized in numerous small round or rod-shaped bodies which appeared to be mitochondria. Unfixed fresh frozen sections showed less intense stain (Plate 3). Control sections incubated without each one of the ingredients showed no stain (Table 2). DNFB completely inhibited enzyme activity. A weak reaction noted in the absence of G-6-PDH was probably due to contamination of commercial HK preparation with various amounts of G-6-PDH. The liver cells showed no staining in the complete medium. In the control experiments for endogenous NADPHz dehydrogenase, heart muscle showed a very faint reaction and the liver cells showed a moderate reaction. PMS inhibited the endogenous NADPHa dehydrogenase. G-6-PDH reaction, in the presence of PMS, was very faint in the heart muscle cells and weak in the liver. Intensity of NADPHa and G-6P dehydrogenase reaction was much weaker than

606 TABLE

N.

BABA,

S. KIM

AND

E. C. FARRELL

2. Light microscopic histochemical evaluation of CPK staining using controls. Liver tissue is used for negative tissue control for its lack of CPK. Numbers in the note column indicate the purpose of control experiments. (1) CPK reaction, (2) control on adehyl kinase (AK). Negative reaction indicates no interference from AK on CPK reaction. (3) Weak reaction is due to interference from contamination of commercial HK preparation with G-6-PDH (see text). (4) Positive CPK reaction without CPK indicated that NADPHa dehydrogenase may be involved in the histochemical reaction without PMS. (5) and (7) NADPHz dehydrogenase can be bypassed both in the heart and liver with PMS. (6) and (8) NADPHz dehydrogenase reaction. No PMS is added to bypass this reaction. (9) and (10) Investigation on the endogenous G-6-PDH. There is weak reaction in the heart and strong reaction in the liver only after a prolonged incubation period. (11) Non-enzymatic hydrolysis of CP does not occur in the tissue. (12) Specific CPK inhibitor abolishes the enzyme reaction. (13) and (14) Possible diffusion of CPK into the liver tissue was examined. Liver showed a positive reaction which, by electron microscopy, was only limited to the surface exposed to exogenous CPK. No significant diffusion of CPK into the tissue block was found. (15) and (16) No specific reduction of NBT occurs in the tissue

Heart Complete medium less CP less ADP less HK less G-BPDH less NADP less PMS less HK and G-6-PDH less HK, G-6-PDH and NADP NADPHs, NBT, PMS, CP, ADP, glut. NADPHa, NBT, CP, ADP, glut. NADPHa, NBT, PMS NADPHs, NBT G6P, NADP, G6P, NADP,

NBT, PMS, CP, ADP, Mgs+ NBT, PMS, Mgs+

Complete medium with : boiled tissue pretreated tissue DNFB fresh tissue preincubated with purified CPK fixed tissue preincubated with purified CPK NBT, PMS NBT

Liver

Note

-

(3)

-

(4)

-

(5)

++++ -,zt +++ + f -7f -,!r ++ ++++ -

” ++

I;;

63)

+ (9) + (10) -

(11)

+

(13)

+ -

(14) (15)

(12)

(16)

HISTOCHEMISTRY

OF CREATINE

PHOSPHOKINASE

607

CPK staining. Therefore, histochemical interference by the endogenous NADPHs and G-6-P dehydrogenases in the heart muscle was considered minimal. Preheating at 42°C reduced the overall stain intensity and the stain remaining after preheating was taken for the localization of ,CPK and MM-CPK. After 10 min of fixation, either glutaraldehyde alone or a mixture of glutaraldehyde and formaldehyde [28] completely abolished CPK reaction. Glyoxalfixed tissue showed a markedly reduced CPK reaction. Therefore, 2% formaldehyde [49] was considered the best fixative.

Electron microscopic studies on normal muscles

The reaction products of the medium A were fine, granular, electron-opaque deposits. The reaction products show a tendency to form coarse granules in some areas. Positive reaction was observed in the mitochondria of the heart and skeletal muscle (Plates 4 and S), and the depositsexisted mostly within the space between the outer and inner membranes or between the cristal membranes [Plate 4(a)]. While a strong CPK activity was present in the SR of the skeletal muscle (Plate S), the reaction in the cardiac SR was inconspicuous. A moderate amount of reaction was scattered throughout the sarcoplasm of the heart and skeletal muscle cells (Plates 4 and 6), some of which appeared to overlap myofilaments. Medium B produced a strong CPK reaction in the terminal cisternae of SR in the skeletal muscle. T-tubules and nuclei were free of enzyme activity. Preheating at 42% reduced cytoplasmic CPK activity (Plate 5). Mitochondrial CPK retained its activity after preheating, suggestingheat resistanceof the mitochondrial isoenzyme. Endogenous NADPHs dehydrogenase was localized primarily along the mitochondrial cristae of cardiac muscle and little cytoplasmic staining was noted. Positive reaction of G-6-PDH required a much more prolonged incubation (over 2 h) than CPK, although the intracellular distribution of G-6-PDH was similar to CPK. Positive CPK reaction was noted only in the outer edges of the pellet of fixed liver mitochondria preincubated with commercial CPK and no reaction was noted within the mitochondria. Diffusion of CPK across the fixed mitochondrial membraneswas, therefore, regarded as minimal.

Comparison between medium A and medium B

With medium B, the reaction product was found to be finely granular with a sufficient electron-opacity, and the distribution of the reaction product was similar to that with medium A (Plates 7 and 8) with the exception of the mitochondrial stain. A marked lossof reaction product following alkaline lead stain proved to be a

608

N.

BABA,

S. KIM

AND

E. C. FARRELL

serious drawback, since organellar membranes could not be delineated satisfactorily without lead stain. One of the major discrepancies between media A and B was the intramitochondrial location of reaction product. With medium B, the deposition appeared to be over the inner membrane mostly on the matrix side of the membrane. With medium A, most of the depostion was on the side of the intermembranous or intracristal space.

Mitochondrialfractions

of the heart muscle

The activity of CPK was demonstrated along the cristal membranes a lesser amount of deposits was observed in the outer compartment chondria.

SRfractions

(Plate 9), and of the mito-

of the skeletal muscle

The microsomal fraction consisted of numerous vesicles of varying size lined by smooth membranes. CPK activity was observed in only a few vesicles in which dense granular deposits of the reaction product overlapped the membranes of the SR vesicles.

Light microscopic histoc&mical

observations on isojroterenol-induced

injuries

As early as 2 h after isoproterenol injection, focal reduction of myocardial CPK activity became evident (Plate 10). Extensive areas with diminished CPK activity were noted at 4 h (Plate 11). The affected areas were much wider than the areas with myocardial degeneration and necrosis recognizable with conventional light microscopy. In the affected areas, individual muscle fibres exhibited a smaller amount of enzyme reaction remaining in the form of dense granularity replacing the normal fibrillar pattern (Plate 12). In the subendocardial regions which underwent myocardial necrosis, the reduction of enzyme activities persisted (Plate 13). Once necrosis of myocardial cells set in, there was no demonstrable CPK activity.

Electron microscopic histochemical observations on isofiroterenol-induced

myocardial injuries

The earliest alteration noted at 2 and 4 h after isoproterenol injection consisted of a considerable reduction of reaction product in the sarcoplasm and the persistence of strong activity in the mitochondria (Plate 14). The relative amount of reaction product in the mitochondria at this stage appeared more abundant than in the control

HISTOCHEMISTRY

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609

PHOSPHOKINASE

rats, which explains granularity of CPK observed with a light microscope. These early changes were gradually followed by the slow reappearance of an increased amount of reaction product in the sarcoplasm and by somewhat reduced activity in the mitochondria after 8 h (Plate 15). In the necrotic cells, there was almost complete loss of enzyme activity from the sarcoplasm. Some intracellular debris, such as fragments of myofilaments, showed a few deposits. The mitochondria, however, were found to retain the reaction product in spite of advanced changes such as swelling and mineral crystallization. Even after they were released from necrotic cells, the mitochondria showed reduced enzyme reaction (Plate 16). Electron-dense CPK reaction deposit in the damaged mitochondria were seen in the sections stained both in the media A and B. The control sections were free from reaction product.

Biochemical studies of serum from isoproterenol-treated

rats

CPK levels in serum were elevated in all animals that received isoproterenol injections. In most animals the elevation was more or lesslinear, reaching a peak between 16 h and 24 h after isoproterenol injection. The maximal value ranged from 4 to 5 times normal (Figure 1).

1200

.= x

600

g .s

500 400

:

300

T-e-1

2oo 1 100 0

0

Nz7 II 2

4

I 6

I 8

I IO

I I2

I I4

I I6

I I8

I 20

I 22

I 24

Hours FIGURE 1. Elevation of serum CPK activity (international proterenol injection (subcutaneous, 75 mg/Kg of body weight). f 1 standard error.

units/l 1 of serum) following isoEach vertical bar shows the range of

610

N. BABA,

S. KIM

AND

E.

C. FARRELL

Agarose electrophoresis of serum collected at 16 and 24 h after injection showed MM- and BB-type CPK isoenzymes (Plate 17). There was some trailing toward the cathode which was not demonstrated in the electrophoresis of whole heart homogenate or serum from the control animals. In the serum from control animals, only the BBtype isoenzyme appeared as a distinct band; barely visible reaction of the MM isoenzyme was present at the origin. 4. Discussion In 1966, the first electron microscopic histochemical method for CPK was reported by Hori [18]. His method was based upon generation of ATP from CP by CPK. ATP was further converted into ADP and phosphate by the endogenous ATPase, and the final lead phosphate precipitation was noted in the sarcoplasm as well as in SR of the skeletal muscle. Because of the reliance on the endogenous ATPase, the validity of his observation may be questioned. The first light microscopic observation was reported in 1967 by Sjiivall using tetrazolium salts [%I. He was the first investigator to suggest the presence of mitochondrial CPK through a histochemical approach. He also noted abundance of CPK in the Type 2 skeletal muscle fibres. In 1968, Kishino et al. [30], by an essentially similar method confirmed Sjovall’s findings. Recently, Khan et al. [29] introduced a NBT film technique preventing undue diffusion of CPK during the procedure. The first immunohistochemical study on CPK was made in 1968 by Mittelbach and Pongratz [38] using anti-CPK antiserum conjugated with flourescein isothiocyanate (FITC). More refined immunohistochemical investigations were reported by Karpati and Sherwin [26] and Sherwin et al. [.54]. Abundance of CPK in the Type 2 skeletal muscle fibres and cross antigenecity of M-subunits of CPK among various mammalian species were confirmed by both groups [26, 38, 541. Turner and Eppenberger [61] immunohistochemically demonstrated MM-CPK in the M-line of chicken muscle fibres, ascertaining the identity of “M-line protein” with CPK. Despite the biochemical importance of CPK, little electron microscopic histochemical study has been reported so far. This is partly because of the technical difficulties in working with soluble and readily diffusible intracellular enzymes. Minimal fixation of tissue as used in the present study appeared to prevent diffusion of CPK during histochemical incubation. When fresh frozen sections were incubated in the reaction medium, a large amount of formazan rapidly precipitated into the medium, while no such precipitation was noted when fixed frozen sections were incubated. Furthermore, even though CPK activity was strongly inhibited by formaldehyde fixation, the fixed tissue was stained more intensely than unfixed tissue, which indicated that the loss of enzyme activity by diffusion from the fresh tissue exceeds the loss from fixation. The importance of minimal tissue

HISTOCHEMISTRY

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PHOSPHOKINASE

611

fixation in a histochemical study of soluble enzymes has been discussed by Fahimi and Amarasingham [15] and us [rJ in relation to lactate dehydrogenase. The second technical problem is the use of the exogenous enzymes (HK and G-6-PDH) and TC-NBT. Since HK and G-6-PDH are macromolecules (102 000 [35] and 240 000 [65] daltons respectively), diffise penetration of these two enzymes into the frozen sections was a critical requirement in the present method. The fact that CPK cytochemical reaction was negative when either one of these two enzymes was omitted from the reaction medium suggests adequate penetration of these exogenous enzymes into cells and cellular organelles (Table 2). When the tissues were incubated in the control medium in which CP and ADP were replaced by ATP, dense reaction product was observed diffusely in the cytoplasm, indicating that the penetration of the two exogenous enzymes (HK and G-6-PDH) into the tissue during incubation was adequate for the electron microscopic study. TC-NBT has been said to penetrate poorly through the cellular membranes because of its electrical charges [52]. It was noted in our experiments that when the tissue sectioner (Smith-Farquhar TC-2) was used for 40 pm sections, the histochemical reaction became much weaker than that in frozen sections. Probably cryogenic membrane damage occurred during the process of frozen sectioning and thus permitted better penetration of highly charged tetrazolium salts. The third problem is specificity of the enzyme reaction. Adenyl kinase (AK) present in muscle tissue may produce a positive reaction by production of ATP from ADP without participation of CP and CPK. According to Oliver [43], the AK activity can be inhibited by adding AMP, which we used in our medium. Without AMP and CP, no positive reaction was obtained in formaldehyde-fixed tissue, even after a prolonged incubation in the substrate-free medium, AK, therefore, appears to be inactivated by formaldehyde fixation. Complete absence of reaction without ADP indicated that endogenous ATP had no participation in the tissue reaction. Another observation supporting the specificity of our method is the complete absence of reaction in the tissue pretreated with a specific CPK inhibitor, DNFB. The fourth problem concerns possible histochemical interference by two endogenous intracellular enzymes : NADPHs dehydrogenase and G-6-PDH. NADPHz dehydrogenase was strongly inhibited by PMS in the medium [15]. Since our reaction medium contained PMS which suppresses NADPHZ dehydrogenase [15], our demonstration of CPK was little affected by endogenous NADPHz dehydrogenase. There was a similarity between intracellular distribution of G-6-PDH and CPK in rat heart. Rat hepatocellular and adipose tissue G-6-PDH have been known also for the similar distribution [3, 191. In the present study, histochemical reaction produced by the endogenous G-6-PDH in rat heart was much weaker than the CPK reaction which was performed with a great quantity of exogenous G-6-PDH. Therefore, we considered that endogenous myocardial G-6-PDH affected little of CPK reaction.

612

N.

BABA,

S. KIM

AND

E.

C.

FARREIL

The fifth problem is that possible diffusion of CPK and intermediate or final reaction product may result in false localization of CPK, since cytoplasmic CPK with a molecular weight of 80 000 daltons [II] exists mainly in the cytosol. However, the absence of reaction product in the T-tubules, nucleus, and beyond the sarcolemmal membrane seems to minimize this possibility. A control experiment with fixed isolated rat liver mitochondria immersed in commercial CPK showed virtually no CPK reaction [58]. There was little reaction within the liver mitochondria. In conclusion, there was little diffusion problem in the current histochemical procedure. Discrepancy in localization of the reaction product was noted between the media A and B in heart mitochondria; with ferricyanide, the deposition was noted on the matrix side of the cristal membrane, and with tetrazolium, the reaction product was seen on the side of the intracristal space. There are intermediary steps in the electron transfer between NADPHg and ferricyanide which include at least one flavoprotein located on the matrix side of the inner membrane [63]. It is possible that such a flavoprotein participated and affected the final localization of CPK reaction with the medium B. Biochemical data [16, 21, 331 suggest the location of mCPK to be in the inner membrane on the side of the intermembranous space and our results with the tetrazolium-PMS technique bypassing diaphorase seem to agree with them. Electrophoretic study in our investigation demonstrated heat stability of ,CPK contrary to the findings by Sobel et al. [58]. Sobel et al. treated mitochondrial extracts with deoxycholate, but we did not. Possible presence of mitochondrial membranous elements in our preparation may have contributed to this difference from Sobel’s observations. Recent biochemical studies have demonstrated that non-mitochondrial cytoplasmic CPK is bound to myofibrils [51, 611 and that myofibrillar isoenzymes possess different Xm and appear to play an important role in muscle contraction by dissipation of CP. Our electron microscopic observations of CPK reaction product overlapping myofibrils may be due to such myofilament-bound CPK. Portions of CPK reaction in A-band may be from CPK bound on myosin filaments [5J, 611. The time course of the elevation of serum CPK in the present study is generally in agreement with the observations by Wexler [64]. The peak enzyme elevation in our experiment, however, was no more than five times normal, as contrasted with an eight-fold increase in Wexler’s series. This difference probably originates from the doses of isoproterenol and the methods of blood collection. The doses in their study ranged between 250 mg and 500 mg per kg of body weight, as opposed to 75 mg in our experiment. In Wexler’s study, blood was collected from the severed neck vessels on decapitation which may have been contaminated with CPK released from the severed muscles of the neck. According to Shell et al. [55], the increased values of serum CPK activity during myocardial injury depend upon the balance between the entry of the enzyme released from the damaged myocardium and the

HISTOCHEMSTRY

OF CREATINE

613

PHOSPHOKINASE

disappearance of the enzyme already present in serum. They proved the disappearance rate of the serum CPK to be monoexponential and computed the quantity of enzymes entering from the ischemic dog heart into the serum. Their data showed that the maximal entry of the enzyme occurs from 3 to 4 h following coronary artery occlusion. When Wexler’s [64] and our data are evaluated in reference to the work by Shell et al. [55], the maximal entry of CPK is found to occur within the first 4 h. The present histochemical study demonstrated a marked initial reduction of CPK activity in wide confluent areas of the rat myocardium following isoproterenol injection. After 4 h, there was gradual return of the enzyme activity in most of the initially affected areas to an almost normal level. The initial rapid elevation of serum CPK, therefore, is a reflection of widespread reversible cellular damage rather than the exact size of myocardial necrosis seen in smaller areas at the later stage of the experiment. Since the mitochondrial changes characterize the early morphological evidence of myocardial degeneration [17, 221, the appearance of ,CPK in serum was initially anticipated. To the contrary, no ,CPK was demonstrated in the sera obtained from rats with isoproterenol-induced cardiac necrosis. This is in accord with the experience with human patients of acute myocardial infarction [26]. Our observations suggested that ,CPK remains in the degenerating mitochondria without ever being released. It is also possible that &PK [16] existed in a totally inactivated form in serum. Further study is needed on the &PK in myocardial necrosis. Braasch et al. [5] demonstrated an early increase of CPK activity in the periinfarct zones during the course of acute myocardial infarction. This finding has been refuted by Kjekshus and Sobel[31] on the basis of the assay technique. Nevertheless, Braasch et al. used 0. IM phosphate buffer and glutathione for tissue homogenization, which would have extracted and preserved the mitochondrial CPK. The findings by Braasch et al. may correspond to our observations of a relatively increased mitochondrial CPK activity in the early stage of myocardial degeneration.

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