Ultrastructural alterations of the mitochondrial ATPase in the calcium paradox as revealed by negative staining

Virchows Arch [Cell Pathol] (1982) 39:267-272 V/reho e/ B �9 Springer-Verlag 1982

Ultrastructural Alterations of the Mitochondrial ATPase in the Calcium Paradox as Revealed by Negative Staining

P.M. Rahamathulla, M. Ashraf, S. Sato and John Benedict Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, OH 45267, USA

Summary. We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P < 0.05) decrease in the number of spheres on the mitochondri- al membranes occurs during the calcium paradox (58.0_+ 4.1/lam vs. control 80.5___6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5+_ 5.0 ~tm). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs.

Key words: Calcium paradox - Mitochondria - ATPase - Negative staining - Diltiazem - Calcium channel blocker - Electron microscopy

Introduction

The calcium paradox described first by Zimmerman and Hulsmann (1966) refers to the irreversible injuries occurring in the hearts that are reperfused with calci- um-containing medium after a perfusion with calcium-free solution. It is general- ly accepted that reintroduction of calcium into the cells previously perfused with calcium-free solution is accompanied by a deterioration of mitochondrial function and decline in tissue stores of adenosine triphosphate (Nayler 1981; Ashraf et al. 1982). An earlier study [Ashraf et al. (in press)] conducted in our laboratory on isolated rat hearts subjected to the calcium paradox have revealed a reduction in ATP level in the cardiac muscle, and this reduction in cardiac ATP was considerably reduced after treatment with a calcium channel blocker, diltiazem (DTZ). The morphological correlation of functional changes in the mitochondria are generally determined either by examination of thin section of the fixed tissue or by isolation of mitochondrial pellets. In thin sections, cell organelles are subject to several chemical treatments and may not represent in vivo configurations of the cellular components. In isolation

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268 P.M. Rahamathulla et al.

procedures involving the use of mi tochondr ia l pellets, the molecular configura- t ion of the biological membranes may also be altered either by chemical or

physical agents. Therefore, we used the negative stain technique on needle biop-

sies to evaluate the mi tochondr ia l ATPase complex of the experimental hearts.

Addi t iona l morphological evidence is provided in the present study on the

characteristic changes in the mi tochondr ia l ATPase complex in the calcium

paradox injury and protective effect of dil t iazem on the mi tochondr ia l ATPase.

Materials and Methods

Sprague-Dawley rats weighing approximately 300 g were lightly anesthetized with ether. The left femoral vein was exposed and 0.4 ml of heparin was injected to prevent blood clotting. After 1 min, the hearts were excised and immersed in ice cold Krebs-Henseleit (KH) medium until the cessation of heart beat. The aortic root was attached to a cannula and immediately perfused for 10 rain with KH medium at 80 mm Hg pressure at 37 ~ C to achieve equilibration. The perfusion medium was changed to calcium-free medium according to experimental schedule given below. Diltiazem (supplied by Marion Labs, Kansas City, MO, USA) when used was added in the perfusion medium at the rate of 2 rag/1. The KH medium consisted of NaCl, 118 raM; NaHCOa, 25 mM ; Kcl, 4.7 nM; KHzPO4, 1.2 mM; CaCI2, 2.5 mM; and glucose, 5.5 mM. In the calcium-free KH medium clacium chloride was omitted. The calcium paradox in the isolated rat hearts was produced by the perfusion of calcium-free KH solution followed by reperfusion with KH solution. Both the media were gassed at 37 ~ C with 95% 02+5% CO2.

Experimental Groups

Group A: Perfusion with KH medium for 30 min. 4 rats. Group B: Perfusion with calcium-free KH medium for 30 rain. 4 rats. Croup C (Calcium Paradox): Perfusion with calcium-free KH medium for I0 min. followed by reperfusion with KH medium for 30 min. 4 rats. Group D: Perfusion with calcium-free KH medium with DTZ for 10 min followed by reperfusion with KH medium with DTZ for 30 min.

Immediately after the experiment, 4 needle biopsies were taken from each rat heart and each biopsy was placed in a drop of negative staining solution containing 1% phosphotungstic acid and 0.005% bovine serum albumin (spreading agent) at pH 7.0. A formvar carbon coated copper grid of 200 mesh was placed over the drop to pick up the tissue fragments and the excess fluid was blotted out. The negatively stained tissue fragments were viewed via a Phillips electron micro- scope operating at 60 KV. A total length of 480 nm mitochondrial inner membrane or cristae was examined from each rat for determining the density of membrane subunits. For thin sections, 1 mm pieces of tissue were fixed in 2.5% glutaraldehyde made in 0.15 M cacodylate buffer, pH 7.3, for 2 h and postfixed in 1% buffered osmium tetroxide for 1.5 h, dehydrated in ethanol and propylene oxide and finally embedded in Spurr medium. Thin sections were made on a Sorvall MT2B Ultramicrotome.

Results

The biopsies f rom the hearts after negative s taining revealed flat tened " s p r e a d "

mi tochondr ia and " n o n s p r e a d " mi tochondr ia . The " n o n s p r e a d " mi tochondr ia

were recognized by their cristae; however, their fine inner structure was not clear. In all the preparat ions, the major i ty of mi tochondr ia were flat tened and spread with their clear visible inner structure.

Group A (Control). The mi tochondr ia from the control hearts perfused with K H solut ion revealed the cristae surface covered with large number s of closely

packed bead-shaped projecting subuni ts or spheres. The head of these subuni ts

Mitochondria and Calcium Paradox 269

Fig, 1. A The mitochondrial cristae from a control heart perfused with KH solution showing large number of closely packed subunits (arrow). • 237,600. B The thin section of the same heart showing intact mitochondria (arrow). x 41,000

Fig. 2 A The mitochondrial cristae from heart perfused with calcium free KH solution. The cristae are covered with many subunits (arrow). x 239,400. B The thin section of the same showing compact mitochondria (arrow). • 62,500. All figures reduced by 30%

measured 7.4_+0.44 nm in diameter. The mean density of these subunits was 80.57_+6.05 per gm. The center-to-center distance between two subunits was 12.35_+0.98 nm (Fig. 1A). The thin section of the hearts examined revealed intact mitochondria (Fig. 1B).

Group B (Calcium-Free). The mitochondria from hearts perfused with calcium- free KH medium for 10 min also revealed large numbers of closely packed subunits as observed in the control. The mean density of subunits was 75.53 + 4.98 per lam. The spherical head measured 7.68 _+ 0.51 nm and the center- to-center distance between two subunits was 13.21+ 1.01 nm (Fig. 2A). The mitochondria observed in the thin sections of these hearts revealed moderate distortion of cristae (Fig. 2B).

270 P.M. Rahamathulla et al.

Fig. 3 A The cristae of mitochondria from heart perfused with calcium free KH solution followed by normal KH medium showing reduced number of subunits (arrow). x 240,900. B The thin section of the same heart showing distorted cristae and electron dense granules in the mitochondria (arrow). x 20,400

Fig. 4. A The cristae of mitochondria from heart perfused with calcium free medium followed by reperfusion with normal KH medium both containing diltiazem. The cristae are covered by a large number of subunits (arrow). • 239,400. B The thin section of the same heart showing slightly disrupted mitochondria (arrow) which are better preserved than Fig. 3B. x41,000. All figures reduced by 30%

Group C (Calcium Paradox). The m i t o c h o n d r i a f rom these hear ts revealed a m a r k e d reduc t ion in the n u m b e r o f subuni ts c o m p a r e d with groups A and B. The mean densi ty o f subuni ts was 58+4 .15 per ~tm and was s ignif icant ly

reduced (P < 0.01) as c o m p a r e d with groups A and B. The spher ical head mea- sured 5 .3_+0.13nm and the center - to-center d is tance two subuni t s was 21.6_+1.4 n m (Fig. 3A). The thin sect ions f rom these hear ts revealed severe d i s to r t ion o f cr is tae and appea rance o f e lec t ron dense depos i t s (Fig. 3B),

Group D (Diltiazem Treatment). The m i t o c h o n d r i a f rom these hear ts were bet ter preserved than G r o u p C. The mean densi ty o f the subuni t s was

Mitochondria and Calcium Paradox 271

75.53 + 5.06 per gm and the spherical heads measured 6.7 + 0.58 nm. The center- to-center distance between two subunits was 13.78+1.2 nm (Fig. 4A). The number of subunits per micron area was significantly higher (P< 0.05) as com- pared with hearts subjected to the calcium paradox without treatment of diltia- zem. The majority of the mitochondria observed in thin sections of these hearts revealed mild distortion of cristae and absence of electron dense deposits (Fig. 4B).

Discussion

The "spread" mitochondria from heart biopsies in the negatively stained prepa- rations revealed the presence of bead-like structures arranged on the inner cristae of the mitochondrial membranes. These mitochondrial subunits are recog- nized as coupling factor 1 or ATPase complex (Kagawa et al. 1966). The mor- phology of these subunits observed in this study is in agreement with earlier reports (Kagawa et al. 1966; Parsons 1966; Beyersdorf et al. 1981). The ultra- structure of subunits remained unaltered in the hearts perfused with calcium-free medium. However, the preparations from the hearts perfused with calcium-free KH medium followed by reperfusion with KH solution (calcium paradox) re- vealed marked differences from other groups. In this group, the center-to-center space between subunits increased substantially, but the diameter of spherical heads was significantly decreased (P < 0.05). This indicates a reduction in number and structural abnormalities. The ATPase in the hearts treated with diltiazem revealed the same morphology as seen in the control and calcium-free groups. It appears from this observation that incorporation of diltiazem in the media restricts the damage to the ATPase. There was no alteration in the ATPase in the preparation from hearts perfused with calcium-free KH solution. The structural alteration of the mitochondria observed on negative staining corre- sponds closely with those seen in the mitochondria of the heart thin sections.

It has been recognized that the ATPase complex is the site of ATP production (Kagawa et al. 1966). Numerous experimental conditions are known to modify the mitochondrial function, including ischemia or hypoxia (Nayler 1981 ; Henry et al. 1974). Jacobs et al. (1956), have shown that raised levels of calcium uncou- ples the oxidation activity of mitochondria. Nayler and Grinwald (1981), conclu- sively demonstrated accumulation of calcium during the calcium paradox inter- feres with the mitochondrial function. The increased amount of calcium perhaps interferes with the ATP production by the mitochondria. In the calcium paradox, the cellular calcium content increases which later overload mitochondria with calcium resulting in a decline of tissue stores of ATP (Ashraf et al. 1982). Accordingly, in the present study overloading of hearts with calcium occurs during the calcium paradox. This increase in calcium is associated with damage to the ATPase complex. Our study (Ashraf et al. 1982) and those of others (Boink etal. 1976; Bulkley et al. 1978) have indicated that ATP content is depleted in the calcium paradox. The present observation demonstrates that this may be due to damage to ATPase, but how exactly this happens is not clear.

The present study has also revealed that incorporation of diltiazem, a calcium antagonist in the medium, is associated with preservation of the ATPase on

272 P.M. Rahamathulla et al.

the cristae of mitochondria. This action appears to be due to reduction of calcium content of cells by diltiazem. Further, Nayler and Grinwald (1981) have also shown that verapamil, another calcium antagonistic drug, does not reduce calcium content in the cardiac tissue during the calcium paradox. Thus, diltiazem appears to have beneficial effect by blocking the intracellular accumula- tion of calcium (unpublished data) thereby preventing injury to the mitochondri- al ATPase. This modified technique of negative staining may be useful for rapid screening of the effects of therapeutic agents against various types of myocardial cell injury.

Acknowledgement. Supported by NIH grant HL 23597. Dr. Ashraf is a recipient of a USPHS Research Career Development Award (KO4 HL00540). We sincerely acknowledge the help of Wilma Christophel in editing this manuscript.

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Received November 30, 1981 /Accepted January 27, 1982