Subpopulations of human heart mitochondria

JOURNAL

OF SURGICAL

RESEARCH

4,495-498

(1986)

Subpopulations of Human Heart Mitochondria’ ERIC S. WEINSTEIN,

M.D.,’

DANIEL

W. BENSON,

M.D.,

AND DONALD

E. FRY, M.D.3

Departments of Surgery. Case Western Reserve University and the Ckveland V.A. Medical Center, Cleveland, Ohio 44106 Presented at the Annual Meeting of the Association for Academic Surgery, Cincinnati, Ohio, November 10-l 3, 1985 Anatomically and biochemically distinct populations of cardiac mitochondria have been isolated from a number of animal species. The physiologic differences between subsarcolemmal (SS) and interlibrillar (IF) mitochondria, coupled with their different location within the cytomatrix, have led to speculation about possible specificities of function within the myocardial cell. To date, these two mitochondrial subpopulations have not been demonstrated in human cardiac tissue. Subsarcolemmal and interfibrillar cardiac mitochondria were isolated from papillary muscle removed from five patients undergoing mitral valve replacement surgery. Mitochondrial respiratory activity was determined polarographically. IF mitochondria had significantly higher state 3 (ADP-dependent) rates of respiration then SS mitochondria (116.7 rt 7.1 versus 86.5 f 8.3 ng atoms or oxygen per minute per milligram mitochondrial protein; P < 0.05 [mean + SE]). These data agree with similar studies performed in other animal species and support the concept of distinct subpopulations of mitochondria within the human myocardial cell. 0 1986 Academic Press, Inc.

INTRODUCTION

Distinct subpopulations of cardiac mitochondria have been demonstrated in several animal species [ 1, 2, 31. Simple, mechanical homogenization of myocardial tissue disrupts the plasmalemma and releases the so-called subsarcolemmal (SS) mitochondria from the cell nuclei and myofibrillar apparatus following differential centrifugation. These mitochondria, which by electron microscopy have been shown to lie immediately below the cardiac cell membrane, differ biochemically from the remaining mitochondria, which lie trapped between the contractile fibers of the cell. These latter mitochondria (the so-called interhbrillar (IF) mitochondria) oxidize substrates faster, exhibit tighter respiratory control, transport calcium more rapidly, and require enzymatic

digestion of the cardiac myofibrils in order to be isolated by centrifugation. The anatomic and biochemical differences between these two subpopulations have led to speculation with regard to possible specificities of function within the cell. Intefibrillar mitochondria may be responsible for supplying the energy necessary for cardiac contraction, while the subsarcolemmal subpopulation fuels the ion pumps in the cell membrane responsible for maintaining cellular homeostasis. To date, no such studies have been performed on human myocardial tissue. The purpose of the following study is to determine if similar differences are present between subsarcolemmal and interfibrillar mitochondria in the human myocardium. METHODS

Subsarcolemmal and interhbrillar mitochondria were isolated from human papillary muscle removed at the time of mitral valve replacement surgery following cardioplegiainduced arrest. All patients had undergone preoperative cardiac angiography and those with significant coronary artery disease, re-

’ Research funding provided in part by a grant from the Veterans Administration. * Dr. Weinstein is a Dudley P. Allen Surgical Research Fellow for the department of Surgery, Case Western Reserve University. 3 To whom reprint requests should be addressed: Chief, Surgical Service, Veterans Administration Medical Center, 1070 1 East Boulevard, Cleveland, Ohio 44106. 495

0022-4804/86 $1.50 Copyright Q 1986 by Academic Press, Inc. AU rights of reproduction in my form resewed.

JOURNAL OFSURGICAL RESEARCH:VOL.40,NO.$MAY quiring concomitant coronary artery bypass grafting, were excluded from the study. SwannGanz catheters were placed immediately prior to the induction of general anesthesia for the determination of cardiac function parameters. Patients who required intraoperative inotropic support prior to removal of the papillary muscle were excluded from the study. Immediately upon removal, the papillary muscle was separated from the chordae and valve leaflets, placed in cold (0-4°C) buffer solution, and transported to the laboratory for isolation of the mitochondria. Mitochondrial isolation. The specimen of papillary muscle was trimmed of all fibrous tissue and calcium deposits, rinsed thoroughly in cold (0-4”C) isolation buffer (100 mMKC1, 50 mM Mo~s,~ 2 mM EGTA, 0.5% BSA, pH = 7.4) blotted dry, and weighed. Employing a modification of the methods of Palmer et al. [l] the tissue was finely minced and then subjected to homogenization in isolation buffer with the Polytron homogenizer at a rheostat setting of 6.5 for 2.5 sec. The wet heart weight to volume of isolation buffer ratio was 1: 10. The homogenate was centrifuged at 5008 for 10 min at 4°C. The resulting supernatant (containing SS mitochondria) was filtered through cheesecloth and kept on ice. The pellet was resuspended in an equal volume of isolation buffer using a Potter-Elvehjem homogenizer and the homogenate was again centrifuged at 500g for 10 min to yield a second supematant of SS mitochondria which was combined with the first. The remaining pellet was resuspended in isolation buffer ( 1: 10) and treated with Nagarse (a bacterial proteinase) by the method of Tomec and Hoppel [4]. Thirty seconds after the bacterial proteinase treatment, an additional volume of isolation buffer was added and the homogenate was centrifuged at 5000g for 5 min. The resulting supematant was discarded and the pellet was resuspended using the Potter-Elvehjem ho4 Abbreviations used: EGTA, ethylene glycol bi@aminoethyl ether) NJ’-tetraacetic acid; Mops, 3-(N-morpholino)propane sulfonic acid; BSA, bovine serum albumin.

1986

mogenizer. As with the isolation of the SSmitochondria, the homogenate was centrifuged at 5008 for 10 min. The supematant containing the IF mitochondria was set aside, the pellet resuspended in isolation buffer, centrifuged at 500g for 10 min and the second IF supernatant was combined with the first. The two groups of supematants were then centrifuged at 3000g for 10 min yielding the mitochondrial pellets. The mitochondrial pellets were subsequently washed twice before being taken up in 0.75 ml of isolation buffer (without BSA). Protein determinations were obtained by the biuret method following cholate digestion [5]. Measurementof mitochondrial respirations. Polarographic studies of isolated mitochondria employed a Clark oxygen electrode and a 1.15 ml reaction chamber at 25°C [6]. Mitochondrial isolates were studied in a salt-based reaction medium (80 mM KCl, 50 mM Mops, 1 rmt4 EGTA, 0.1% BSA, pH 7.0). Approximately 1.0 mg of mitochondrial protein was added to the reaction media and respiration was initiated with 200 nmole of ADP. The substrates studied were 20 mM glutamate or 10 mM succinate with 3 @V rotenone. State 3 (ADP-dependent) and state 4 (ADP-independent) respiratory rates were measured. The respiratory control index (RCI) was calculated as the state 3/state 4 rate ratio, a sensitive indicator of mitochondrial respiratory integrity. Statistics.Statistical comparisons of results between groups were obtained by Student’s t test. RESULTS

Mitochondrial isolations were performed on the papillary muscle from five patients undergoing mitral valve replacement surgery. Four of the patients were female. Their ages ranged from 50 to 76 years. Three patients had severe mitral stenosis and two patients had severe mitral insufficiency documented by preoperative angiography. Cardiac indices (l/min/m2) and pulmonary capillary wedge pressure (mm Hg) determined immediately prior to induction of anesthesia were 1.9 f 0.3 and 23.4 + 3.9, respectively

WEINSTEIN,

BENSON, AND FRY: HEART

(mean f SE). The interval from aortic crossclamp application until the specimen was removed was between 8 and 10 min in all patients. Mitochondrial isolations were begun within 30 min of aortic cross-clamp application. Approximately 1.O g of papillary muscle was used for the isolation of mitochondria.

Mitochondrial Respirations The respiratory data for human cardiac subsarcolemmal and interfibrillar mitochondria are summarized in Table 1. Human interfibrillar mitochondria had significantly higher state 3 rates of respiration than did subsarcolemmal mitochondria when glutamate was used as the substrate (116.7 f 7.1 versus 86.5 + 8.3; P c 0.05). A similar trend was noted with succinate, although this was not significant at n = 5. The differences between the two mitochondrial subpopulations in human myocardial tissue were similar to those seen in other animal species. Respiratory control indices (RCI) for the interflbrillar subpopulation tended to be higher with glutamate serving as the mitochondrial substrate (24.9 f 1.8 versus 20.2 + 0.9), although this was not significant at n = 5. DISCUSSION

The hypothesis of subpopulations of mitochondria within cells is not new. In 1967, Hulsman et al. [7] described a method for isolating two types of mitochondria from skeletal muscle using loose and tight fitting pestles in a Dounce homogenizer. Because of the rela-

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497

tively low yields of mitochondrial protein obtained by this method in both skeletal and cardiac muscle, the concept of separate mitochondrial populations within cells lay dormant until 1977 and the work of Palmer et al. [ 11. These investigators used the bacterial proteinase Nagarse to selectively lyse the contractile proteins of the cell, thus freeing the interfibrillar mitochondria for differential centrifugation in the sequential fashion described above. Interlibrillar mitochondria were shown to oxidize substrates at greater rates and exhibit increased activity of several respiratory enzymes when compared to subsarcolemmal mitochondria. These observations led to the hypothesis of a s&compartmentalization of bioenergetic processes within the cell. This concept of distinct subpopulations was questioned, however, by Matlib et al. [8], who suggested that the observed differences between SS and IF mitochondria were the result of artifacts produced by the isolation process itself. Indeed, the SS population is subjected more directly to the effects of mechanical homogenization, while the IF mitochondria are subjected to proteinase treatment. In addition, Palmer et al., in an apparent attempt to improve mitochondrial yield, chose to isolate the two subpopulations in separate types of isolation buffer: a sugar based buffer for the SS population and a salt based buffer solution for the IF mitochondria. Previous work from our laboratory examined the effects of both types of isolation buffer on each mitochondrial subpopulation and demonstrated consistent differences in mito-

TABLE 1 Glutamate

ss IF

Succinate

State 3

State 4

RCI

State 3

State 4

RCI

86.5 f 8.3 116.7 + 7.1*

4.4 f 0.5 4.9 + 0.6

20.2 + 0.9 24.9 + 1.8

50.4 + 7.1 69.7 f 6.6

11.2 f 1.4 16.4 + 1.7

4.4 f 0.2 4.3 370.2

Nofe. SS: subsarcolemmal mitochondria; IF: interhbrillar mitochondria. Respiratory rates expressed as nanogram atoms O/min/mg mitochondrial protein (mean + SE). RCI: respiratory control index = state 3/state 4. *P < 0.05 greater than SS.

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chondrial respiratory activity [9, lo]. Further studies by Palmer et al. [ 1 I] subjected each population to the other’s mechanical treatment and demonstrated that significant differences in respiratory activity remained between SS and IF mitochondria. Other investigators have confirmed the respiratory and enzyme differences between SS and IF mitochondria and have shown differences in rates of protein synthesis [ 121 and calcium transport [ 131 as well. The significance of the differences between these two populations becomes more apparent when one examines their individual responses to certain physiologic and disease states. Subsarcolemmal mitochondria have been shown by several investigators to be more susceptible to ischemia than intefibrillar mitochondria in the nonworking heart; a situation in which the cell’s homeostatic mechanisms would be under maximum stress [ 13, 141. Hoppel et al. [2] demonstrated a defect in interfibrillar mitochondrial function in the congenitally cardiomyopathic hamster which develops severe congestive heart failure at approximately 3 months of age. Human cardiac mitochondria appears to have similar differences between subsarcolemmal and interlIbri1k.u mitochondria. Although previous animal data from our laboratory have identified not only increases in mitochondrial state 3 rates, but significantly higher RCIs in the interfibrillar mitochondria, the fact that an increased state 3 rate can only be demonstrated in this study may be related to the low number of patients examined. In conclusion, significant respiratory differences between cardiac subsarcolemmal and internbrillar mitochondria have been demonstrated in human tissue that are consistent with previous observations made in animal models. Patterns of adaptation and injury to pathophysiologic stimuli demonstrated in animal models may be applicable to human studies and may provide a framework from

which to direct specific cellular therapy in human myocardial disease. REFERENCES 1. Palmer, J. W., Tandler, B., and Hoppel, C. L. Biochemical properties of subsarcolemmal and interfrbrillar mitochondria from rat cardiac muscle. J. Biol. Chem. 252: 8731, 1977. 2. Weinstein, E. S., Spector, M. L., Adams, E. M., et al. Mitochondrial and myocardial performance: Response to ischemia and reperfusion. Arch. Surg., in press. 3. Hoppel, C. L., Tandler, B., Parland, W., et al. Hamster cardiomyopathy: A defect in oxidative phosphorylation in the cardiac intertibrillar mitochondria. J. Biol. Chem. 257: 1540, 1982. 4. Tomec, R., and Hoppel, C. L. Camitine palmitoyltransferase in bovine fetal heart mitochondria. Arch. Biochem. Biophys. 170: 716, 1975. 5. Gomal, A. G., Bardawill, C. J., and David, M. M. Determination of serum proteins by means of the Biuret reaction. J. Biol. Chem. 177: 75 1, 1949. 6. Chance, B., and Williams, G. R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17: 65, 1956. Hulsman, W. C., De Jong, J. W., and Van Tol, A. Biochem. Biophys. Acta 162: 292, 1968. Matlib, M. A., Wilson, D., Rouslin, W., et al. B&hem. Biophys. Res. Commun. 84: 482, 1978. Weinstein, E. S., Benson, D. W., Ratcliffe, D. J., et al. Cardiac mitochondtial subpopulations: Implications for shock research. Circ. Shock 13: 93, 1984. 10. Weinstein, E. S., Benson, D. W., Ratcliffe, D. J., et al. Cardiac mitochondrial subpopulations: Evidence for compartmentalization of bioenergy metabolism. Surg. Forum 35: 261, 1984. 11. Palmer, J. W., Tandler, B., and Hoppel, C. L. Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: Effects of procedural manipulations. Arch. Biochem. Biophys. 236: 691, 1985. 12. Mayer, L. R., Banner, H. W., Farrar, R. P. Myocardial mitochondrial synthesis in response to various workloads. Res. Comm. Chem. Path. Pharm. 34(l): 157, 1981. 13. Wolkowicz, P. E., Michael, L. H., Lewis, R. M., et al. Sodium-calcium exchange in dog heart mitochondria: Effects of &hernia and verapamil. Amer. J. Physiol. 244: H644, 1983. 14. Weinstein, E. S., Benson, D. W., Ratcliffe, D. J., et al. Experimental myocardial ischemia: Differential injury of mitochondrial subpopulations. Arch. Surg. 120: 332, 1985.