Basic Research in

Cardiology

Basic Res Cardio183:250-257 (1988)

Rapid changes in myofibrillar proteins after reperfusion of ischemic myocardium in dogs H. Hashizume*) and Y. Abiko Department of Pharmacology, Asahikawa Medical College, Asahikawa 078, Japan

Summary: The effect of reperfusion on cardiac myofibrillar proteins in the irreversibly injured ischemic myocardium was studied in dogs. Ischemia of the myocardium was produced by complete occlusion of the left anterior descending coronary artery for 90 min (the group of 90I). Occlusion of the coronary artery was then released (reperfusion) for 0.5 rain (the group of 90I + 0.5R), 5 rain (the group of 90I + 5R), or 20 rain (the group of 90I + 20R). In control dogs, the coronary artery was not occluded (the group of no ischemia). Myofibrils (Mfp) were prepared from the myocardium (with centrifugation) in each of the groups, and subjected to electrophoretic analysis in terms of myofibrillar proteins. The yield of Mrs in the groups of 90I, 90I + 0.5R, 90I + 5R, and 90I + 20R was lower than that in the group of no ischemia. There were no marked differences, however, in the electrophoretic pattern of Mfp among the five groups. These results suggest that myofibrils are broken down during reperfusion after ischemia. Therefore, supernatant solution after the first stage of homogenization during the course of preparation of myofibrils (mrs) was also examined. There were many unidentified bands in Mfs, being assumed to be originated from myofibrillar proteins, in the groups of both 90I + 0.5R and 90I + 5R, although these bands were not observed in the group of 90I. These results indicate that degradation of myofibrillar proteins occurs rapidly after reperfusion of the irreversibly injured myocardium. It is uncertain, however, whether reperfusion has a detrimental effect on the reversibly injured myocardium, too. Key words: m__yocardialischemia; _reperfusionon myofibrillar proteins; electrophoresis of myofibrils

Introduction Occlusion of the coronary artery produces ultrastructual changes in the myocardial cells that were perfused by the coronary artery; coronary occlusion inflicts ischemic injury on the myocardium. Release of the occluded coronary artery (reperfusion) produces either disappearance or continuation (or exacerbation) of the ultrastructural changes; in the former, the ischemic injury is reversible, and in the latter, irreversible. Thus reperfusion often aggravates the ischemic injury (repeffusion injury). The ultrastructural changes of the ischemic myocardial cells include clearing of mitochondrial matrix, fragmentation of mitochondrial cristae, appearance of amorphous dense bodies in mitochondria, and relaxation of myofibrils (3, 7, 17). In the myocardium with reperfusion injury, there is a progressive increase in these ischemic changes in the mitochondria, and, in addition, there is development of contraction bands of myofibrils (3, 4, 7, 14, 16-18), which causes overextension and disruption of adjacent myofibrils and is

*) Maiden name: H. Sashida 489

Hashizume and Abiko, Myofibrillar proteins in repcrfused hearts

251

possibly related to cell death (2), indicating that myofibrils are severely affected by reperfusion. Biochemical studies have revealed that cardiac myofibrillar proteins (such as myosin, actin, and troponin) break down after p e r m a n e n t occlusion of the coronary artery for a period of 3 h or more, which causes irreversible ischemic injury (5, 6, 10, 13, 15, 20). It is reasonable then to assume that myofibrillar proteins would break down to a greater extent after reperfusion of the irreversibly injured ischemic myocardium. The purpose of the present study, therefore, was to examine the effect of reperfusion on the myofibrillar proteins in the irreversibly injured ischemic myocardium.

Materials and Methods

Animal preparation Mongrel dogs of either sex weighing 11.2_+0.8 kg (mean +_SE) were anesthetized with sodium pentobarbital (30 mg kg -1, i.v.). Under artificial respiration, a left thoracotomy was performed in thc 5th left intercostal space, and the left ventricular wall was exposed. The left anterior descending coronary artery (LAD) was freed from the adjacent tissues in order to occlude the LAD later. Dogs were divided into five groups as follows; (1) no ischemia, (2) 90 rain ischemia (90I), (3) 90 min ischemia plus 0.5 min reperfusion (901 + 0.5R), (4) 90 min ischemia plus 5 min reperfusion (90I + 5R), and (5) 90 min ischemia plus 20 min reperfusion (90I + 20 R). In each of the five groups, five animals were employed, the total number of dogs being 25. The LAD was completely occluded and then released. In order to confirm the presence of ischemia induced by LAD occlusion, an epicardial electrocardiogram was taken from the surface of the LAD area. At the end of experiments, cardiac muscle was taken from the center of the ischemic area and was immediately plunged into liquid nitrogen to prepare samples of isolated cardiac myofibrils.

Preparation of isolated cardiac myofibrils The outline of the method for preparation of the isolated cardiac myofibrils is shown in Fig. 1. Cardiac myofibrils were prepared according to the method of Zak et al. (21) with some modifications. Throughout the preparation of isolated cardiac myofibrils, centrifugation was carried out at 3,200 g for 5 rain after each wash, and the pellet was collected. The cardiac myofibrils were treated with 1% Triton X-100 for 30 min at 4~ for elimination of membrane materials (19), the resultant myofibrils being cardiac myofibrils centrifuged at 3,200 g (represented by Mfp in Fig. I). The details of the method are described in a previous paper (15). In the present study, supernatant solution at the first stage of homogenization (see Fig. 1) of the cardiac muscle was also preserved, and was centrifuged again at 20,000 g for 15 rain after the treatment with 1% Triton X-100. The resultant pellet was collected, being myofibrillar fragments in the supernatant solution (represented by Mfs in Fig. 1). In order to inhibit degradation of myofibrillar proteins during the course of preparation, 0.1 mM phenylmcthylsulfonyl fluoride was added to each of the four buffer solutions shown in the legend of Fig. 1. The concentration of the total myofibrillar protein in Mfp was determined by the buiret method, and the yield of cardiac myofibrils expressed at the amount of the total myofibrillar protein in Mfp to wet weight of cardiac muscle used. In preparing samples for an electrophoretic study, the concentration of protein was also determined by means of Bradford's microassay (1, 12).

Electrophorctic study Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in slab gels (12,0 x 9.0 x 0.1 cm) with 4.5 % stacking and 12 % separating gels using the buffer system of Laemmli (8). The gels were stained with 0.25 % Coomassie Brilliant Blue R 250. The following proteins were used as the marker proteins for SDS-PAGE; phosphorylase b_ (94,000 Da), bovine serum albumin (67,000 Da), ovalbumin (43,000 Da), carbonic anhydrase (30,000 Da), soyabean trypsin inhibitor (20,100 Da), and ct-lactalbumin (14,400 Da).

252

Basic Research in Cardiology, Vol. 83, No. 3 (1988) frozen cardiac muscle (1.0g) 3,200g 5 min

slice I

wash

shear

(Omni mixer )

1% Triton X - I O 0 30 min

, 1% Triton X-IO0 3 0 rain r 20,O00g 15 rain cardiac myofibrils

(Mfs)

cardiac myofibrils

(Mfo) Fig. 1. Method of preparation of cardiac myofibrils. (Mfp), cardiac myofibrils centrifuged at 3,200 g for 5 min; (Mrs) cardiac myofibrillar fragments in supernatant solution at the first step of homogenation of cardiac muscle. Both Mfp and Mrs were treated with 1% Triton X-100 for 30 rain in order to eliminate the membrane materials. Buffer I: 0.1 M KCI, 5 mM MgCI2, 5 mM ethylene glycol-bis(13-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM Tris-maleate (pH 6.8); buffer If: 0.25 M sucrose, 5 mM MgCI2, 5 mM EGTA, 1 mM pyrophosphate, and 0.1 mM PMSF (pH 6.8); buffer III: 0.l M KCI, 5 mM MgCI2, 5 mM EGTA, 0.1 mM PMSF, and 10 mM Tris-maleate (pH 6.8); buffer IV: 0.1 M KCI, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM PMSF, and 10 mM Tris-acetate (pH 7.0).

Statistical analysis Values are expressed as means + SEM. Differences among means were tested by one-way analysis of variance followed by Tukey's multiple comparisons test. A P-value of 0.05 or less was considered significant.

Results The ST segment of epicardial electrocardiogram was elevated by occlusion of the LAD, and the elevation of the ST segment 10 rain after occlusion in the groups of 901,901 + 0.5R, 901 + 5R, and 901 + 20R was 20.3 + 4.2, 22.4 + 5.7, 15.4 + 3.3, and 17.4 + 5.4 mV, respectively. The degree of elevation of ST was not significantly different among the four groups. The myocardial samples in the four groups were taken from the center of the ischemic area, in which an increase in ST segment was prominent. Figure 2 shows a representative slab gel electrophoretogram of the isolated cardiac myofibrils (Mfp) in the groups of no ischemia, 901, 901 + 0.5R, 901 + 5R, and 901 + 20R, and also shows an electrophoretic pattern of marker proteins (94,000 Da, 67,000 Da, 43,000 Da, 30,000 Da, 20,100 Da, and 14,400 Da). In each electrophoretogram in all five groups there were bands corresponding to the myosin heavy chain (MHC), ct-actinin (AN), the

Hashizume and Abiko, Myofibrillar proteins in reperfused hearts

253

~q'lC AN

~

~

=

~

94,000

67,000 55K

----

.-.--

.-----

9u~'

A

43,000

BIB, IBB, UBp qBiW 4Lmj,

TM

~.,~

=m~

,=~,~

~=,~

MLC1

~

~ =

,==,,,

~

MLC2

- " "'=

~

~ ~

~ ,,

~,,~m 30,000

;;~

,.._. -

,--,,.,

20,100 14,400

Fig. 2. Electrophoretogram of isolated cardiac myofibrils (Mfp). SDS-PAGE was carried out in Laemmli's buffer system. The myofibrillar protein (about 30 ~g) was applied on each well. Groups: no ischemia; 901, 90 rain ischemia; 90I + 0.5R, 90 min ischemia and 0.5 min reperfusion; 901 + 5R, 90 min ischemia and 5 min reperfusion; 90I + 20R, 90 min ischemia and 20 min reperfusion; MP, marker proteins of which the molecular weights (MW) are shown in the right side; MHC, myosin heavy chain; An, a-actinin; 55 K, the 55,000 Da protein; A, actin; TM, tropomyosin; MLC1, myosin light chain 1: MLC2, myosin light chain 2. 55,000 Da protein (55 K), actin (A), tropomyosin (TM), myosin light chain I ( M L C I ) , and myosin light chain 2 (MLC2). There was no marked difference in the electrophoretic pattern of the Mfp among the five groups, whereas the yield of Mfp differed among different groups (Table 1). The yield of Mfp in the group of 90I was lower than that in the group of no ischemia, and, in addition, the yield of Mfp in the groups of 90I + 0.5R, 90I + 5R, and 90I + 20R was lower than that in the group of 90I. Nevertheless, the elecrophoretic pattern of Mfp these ischemic (with or without reperfusion) groups was essentially the same as that in the group of no ischemia. These findings suggest that some of the myofibrils were broken into small fragments in the groups of 901, 90I + 0.5R, 90I + 5R, and 901 + 20R and that only normal or almost normal myofibrils were collected, the fragmented myofibrils remaining in the supernatant solution. Therefore, next we analyzed the supernatant solution in terms of myofibrillar proteins. Figure 3 shows a representative slab gel electrophoretogram of the supernatant fraction (Mrs) and marker proteins. Because the supernatant solution should include membrane materials as well as fragmented myofibrils, we compared the Mfs electrophoretogram in the presence of Triton X-100 (Triton ( + ) ) with that in the absence of Triton X-100 (Triton ( - ) ) . For this purpose, the supernatant solution in each of the groups was divided into halves, one of which was treated with Triton X-100 and the other was not. Each of the supernatant

Basic Research in Cardiology, Vol. 83, No. 3 (1988)

254

Table 1. Yield of isolated cardiac myofibrils. Group

No ischemia

90I

901 + 0.5R

90I + 5R

90I + 20R

Yield

45.6 + 0.7

34.0 + 1.9"

22.9 + 1.5 *a

24.9 + 1.1 *~

15.1 + 1.3 *A

% %

100.0

74.6 100.0

50.2 67.4

54.6 73.2

33.1 44.0

5

5

5

5

mg/g wet tissue

Dogs (n) 5

Values are means + SEM. 901, 90 rain ischemia group; 90I+ 0.5R, 90 min ischemia and 0.5 min reperfusion group; 90I+5R, 90 min ischemia and 5 min reperfusion group; 90I+20R, 90 min ischemia and 20 min reperfusion group. * P < 0.01 for differences with respect to no ischemia. P < 0.01 for differences with respect to 901. solutions was t h e n c e n t r i f u g e d at 20,000 g for 15 m i n , a n d the r e s u l t a n t pellet was a n a l y z e d by S D S - P A G E . A s s h o w n in Fig. 3, t h e r e was a m a r k e d difference in the e l e c t r o p h o r e t i c p a t t e r n o f Mrs b e t w e e n t h e p r e s e n c e a n d a b s e n c e of T r i t o n X-100 in t h e g r o u p s of n o ischemia a n d 901 + 0.5R. Similar results were o b t a i n e d in o t h e r groups. T h e e l e c t r o p h o r e t i c p a t t e r n of Mrs ( s h o w n in Fig. 3) in the p r e s e n c e of T r i t o n X-100, h o w e v e r , was essentially

/

/

Triton,+,

/Triton(--,/

//.."/,. w, Idl-lC AN

A

~

g4,000

=

67,000

~

,Dump ~

43,000

m

~

~

30,000

~

~

m

~

~

---- ~

---~

~,~ -

"

20,100 14,400

Fig. 3. Electrophoretogram of cardiac myofibrillar fragments in supernatant solution (Mrs) at the first stage of homogenation of cardiac muscle. Mfs was treated either with 1% Trition X-100 for 30 min (Triton (+)) or not treated (Triton ( - ) ) . Other symbols are as given in Fig. 2. The arrows indicate unidentified bands.

Hashizume and Abiko, Myofibrillar proteins in repeffused hearts

255

the same as that of Mfp (shown in Fig. 2) in all the groups, although there were some unidentified bands in the electrophoretogram of Mfs. These results suggest that Mfs contains membrane materials if it is not treated with Triton X-100. Therefore, in the following, only the results (regarding Mrs) in the presence of Triton X-100 will be described. There was no significant difference in the electrophoretic pattern of Mrs between the groups of no ischemia and 901. There was, however, a remarkable difference in the electrophoretic pattern between the group of no ischemia and those of 9 0 I + 0.5R and 901 + 5R; there were many unidentified bands between the bands corresponding to MHC and A (indicated by arrows in Fig. 3). In the group of 901 + 20R, however, the number of unidentified bands were lower. Another finding was that the intensity of the band corresponding to 55 K in the group of 901 + 20R was markedly lower than that in the group of no ischemia.

Discussion

It has been demonstrated that myocardium is irreversibly injured by coronary occlusion for 40--60 min (2, 3, 7) or 90 min (17). In the present study, the L A D was completely occluded for 90 rain in all the groups except for the group of no ischemia. Therefore, it is possible that the ischemic injury induced by the occlusion is irreversible in the groups of 90I, 901 + 0.5R, 90I + 5R, and 90I + 20R. One of the most interesting findings in the present study is that the myofibril yield in the groups of 901 + 0.5R, 901 + 5R, and 90I +20R was lower than in the group of 901 (Table 1). The content of the non-ischemic myofibrils (45.6 mg/g wet tissue) is relatively low compared with that in other studies (10, 17), suggesting that the myofibril yield in the present study underestimates the total content of myofibrils. It should be considered therefore that the data in Table 1 are not an accurate estimate of myofibrillar content in the myocardium but a result of a pilot study. Nevertheless, the date in Table 1 clearly show that the myofibril yield decreases with an increase in duration of the reperfusion period. The cardiac myofibrils we prepared were obtained by ordinary centrifugation at 3,200 g for 5 min, the pellet being called Mfp. The phase and electronmicroscope examinations have revealed that Mfp are composed almost entirely of myofibrils (15). If the myofibrils were fragmented, however, they would not be centrifuged at 3,200 g because of light weight. This view and the findings in the present study suggest that during ischemia and/or reperfusion myofibrils are broken into small fragments. This suggestion can be supported by the fact that there are some fragmented myofibrils, when examined with a phase microscope, in Mfp prepared from the ischemic myocardium. In order to collect the fragment myofibrils, the supernatant solution that bad been obtained with ordinary centrifugation was centrifuged again at higher gravity for a longer period (20,000 g for 15 min), the pellet being called Mfs. In the present study, both Mfp and Mrs were analyzed in terms of myofibrillar proteins by means of an electrophoretic technique. The electrophoretic patterns of Mfp in all the groups of no ischemia, 901,901 + 0.5R, 90I + 5R, and 90I + 20R were similar (Fig. 2). This means that Mfp contains only normal or almost normal myofibrils, and therefore the fragmented myofibrils during ischemia and/or reperfusion are considered to be in the supernatant solution (i.e., in Mrs), The electrophoretic pattern of Mrs (Triton (+)) in all the groups of no ischemia and 90I was similar to that in Mfp, except for some unidentified bands which were especially evident in the groups of 901 + 0.5R and 901 + 5R (Fig. 3). These unidentified bands may not be due to contamination of the cell membranes, because these bands were still present even after treatment with Triton X-100, but perhaps due to degradation products of myofibrillar

256

Basic Research in Cardiology, Vol. 83, No. 3 (1988)

proteins. In the group of 90I + 20R, however, the unidentified bands were not prominent. This is probably because degradation products were washed out of the myocardium into the coronary venous effluent during a longer period (20 rain) of reperfusion. Alternatively, myofibrils were so severely fragmented or damaged that we could not collect them, even when greater centrifugation (20,000 g) was employed. It is of special interest to note that there were many unidentified bands in the group of 90I + 0.5R, whereas there were less in the group of 90I. This indicates that reperfusion for only 30 s has a detrimental effect on myofibrillar proteins in the irreversibly ischemic myocardium, although it is uncertain whether reperfusion has a detrimental effect in the reversibly ischemic myocardium, too. Another point of interest is that the intensity of the band corresponding to 55 K protein, possibly one of the Z-line proteins (5, 6), was markedly low in the group of 90I + 20R. This evidence accords with the results in our previous paper (15), in which 3-h coronary occlusion significantly decreased the 55 K protein and ct-actinin, another Z-line protein, suggesting that Z-line proteins are easily broken down after ischemia, whether followed or not by reperfusion. It has been reported that supercontraction and randomization of myofibrils occur in the area of reperfusion injury (7, 11), which may be related to cell disruption induced by calcium, increased in the myocardium during reperfusion (9). These findings indicate t h a t the increased level of calcium may be primarily responsible for changes in myofibrillar proteins after reperfusion following ischemia. Acknowledgements The authors wish to thank Mr. Tadahiko Yokoyama for his technical assistance and Miss Hitomi Nishidate for her secretarial work.

Re[erences 1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 2. Ganote CE (1983) Contraction band necrosis and irreversible myocardial injury. J Mol Cell Cardiol 15:67-73 3. Jennings RB, Ganote CE (1974) Structural changes in myoeardium during acute ischemia. Circ Res 34, 35 (Suppl III):156-172 4. Jenniugs RB, Reimer KA (1983) Factors involved in salvaging ischemic myocardium: effect of reperfusion of aterial blood. Circulation 68 (Suppl I):25-36 5. Katagiri T (1977) Changes of cardiac structural proteins in myocardial infarction. Jpu Heart J 18:711-721 6, Katagiri T, Kobayashi Y, Sasai Y, Toba K, Niitani H (1981) Alterations in cardiac troponin subunits in myocardial infarction. Jpn Heart J 22:653-664 7. Kloner RA, Ganote CE, Whalen Jr DA, Jennings RB (1974) Effect of a transient period of ischemia on myocardial cells. II. Fine structure during the first few minutes of reflow. Am J Pathol 74:399-422 8. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680-685 9. Nag AC, Fischman DA, Aumont MC, Zag R (1977) Studies of isolated adult rat heart cells: The surface morphology and the influence of extracellular calcium ion concentration of cellular viability. Tissue & Cells 9:419-436 10. Reddy YS, Wyborny L, Lewis RM, Schwartz A (1976) Biochemical and morphological correlates of cardiac ischaemia: contractile proteins. Cardiovascular Res 10:129-135 11. Reimer KA, Lowe JE, Ramussen MM, Jennings RB (1977) The wavefront phenomenon of ischemic cell death. 1. Myocardial infarct size vs duration of coronary occlusion in dogs. Circulation 56:786-794

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12. Rubin RW, Warren R (1977) Quantitation of microgram amounts of protein in SDS-mercaptoethanol-Tris electrophoresis sample buffer. Anal Biochem 83:773-777 13. Sashida H, Abiko Y (1985) Inhibition with NCO-700, a protease inhibitor, of degradation of cardiac myofibrillar proteins during ischemia in dogs. Biochem Pharmacol 34:3875-3880 14. Sashida H, Abiko Y (1986) Protective effect of diltiazem on ultrastructural alteration induced by coronary occlusion and reperfusion in dog hearts. J Mol Cell Cardiol 18:401-411 15. Sashida H, Uchida K, Abiko Y (1984) Changes in cardiac ultrastructure and myofibrillar proteins during ischemia in dogs, with special reference to changes in Z lines. J Mol Cell Cardiol 16:1161-1172 16. Schaper J, Hehrlein F, Schlepper M, Thiedemann K-U (1977) Ultrastructural alterations during ischemia and reperfusion in human hearts during cardiac surgery. J Mol Cell Cardiol 9:175--189 17. Schaper J, Mulch J, Winkler B, Schaper W (1979) Ultrastructural, fuctional, and biochemical criteria for estimation of reversibility of ischemic injury: A study on the effects of global ischemia on the isolated dog heart. J Mol Cell Cardiol 11:521-541 18. Shen AC, Jennings RB (1972) Myocardial calcium and magnesium in acute ischemic injury. Am J Pathol 67:417--440 19. Solaro RJ, Pang DC, Briggs FN (1971) The purification of cardiac myofibrils with Triton X-100. Biochem Biophys Acta 245:259-262 20. Toyo-oka T, Ross Jr J (1981) Ca ~+ sensitivity change and troponin loss in cardiac natural actomyosin after coronary occlusion. Am J Physiol 240 (Heart Circ Physiol 9):H704-H708 21. Zak R, Etringer J, Fishman DA (1972) Studies on the fractionation of skelctal and heart muscle. In: Banker BQ, Przybylski RJ, Van der Meulen JP, Victor M (eds) Research in Muscle Devclopment and the Muscle Spindle, Excerpta Mcdica, Amsterdam, 163-175 Received June 22, 1987 Authors' address: Dr. Hiroko Hashizume, Department of Pharmacology, Asahikawa Medical College, Asahikawa 078, Japan (at present: Department of Experimental Cardiology, Max Planck Institute, 6350 Bad Nauheim,

F.R.G.).